In vitro, by measuring the protein levels of several key components involved in Wnt/β-catenin signaling, we confirmed that this signaling pathway was activated in the myofibroblast differentiation of LR-MSCs. Targeted inhibition of Wnt/β-catenin signaling by a small molecule, ICG-001, dose-dependently impeded the proliferation and transforming growth factor-β1 (TGF-β1)-mediated fibrogenic actions of LR-MSCs. In vivo, ICG-001 exerted its lung protective effects after bleomycin treatment through blocking mesenchymal-myofibroblast transition, repressing matrix gene expression, and reducing cell apoptosis. Moreover, delayed administration of ICG-001 attenuated bleomycin-induced lung fibrosis, which may present a promising therapeutic strategy for intervention of IPF. Interestingly, these antifibrotic actions of ICG-001 are operated by a mechanism independent of any disruption of Smad activation. In conclusion, our study demonstrated that Wnt/β-catenin signaling may be an essential mechanism underlying the regulation of myofibroblast differentiation of LR-MSCs and their further participation in the development of pulmonary fibrosis.An emerging paradigm proposes a crucial role for lung resident mesenchymal stem cells (LR-MSCs) via a fibroblastic transdifferentiation event in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Aberrant activation of Wnt/β-catenin signaling occurs in virtually all fibrotic lung diseases and is relevant to the differentiation of mesenchymal stem cells (MSCs).  The traditional therapeutic drugs empirically employed in the clinic are limited to nonspecific antioxidants, immunosuppressants with undesirable treatment effects and low curative rates3. Recently, two new drugs, pirfenidone and nintedanib, have shed some light on reducing lung function decline, but they fail to halt or reverse the progression of the disease5. Lung transplantation is considered to be the only definitive therapy that prolongs survival, although the post-transplantation 5-year survival for IPF patients does not exceed 50%6. In this context, it is crucial to explore the pathogenesis of pulmonary fibrosis and find out appropriate therapeutic approaches.Idiopathic pulmonary fibrosis (IPF) is an agnogenic disease characterized by diffuse alveolar inflammation and a fibrotic lung disorder. Its clinical manifestation is dyspnea on exertion, respiratory deterioration, and heart failure, eventually leading to death7. Myofibroblasts, a specialized subset of fibroblasts, embody the key features of active fibrosis by their ability to synthesize high levels of extracellular matrix (ECM), making them the key cells that represent the hallmark of IPF9. Accordingly, defining the sources of these myofibroblasts and abrogating their proliferation and accumulation could serve as a potential treatment strategy for pulmonary fibrosis.Although the pathogenic mechanism of IPF is unclear, considerable evidence indicates that the disease pathogenesis is initiated through alveolar epithelial cell injury and apoptosis, which results in the accumulation of stem cells responsible for the expansion of the fibroblast and myofibroblast populations in the lungs10. It is believed that tissue resident MSCs exist in most mammalian organs such as the lung, brain, and liver, and their resident tissue niche specifies their function during the development of disease12. Recent evidence suggests that lung resident mesenchymal stem cells (LR-MSCs) express mRNAs encoding contractile and ECM proteins indicative of a myofibroblast progenitor cell phenotype13. The behavior of these LR-MSCs is highly sensitive to the microenvironment to which they are exposed, and they can be driven by local factors to differentiate into myofibroblasts that contribute to lung remodeling at the expense of functional tissue repair14. Generally acknowledged as one of the primary causative agents of myofibroblast differentiation, transforming growth factor-1 (TGF-β1) is extensively overexpressed by cells in fibrotic lungs, resulting in the dysregulation of normal lung homeostasis by enhancing ECM accumulation and altering the balance of matrix metalloproteinases (MMPs) and their inhibitors15.Mesenchymal stem cells (MSCs) are pluripotent stem cells with properties of potential multi-directional differentiation, immune regulation and self-replication16. We have previously implicated the participation of Wnt/β-catenin signaling in lung repair and pulmonary fibrosis via regulating the differentiation of mouse MSCs into specific cells18. However, LR-MSCs are located in the interstitial perivascular regions and exhibit the ability to differentiate into cells capable of deleterious remodeling not only in mouse but also in human lungs19. To date, it remains unknown whether Wnt/β-catenin signaling works on the transdifferentiation of human LR-MSCs to invasive myofibroblasts. Unraveling this puzzle may have wide-ranging clinical significance.Wnt/β-catenin signaling is an evolutionarily conserved signal pathway that has been suggested to be crucial in regulating embryonic development and tissue homeostasis. At the cellular level, this signaling controls cell proliferation and motility, generation of cell polarity, and self-renewal of stem cells21. Along this line, inhibition of the β-catenin-mediated transcriptional complex assembly could be exploited as a potential target to restrain abnormal activation of Wnt/β-catenin signaling in IPF. In this study, we investigated the effect of disrupting Wnt/β-catenin signaling by ICG-001, a peptidomimetic small-molecule inhibitor, on myofibroblast differentiation of mouse LR-MSCs in vitro. In the bleomycin-induced mouse pulmonary fibrosis model, the disease pathogenesis is correlated to endogenous LR-MSCs abnormal differentiation and abrogated by ICG-001. More importantly, our finding that ICG-001 also abolished human transforming growth factor (hTGF-β1)-mediated fibrogenic action in human LR-MSCs further suggests that inhibiting Wnt/β-catenin signaling may be an effective strategy for treating fibrotic lung diseases.The hallmark of the activation of Wnt/β-catenin signaling is β-catenin accumulation in the cytoplasm and its translocation into the nucleus, where it binds to T-cell factor (TCF)/lymphocyte enhance factor (LEF). Nuclear β-catenin/TCF subsequently recruits the co-activators cyclic AMP response-element binding protein (CBP) and other components of the basal transcription machinery, to mediate transcription of target genes22. These lung-derived adherent cells displayed the morphology of a long spindle, spiral and radial arrangement   Fig. . The purCs) Fig. . These rClassic MSCs are defined by their capacity for adipogenic and osteogenic differentiation. In assessing this potential in human and mouse LR-MSCs, we found that both cells could be induced to undergo osteogenesis and adipogenesis Figs  and 2C. 25. Following treatment with the profibrotic cytokine hTGF-β1 at 10 ng/ml for 1, 3 and 5 days respectively, human LR-MSCs displayed increased expression of α-smooth muscle actin (α-SMA), collagen I and fibronectin, the major markers of myofibroblasts  has previously been considered as a marker for LR-MSCs in both murine and human lung tissues, and loss of active ABCG2 expression in vivo likely correlates to an altered phenotype and differentiation status of LR-MSCs19. Here, we found that ICG-001 abolished the transition of LR-MSCs from an ABCG2-expressing stem cell phenotype to a α-SMA-expressing myofibroblast phenotype in bleomycin-treated mice , the recruitment of circulating fibrocytes, and differentiation of resident mesenchymal cells31. In experimental pulmonary fibrosis, approximately 33% of fibroblasts are derived from EMT, and about 20% fibroblasts originate from the bone marrow progenitor influx32. We can speculate that differentiation of local mesenchymal cells or cells from another undisclosed source accounts for the remaining half of the fibroblasts. While the function of EMT as well as fibrocyte traffic from extra-pulmonary sources in lung fibrosis has been well investigated, whether myofibroblast transition of mesenchymal cells especially MSCs facilitates fibrotic disease progression remains to be determined. In this study, we focused on LR-MSCs with respect to their role in the development of pulmonary fibrosis.IPF, a devastating lung disease with unknown etiology, is featured by progressive deterioration of respiratory functions and poor outcomes. The key histological feature of IPF is the presence of fibroblastic foci in the background of ECM proteins34. By isolating human LR-MSCs following incubation with hTGF-β1, we demonstrate that Wnt/β-catenin signaling was activated and was able to drive the differentiation of human LR-MSCs into the myofibroblast lineage, evidenced by the expression of specific molecular markers of myofibroblasts. The Wnt/β-catenin signaling pathway affects cellular functions by regulating β-catenin levels and subcellular localization, and GSK-3β plays a pivotal role in this process. With the phosphorylation occurring at a serine residue in the N-terminus, the GSK-3β activity is inhibited leading to the hypophosphorylation of β-catenin, which allows translocation of this cytoskeletal protein into the nucleus35. Subsequent binding of β-catenin to the TCF/LEF family of transcription factors converts them from transcriptional repressors to activators36.Because current treatment approaches in clinical settings are limited and often ineffective, novel therapeutic agents that target key signal pathways involved in the pathogenesis of pulmonary fibrosis to halt disease progression are urgently needed. It is documented that the Wnt/β-catenin signaling pathway is up-regulated and the levels of Wnt isoforms are increased in the lungs of IPF patients and bleomycin-treated mice37. In addition, through the EdU incorporation and CCK8 assay of ICG-001, we confirmed that β-catenin/CBP-driven gene transcription is critical for inducing a proliferative state of MSCs.Considering a crucial role of Wnt/β-catenin signaling in MSC fate decision and the development of pulmonary fibrosis, we reasoned that inhibiting this signaling might result in the attenuation of mesenchymal-myofibroblast transition. Indeed, blocking Wnt/β-catenin signaling by ICG-001—the specific inhibitor of β-catenin/CBP-driven transcription—impedes fibrogenic action in both human and mouse LR-MSCs. Mechanistically, the action of ICG-001 in disrupting β-catenin-mediated gene transcription is unique, for it selectively prevents β-catenin/CBP complex formation by binding to CBP rather than β-catenin itselfIn vivo, our observation that targeted inhibition of Wnt/β-catenin signaling by ICG-001 protects lungs from bleomycin-induced injury is in favor of the concept that hyperactive Wnt/β-catenin signaling is involved in the pathogenesis of IPF. The antifibrotic efficacy of the Wnt/β-catenin inhibitor ICG-001 may be attributed to several potential mechanisms. First and foremost, we observed that LR-MSCs underwent bleomycin-induced phenotypic changes to become α-SMA+ myofibroblasts, suggesting that endogenous LR-MSCs failed to effectively repair lung injury and were involved in pulmonary fibrosis instead. As Wnt/β-catenin signaling is activated in the course of mesenchymal-myofibroblast transition in vitro, blocking this signaling would inhibit myofibroblast differentiation of LR-MSCs in the fibrotic lungs. This notion is consistent with the findings that ICG-001 repressed LR-MSCs to acquire a fibroblastic phenotype in bleomycin-treated mice. Secondly, bleomycin alone may not be sufficient to induce myofibroblast transition in LR-MSCs; down modulators of injury and fibrosis, such as MMPs and TGF-β1, clearly play a role in the maladaptive differentiation of LR-MSCs38. Elevated concentrations of MMP-2 were reported in the fibroblastic foci of IPF, and dysregulation of MMP activity accounts for functional alterations and tissue damage39. MMP-mediated degradation of ECM has also been implicated as a physiological mechanism for the release and activation of TGF-β, which leads to enlarged ECM deposition in fibrosis38. Consequently, inhibiting Wnt/β-catenin signaling by ICG-001 can elicit lung protective effects by repressing the expression of MMP-2, a member of the MMP family involved in the deregulation of synthesis and degradation of ECM proteins. Thirdly, ICG-001 may exert its antifibrotic action through preventing the expression of FSP-1 and fibronectin, two Wnt/β-catenin signaling target genes associated with EMT or ECM deposition41. Moreover, induction of fibrotic cytokine CTGF paralleled the progression of fibrogenesis. As a mediator of TGF-β1 expression and an enhancer of ECM deposition, the presence of CTGF promotes formation of myofibroblasts through transdifferentiation of other cells including epithelial cells, fibrocytes and MSCs42. It is therefore conceivable that blocking Wnt/β-catenin signaling by ICG-001 can inhibit the CTGF induction and further reduce ECM production.43. To avoid disrupting inflammation and be more relevant to clinical settings, we injected ICG-001 intraperitoneally 7 days after bleomycin administration. It is known that bleomycin recognize and noncovalently bind to specific sequences in DNA in the presence of ferrous ion and then to perform chemistry on a specific deoxynucleoside residue that can ultimately lead to strand scission. Inhibition of Wnt/β-catenin signaling in advance, the apoptosis at the single-cell level was reduced in the lungs of bleomycin-treated mice. More importantly, late administration of ICG-001 also suppressed the fibrogenic action and DNA strands breaks of cells induced by bleomycin, for it interrupted the continuous activation of Wnt/β-catenin signaling. Besides, these therapeutic actions are operated by a mechanism independent of any disruption of Smad activation.The pathological processes involved in bleomycin-induced rodent pulmonary fibrosis can be represented by biphasic responses, i.e. the early inflammatory reaction phase (7 days) and the subsequent fibrosis phase (apparently on day 14)in vitro and in vivo evidence, unraveled a mechanism by which Wnt/β-catenin signaling regulates the differentiation of LR-MSCs into myofibroblasts. Targeted inhibition of Wnt/β-catenin signaling by ICG-001, a small molecule, could attenuate the capability of LR-MSCs for acquiring fibroblastic properties. In addition, ICG-001 was capable of ameliorating fibrotic lesions through blocking the expression of a number of fibrogenic genes and cytokines in a bleomycin-induced lung injury model. We also identified that the antifibrotic effect of ICG-001 did not involve interference with Smad signaling. Despite the fact that more investigations are needed to thoroughly dissect the mechanisms, our study fills a gap in connecting LR-MSCs with their role in the development of pulmonary fibrosis via the Wnt/β-catenin signaling pathway, which may have an implication for the treatment of fibrotic lung diseases.In summary, the present work, supported by multiple lines of The animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals  and all experimental protocols have been approved by the Animal Care and Use Committee of Nanjing University. Lung samples from patients who provided informed and written consent following permission from the ethical committee of Nanjing University were used for preparation of human LR-MSCs, and all methods were performed in accordance with the guidelines and regulations.22. Briefly, fresh human specimens were obtained from the distal lung of 10 patients undergoing pneumonectomy for pneumothorax. These individuals are generally young, healthy, non-smokers who require the resection of a congenital sub-pleural bleb to prevent pneumothorax recurrence. The minced lung parenchyma tissue was placed in an enzyme mixture containing 0.2% collagenase I , 2.4 U/ml dispase (Sigma) for 1 h at 37 °C with shaking. This suspension was filtered followed by centrifugation. Next, lung cells were seeded in DMEM containing 10% fetal bovine serum, 4% L-glutamine, 1% nonessential amino acids, and 1% penicillin and streptomycin, and maintained in a humidified atmosphere of 95% air, 5% CO2 at 37 °C. Single separated fibroblastoid colonies were identified at a mean interval of 2 weeks after initial plating. A homogeneous population of MSCs was obtained from all colonies of an individual patient.Human LR-MSCs were isolated and cultured as previously reported44. In brief, mouse lung parenchyma tissues were digested after cutting with a razor blade. The digested material was filtered and depleted of red blood cells by Ammonium-Chloride-Potassium (ACK) lysis buffer. Cells were resuspended in PBS and stained for Sca-1 and CD45 followed by sorting using the magnetic-activated cell sorting (MACS) . Freshly isolated LR-MSCs were cultured with DMEM containing 10% fetal bovine serum, 4% L-glutamine, 1% nonessential amino acids, and 1% penicillin and streptomycin, and maintained in a humidified atmosphere of 95% air, 5% CO2 at 37 °C. Both human and mouse LR-MSCs were passaged 1:2 using 0.25% trypsin when they reached 70–90% confluence.C57BL/6 mice aged 4-6 weeks were purchased from the Medical School of Yangzhou University . Mouse LR-MSCs were isolated and cultured as previously reported by us and others6 cells/ml and were cultured for 12 h followed by treatment with various concentrations of ICG-001  for 24 h. Cells were collected after centrifugation at 300 g for 5 min. Next, cells were washed twice with cold PBS and were resuspended in 500 μl binding buffer. Each cell sample was then stained with 5 μl Annexin V-FITC and 5 μl PI and incubated in the dark at 25 °C for 15 min. Flow cytometry was performed on a FACS CaliburTM flow cytometer (Becton Dickinson) and the data were analyzed with FlowJo software .To determine the cell-surface antigen phenotypes, cells were incubated with fluorescent antibodies at 37 °C for 1 h in the dark followed by two washes with PBS. The antibodies used for human LR-MSCs included: FITC–conjugated anti-CD34, CD45; PE-conjugated anti-CD90, CD105, CD146 , CD14, CD31, CD73, HLA-ABC, HLA-DR  and CD44 . The antibodies used for mouse LR-MSCs included: FITC-conjugated anti-Ly-6A/E (Sca-1); PE-conjugated anti-CD34, CD29, CD73, CD31 , CD44 and CD45 (eBioscience). Cell apoptosis was analyzed by an annexin V-FITC and propidium iodide (PI) staining kit  according to manufacturer’s instructions. Human LR-MSCs were transplanted to six-well culture plates at a density of 1 × 103 cells/cm2 on 12-well plates in standard growth medium until they reached 80% confluence. The cells were then incubated in osteogenic induction medium, which is basic medium (DMEM low glucose with 10% FBS) supplemented with 100 nM dexamethasone, 50 μg/ml ascorbic acid, and 10 mM β-glycerolphosphate . For alkaline phosphatase staining, after 7-day osteogenic culture, cells were fixed in 4% formaldehyde for 5 min, washed twice using 20 mM Tris buffer and stained by alkaline phosphatase staining solution  for 20 min at room temperature, then cells were washed with buffer. For alizarin red staining, after 21-day osteogenic culture, cells were fixed in 70% ice ethanol, washed twice using ddH2O and dyed for 30 min with alizarin red solution (Millipore) at room temperature, then rinsed with ddH2O for four times and the images were taken.Human and mouse LR-MSCs were seeded at a density of 4 × 103 cells/cm2 on 12-well plates in standard growth medium until they reached 80% confluence. The medium was then replaced with adipogenic induction medium, which includes basic medium (DMEM low glucose with 10% FBS), 0.1 μM dexamethasone, 0.5 mM isobutylmethylxanthine, 50 μM indomethacin, and 5 μg/ml of insulin (Invitrogen). The cells were cultured for another 21 days. Then, cells were fixed with 4% formaldehyde for 40 min at room temperature, following washed twice in PBS and stained with oil red O Solution (Millipore). After a 50-min incubation at room temperature, cells were washed twice using ddH2O and the images were taken.Human and mouse LR-MSCs were seeded at a density of 4 × 1026. Both human and mouse LR-MSCs were pretreated with ICG-001  for 1 h, followed by incubation with 10 ng/ml hTGF-β1 or TGF-β1  for 24 h. The cells were collected for Q-PCR, Western blot analysis, and immunofluorescence staining.Human LR-MSCs were treated with or without hTGF-β1  at 10 ng/ml for indicated periods of time, and then harvested for analysis of the myofibroblast markers and the key components of Wnt/β-catenin signaling. A small-molecule inhibitor, ICG-001, which specifically disrupts β-catenin signaling in a CBP-dependent fashion has been reported in previous literatureCell proliferation was assessed by 5-ethynyl-2′-deoxyuridine (EdU) incorporation using the Click-iT EdU Alexa Fluor 488 cell proliferation assay kit (Invitrogen). Human LR-MSCs were grown on glass coverslips in DMEM containing 10% FBS, 4% L-glutamine, 1% nonessential amino acids, and 1% penicillin and streptomycin. Then, human LR-MSCs were treated with 10 μM ICG-001 for 24 h in the presence or absence of hTGF-β1 (10 ng/ml). The EdU solution was added to the medium during the last 4 h of the 24-h incubation period. After labeling, cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Then cells were washed with 3% BSA in PBS and were incubated with 0.5% Triton X-100 in PBS for 20 min at room temperature. EdU-labeled cells were visualized using Click-iT reaction cocktails (Invitrogen). Cells were incubated with Hoechst 33342 to visualize nuclei, then analyzed using a confocal fluorescence microscope .Cell Counting Kit-8 , which measures cell viability, is based on the conversion of an orange-colored product from water-soluble tetrazolium salt (WST-8) by dehydrogenases in live cells. Human LR-MSCs were treated with various concentrations of ICG-001  in the presence or absence of hTGF-β1 (10 ng/ml) in 96-well plates for 24 h followed by the CCK-8 assay according to the instructions from the manufacturer.Total RNA was extracted from cultured cells or lung tissues using Column Animal RNAout according to the manufacturer’s instructions. The purity of RNA was determined with a spectrophotometer . An Easyscript first-strand cDNA synthesis super mix kit (Vazyme) was used for reverse transcription polymerase chain reaction (RT-PCR). Q-PCR was conducted using the SYBR Green Q-PCR kit  on a ViiA 7 Q-PCR system . Each sample was run in triplicate, and PCR reactions without the addition of the template were used as blank controls. The relative quantification of the expression of the target genes was measured using glyseraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control. The sequences of primer pairs used in this assay are listed in Table S1.35. Proteins were separated using 10% SDS-PAGE and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes. The primary antibodies employed were: rabbit anti-glycogen synthase kinase-3β (GSK-3β), rabbit anti-p-GSK-3β, rabbit anti-smad2/3 , rabbit anti-β-catenin, rabbit anti-fibronectin, mouse anti-matrix metalloproteinase-2 (MMP-2), mouse anti-β-actin, rabbit anti-p-smad2, rabbit anti-p-smad3 , rabbit anti-collagen I, and mouse anti-α-SMA. Species-matched horseradish peroxidase-conjugated IgG  was used as the secondary antibody. The chromogenic signal intensity was detected using an Odyssey Scanning System  and quantified using image J software .Proteins were purified from either cells or lung tissues. Western blot analysis was performed as previously describedC57BL/6 mice weighing approximately 20 to 22 g were maintained under standard conditions with free access to water and laboratory rodent food. After anesthesia with pentobarbital sodium (3 mg/kg), mice received a single, slow intratracheal injection of 5 mg/kg bleomycin  dissolved in 50 μl of saline with MicroSprayer . Sham control mice received 50 μl of saline instead. In the preventive study Fig. , PBS or The mouse lungs were fixed in 4% neutral phosphate-buffered paraformaldehyde overnight, dehydrated, transparentized and embedded in paraffin before sectioning into 5 μm-thick slices. The sections were stained with hematoxylin-eosin (H&E) for structured observation, or with Masson trichrome stain for detection of collagen deposits, or were subjected to TUNEL assay and immunohistochemistry/immunofluorescence analyses.2O2. After blocking with 3% BSA, the sections were incubated with mouse anti-α-SMA overnight at 4 °C. The secondary antibodies incubated were horseradish peroxidase-conjugated goat anti-mouse IgG (Boster). The DAB Substrate System (DAKO) was used to reveal the immunohistochemical staining.Five μm-thick slides were deparaffinized with xylene before rehydration using an ethanol gradient. Next, quenching of endogenous peroxidase activity was achieved by incubation with 3% HTUNEL assay as described by the manufacturer , and the nuclei were stained with 4′ 6-diamidino-2-phenylindole (DAPI) (Sigma). The images were captured using a laser scanning confocal fluorescence microscope (Olympus).27. The following primary antibodies were employed: rabbit anti-β-catenin, mouse anti-α-SMA, rabbit anti-collagen I, rabbit anti-fibronectin, and rabbit anti-ATP binding cassette transporter subfamily G member 2 (ABCG2) (Abcam). Alexa Fluor 488-conjugated goat anti-rabbit antibody or Alexa Fluor 594-conjugated goat anti-mouse antibody (Invitrogen) was used as the secondary antibody. Nuclei were stained with DAPI (Sigma). The images were captured using a confocal fluorescence microscope (Olympus).The immunofluorescence analysis of LR-MSCs or lung tissues were performed as described previouslyExperimental results were expressed as means ± standard deviation. Differences were analyzed for significance (P < 0.05) by one-way ANOVA using SPSS for windows version 11.0 .Supplementary Information


Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. β-catenin is widely thought to be a major oncogene in HCC based on the frequency of mutations associated with aberrant Wnt signaling in HCC patients. Challenging this model, our data reveal that β-catenin nuclear accumulation is restricted to the late stage of the disease. Until then, β-catenin is primarily located at the plasma membrane in complex with multiple cadherin family members where it drives tumor cell survival by enhancing the signaling of growth factor receptors such as EGFR. Therefore, our study reveals the evolving nature of β-catenin in HCC to establish it as a compound tumor promoter during the progression of the disease. Aberrant Wnt/b-catenin signaling is thought to be a major driver of hepatocellular carcinoma. Here, the authors show that β-Catenin is predominantly integrated within the AJ complex during the early stages of this cancer and enhance EGFR signaling to promote tumour survival. Most HCC patients are not eligible for curative surgery due to the advanced stage of the disease at the time of diagnosis. Therapeutic options are limited in their availability and efficacy, with multi-kinase inhibitor Sorafenib showing survival benefit of only 3 months3. Therefore, there is a critical need for new HCC treatments. Large sequencing efforts have shown that most recurring driver mutations in HCC  are shared with other cancer types4. However, a recent global cancer gene expression analysis also reveals that HCC transcriptome is strikingly different from other cancers5. From this discovery emerges the concept that common driver mutations may have unique functional consequences in the context of HCC. However, the mechanisms of action of the principal drivers of HCC initiation and progression remain elusive.Hepatocellular carcinoma (HCC) is the second cause of cancer-related deaths worldwide6. In the absence of a Wnt ligand, β-catenin is rapidly phosphorylated by the destruction complex (comprising glycogen synthase kinase 3β (GSK3β), casein kinase Iα (CKIα), Axin, and adenomatosis polyposis coli (APC)) on four key serine/threonine residues, which primes β-catenin for degradation7. Wnt ligand binding destabilizes the destruction complex, thus promoting β-catenin stability and its translocation to the nucleus where it serves as a co-factor for TCF/LEF (T cell factor/lymphoid enhancer-binding factor) transcription factors. Second, β-catenin at the membrane is a component of the adherens junction (AJ) complex in association with cadherins. Within the AJ complex, β-catenin acts as a bridge between α-catenin and cadherins to maintain proper cytoskeleton structure, cell–cell interaction, and cell signaling8. Earlier works suggest that the AJ complex-associated β-catenin represents a reserve pool of β-catenin that can potentially feed into Wnt signaling activity, thereby allowing E-cadherin to modulate Wnt signaling by physically sequestering β-catenin at the membrane11. However, the mechanism of initial repartition and subsequent transition between these two pools remains poorly understood.β-Catenin is a protein that serves two distinct roles depending upon its subcellular location. First, β-catenin in the nucleus is a critical effector of the Wnt signaling pathway14. Similar mutations targeting Wnt pathway components are also frequent in HCC (15–33% of HCC patients carry activating mutations in ctnnb1 (coding for β-catenin)15, while 17% have inactivating mutations in axin or apc16) and have led to the conclusion that these mutations also act as dominant event to trigger oncogenic Wnt/β-catenin signaling in HCC. However, transgenic expression of degradation-resistant β-catenin in the liver is not sufficient to initiate HCC17, and it is therefore unclear whether β-catenin activation is a driving mutation or a cooperating event that supports HCC progression initiated by other oncogenic events19. In addition, loss of β-catenin in chemically induced models of HCC increases tumor progression21, suggesting a paradoxical effect for β-catenin inactivation in HCC. Finally, although the kinetics of Wnt pathway-activating mutations during the course of the disease are poorly known, aberrant Wnt signaling activity is restricted to the advanced stages of the disease22. Collectively, these data suggest that the role of β-catenin in HCC is more complex than currently envisioned based on the mutational status of the Wnt pathway in HCC patients.Mutations targeting components of the Wnt pathway are very frequent in colorectal cancer and hepatoblastoma, and aberrant Wnt signaling activation has been established as a dominant oncogenic driver in these tumors23, and initial studies drew a correlation between E-cadherin loss of expression and Wnt signaling activation in HCC24. These results led to the conclusion that E-cadherin exerts a dual tumor suppressor function in HCC: first by sequestering β-catenin away from Wnt signaling and second by maintaining cell–cell contact to prevent epithelial–mesenchymal transition (EMT) and metastasis25. However, this view may be too simplistic as E-cadherin expression in human HCC is largely variable and is elevated in 40% of HCC cases27. In addition, studies on other types of solid tumors have shown that E-cadherin can exert pro-tumorigenic functions29. These findings suggest that the role of E-cadherin in HCC is more complex than previously thought.β-Catenin accumulates at the plasma membrane of HCC cells in the absence of significant Wnt target gene activation31. We show that, despite the early occurrence of mutations targeting Wnt signaling components in human HCC, membrane localization of β-catenin is a dominant feature of HCC until the advanced stages of the disease when β-catenin progressively engages in Wnt signaling activation. At the plasma membrane, β-catenin interacts with multiple cadherin family members to promote the signaling of growth factor receptors such as epidermal growth factor receptor (EGFR) and support HCC cell survival. Overall, our study reveals the evolutive nature of β-catenin tumor-promoting functions during HCC progression.To elucidate the role of β-catenin during HCC progression, we have integrated patient data analysis with mechanistic studies using a pre-clinical model of HCC initiated upon functional inactivation of the Rb family, an almost universal event during HCC development4. However, when these mutations occur during the course of the disease and whether they directly translate into the activation of the Wnt signaling are not known. To determine the mutational status of Wnt signaling during HCC development, we took advantage of a The Cancer Genome Atlas (TCGA) HCC dataset representing patients from stages I to III. Analysis shows that mutations targeting Wnt signaling are present in ~35% of patients, independent of their tumor stage . These results indicate that nuclear localization of β-catenin is not an immediate consequence of mutations targeting Wnt pathway components, suggesting that additional events are required for aberrant Wnt signaling activation.Wnt pathway components are mutated in 30–40% of HCC patientsage Fig. , indicatage Fig. , which c36. Therefore, human/mouse cross-species analyses show that β-catenin is commonly found at the cellular membrane in HCC.Localization of β-catenin at the membrane has been observed in multiple mouse models of HCC, either chemically induced by DEN or triggered by single or combined targeted genetic alterations 31. Rb-family proteins  are key regulators of cell cycle activity and their functional alteration is almost universal during HCC initiation. Conditional inactivation of Rb gene family  in the adult mouse liver generates a well-differentiated type of HCC (TKO HCC) that recapitulates multiple features of the human disease31. Whereas β-catenin is expressed in different subcellular compartments of hepatocytes around the central vein (CV) in control liver (CTRL), it is exclusively detected at the membrane of tumor cells in TKO HCC  confirmed that β-catenin is expressed at the plasma membrane of cell lines previously derived from individual primary TKO HCC tumors (collectively designated as TKO HCC cells)31 Fig. . Stable 31 Fig.  identifi31 Fig. . In vitr31 Fig.  and redu31 Fig. . Impaire31 Fig. . In addi31 Fig. . To furtity Fig. . Collect38. Therefore, we cannot rule out the contribution of Wnt signaling in HCC cell survival even though β-catenin is undetectable in the nucleus of early-stage human HCC and TKO HCC cells by IHC. Protein translation inhibition using cycloheximide (CHX) revealed that β-catenin protein expression is stable in TKO HCC cells  expression  and canonical  Wnt target genes in TKO HCC compared to controls  and GS/glul  43) Fig. , but fai43) Fig. . Collectctnnb1 is wild type and β-catenin is phosphorylated by the destruction complex in serially transplanted TKO HCC tumors  as a readout for Wnt signaling activity in the TCGA dataset of HCC patients and found that E-cadherin expression negatively correlates with Wnt signaling activity  expression . Efficient E-cadherin KD did not repress the proliferative capacity of TKO HCC cells, despite an alteration in cell morphology and cell–cell contact at low cell density Fig. . IF anal–2. EfficThese data indicate that compound integration of β-catenin by several members of the cadherin family in the AJ complex is crucial for β-catenin retention at the membrane and HCC cell survival.34. Collectively, these data suggest that the long-term consequences of disrupting the AJ complex, which include increased proliferation, heterogeneous nuclear size, and ploidy, as well as Wnt signaling activation, are beneficial for tumor growth.To determine the long-term consequences of disrupting the AJ complex in HCC development, TKO HCC cells with E/N-cadherin single KD or DKD were subcutaneously transplanted into the flank of immunocompromised nonobese diabetic/severe combined immunodeficiency (nod-scid) mice. In contrast to the growth inhibition observed in an in vitro short-term context, DKD did not impair tumor formation in vivo Fig.  and actu8. α-Catenin KD altered the morphology (but not the cell–cell contact) of TKO HCC cells , but retains the transmembrane domain (for anchoring E-cadherin to the plasma membrane) and the cytoplasmic domain  in TKO HCC. We first disrupted AJ complex interaction with the intracellular cytoskeleton by repressing the expression of α-catenin, which acts as a bridge between the AJ complex and F-actin filaments45. Among these RTKs, E-cadherin interacts with EGFR through its ectodomain46. EGFR is overexpressed in 40–70% of HCC and treatment of HCC cell lines, xenograft, and mouse models with EGFR inhibitors represses cell growth48, indicating that EGFR signaling is critical for HCC cell proliferation. To determine whether EGFR signaling supports TKO HCC cell survival, we first silenced EGFR expression by short hairpin RNA (shRNA). Repression of EGFR expression decreases TKO HCC cell growth in a dose-dependent manner. Analysis of other EGFR family members shows no compensation by EGFR2, while EGFR3 was undetectable by qPCR and immunoblot analysis , Afatinib® (EGFR family inhibitor50), and Sorafenib® (a multi-kinase inhibitor used as a control51). In these conditions, we found that EGFR inhibition by both Erlotinib and Afatinib trigger apoptosis of TKO HCC cells and TKO HCC organoids , with varying and sometimes conflicting consequencessis Fig. . Next, wids Fig. . Interesegfr mRNA was not altered upon silencing β-catenin   Fig. . Overalles) Fig. . HoweverAnalysis of the control liver, and primary and serially transplanted TKO HCC samples indicates that EGFR expression decreases in TKO HCC vs. control livers  (Supplementary Data File), and exclusion of these outlier patients restores a significant correlation between activated EGFR and E-cadherin within the female group  such as escaping immune surveillance14, the functional consequences of aberrant Wnt signaling for HCC remain obscure, as illustrated by its association with either good or bad prognosis59. Our data, as well as others, indicate that aberrant Wnt signaling is a feature of advanced HCC, characterized by the activation of multiple other oncogenic pathways 22. This molecular context raises the question of the exact contribution of Wnt signaling for late-stage HCC maintenance and progression. In particular, the mosaic expression of GS , long-term consequences are multiple and appear to be mutually exclusive such as altered DNA content and Wnt signaling activation. In contrast to colorectal cancer and hepatoblastoma, where Wnt signaling exerts a well-established driver role GS Fig. , a key mIn conclusion, our results challenge the current view on Wnt/β-catenin signaling in HCC and reveal an unexpected role for β-catenin in HCC development, with emphasis on its complex and evolving tumor-promoting function during the course of the disease. Future research will specifically focus on targeting AJ complex-associated tumor-promoting functions of β-catenin to impact HCC development.31. All experiments with mice were approved by the Children’s Hospital of Philadelphia Institutional Animal Care and Use Committee (CHOP IACUC) (protocol #969). All animal studies were conducted in compliance with the ethical regulations for animal testing and research. For intrasplenic injection, mice were anesthetized and surgically opened on the upper left flank to inject adenovirus into the spleen. For subcutaneous serial transplantation, independent HCC primary tumors from the parental TKO mice were dissociated into single cells. The tumor cells (ranging 1 × 103–106) were resuspended in Matrigel and injected subcutaneously into the flanks of nod/scid mice. The resulting subcutaneous tumors [Generation 1] were harvested, dissociated, and subcutaneously transplanted [Generation 2]. For subcutaneous TKO HCC cell injection, TKO cells (ranging 5 × 104–105) were sorted and injected subcutaneously into nod/scid mice. Experiment endpoint was determined by the first appearance of tumors >1 cm2.Rb-family cTKO mice were maintained on a mixed 129Sv/J;C57/BL6 background30. Experiments have been performed in both cell lines with similar results and they are collectively referred to as TKO HCC cells. TKO HCC, HepG2 (ATCC® HB-8065), Hep3B (ATCC® HB-8064), and Huh-7 (JCRB0403 HuH-7) cells were cultured in DMEM (Mediatech), supplemented with 10% fetal bovine serum (FBS)/1% penicillin–streptomycin–glutamine (Invitrogen). SNU449 (ATCC® CRL-2234) cells were cultured in RPMI, supplemented with 10% FBS/1% penicillin–streptomycin–glutamine. L cells (ATCC® CRL-2648) were cultured in DMEM with 10% FBS ). Cell lines were tested for mycoplasma detection using an in-house PCR-based methods. For organoid culture, primary TKO HCCs were harvested and digested with an enzymatic cocktail to obtain a single-cell suspension. Organoids were generated and maintained as recently described37. For viral infection, shRNA sequences were generated using psicoligomaker63 and cloned into the pSicoR-PGK-RFP and psicoR-CMV-GFP vectors. Viral infection was performed as previously described31. To determine protein stability, cells were treated with 25 μg/ml CHX (Millipore) 24 h after plating, and harvested after the indicated treatment time period prior to immunoblotting. For Axin stabilization, TKO cells were treated with 5 μM XAV939 (Selleckchem) for 48 h39. For Wnt3A media treatment, the recipient cells were treated with media from L-Wnt3A cells mixed with fresh media at 1:1 ratio. For EGF treatment, serum-starved cells were stimulated with 20 ng/ml EGF for 15 min–1 h. For EGFR inhibition/multi-kinase inhibition, the cells were plated in a 6-well plate at 5 × 105 cells/well in the presence of DMSO or 0.1, 1, 10, or 50 μM and Afatinib, Erlotinib, or Sorafenib for 24 h and were harvested for analysis. For orthovanadate treatment, the cells were treated with 0.5 mM orthovanadate for 2 h and were harvested for analysis. To generate the growth curves, the cells were plated in triplicate in a 6-well plate at 5 × 104 cells/well and counted every 24 h for 5 days. Cells collected on days 0 and 5 were processed for RNA analysis and immunoblotting. For AnnexinV staining, the cells were plated in triplicate in a 6-well plate at 1 × 105 cells/well and cultured for 3 days prior to AnnexinV (Roche) analysis. For BrdU labeling, BrdU (1:100) were added to the media 1 h prior to harvest.Rb-family TKO 1.1 and 2.1 cell lines were derived from independent TKO HCC tumors42. Human Flag-β-catenin pcDNA3, for human β-catenin overexpression, was a gift from Eric Fearon (Addgene plasmid #16828)64. pWZL Blast DNE, for DNE overexpression, was obtained from Addgene (plasmid #18800)44; DNE sequence was further subcloned into MSCV-IRES-GFP (MigR1) vector. MigR1-GFP vector was a gift from Warren Pear (Addgene plasmid #27490)65. pBABE Flag-Ubiquitin, for Flag-Ubiquitin overexpression, was a gift from Donita Brady.EdTC, for dnTCF4 overexpression, was a gift from Roel Nusse (Addgene plasmid #24310)15. Primer sequences are available in Supplementary Tables Total RNA was extracted, purified, and reverse transcribed as previously described. qPCR was performed with SYBR Green PCR Master Mix (Life Technologies) on the Viia7 Real-Time qPCR system (Life Technologies). Data were normalized using gapdh as a reference gene. For sequencing of β-catenin, total RNAs were isolated from frozen samples and reverse transcribed to obtain complementary DNAs used as a template to amplify total β-cateninFor immunoprecipitation, the cells were harvested in Triton-X IP Lysis Buffer and incubated with relevant antibodies for 1 h at 4 °C, followed by 1 h incubation with A/G agarose beads. For immunoblotting, frozen tumors, control livers, and cells in culture were lysed in 1% sodium dodecyl sulfate (SDS) or radio-immunoprecipitation assay. Selected uncropped immunoblots can be found in the Supplementary Fig. Mouse immunoglobulin G (IgG) , rabbit IgG , α-catenin , APC , Axin1 , Axin1 , β-actin , β-catenin , phospho-β-catenin , Claudin7 , E-cadherin , phospho-EGFR(Tyr1068) , EGFR , EGFR1 , EGFR2/HER2 , Flag , GS , GSK3α/β , horse radish peroxidase-conjugated Flag , K-cadherin , Ki67 , N-cadherin , Vimentin , and Zeb1 . Otherwise noted, antibody concentration listed is for immunoblotting.Human HCC tissue array (HLiv-HCC060CD-01) was purchased from US Biomax. HCC stages were classified according to the AJCC Clinical Staging System (v7). β-Catenin staining pattern and signal intensity were assessed by E.E. Furth, a board-certified GI tract pathologist.https://genome-cancer.ucsc.edu/proj/site/hgHeatmap/?datasetSearch = TCGA-COAD). For protein and mRNA expression, processed files (level 3) were used for further analyses and HCC patient data were separated into two groups based on protein expression. Non-parametric t test was performed to access significance between the protein and mRNA expressions of corresponding samples . We used a two-sample Kolmogorov–Smirnov test to determine whether the distribution between both samples is different , and plotted using the R packages ggplot. Spearman’s ρ statistic was used to test for association (Supplementary Fig. Genomic DNA sequences as well as the protein and mRNA expression Figs. , 4g, h wles Fig. . Analysis. Figs.  The box s. Figs. . Pearsons. Figs. . Data weent Fig.  Protein Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


KIC) of cured modified epoxy systems with the additives are significantly higher than those of unmodified epoxy systems, without sacrificing the processability (Tg) and flexural strength. The toughening mechanism is understood as a synergism combination among the phase separation mechanism, the in situ homogeneous toughening mechanism, and the particle cavitation mechanism.We synthesized PPG-terminated tetra-carbamates as a new toughening additive for epoxy thermosets through facile addition reaction of hexamethylene diisocyanate (HDI) with poly(tetra-methylene glycols) (PTMG) and poly(propylene glycols) (PPG). The effects of prepared tetra-carbamates on the rheological behavior of neat epoxy resin were studied along with the various cured properties of their modified epoxy systems. Four carbamate groups (–NHCOO–) endow the prepared additives not only with good intramolecular interactions, but also with optimal intermolecular interactions with epoxy polymers. This results in the suitable miscibility of the additives with the epoxy matrix for the formation of the typical biphasic structure of microparticles dispersed in the epoxy matrix via polymerization-induced microphase separation. The impact strength and critical stress concentration factor ( Epoxy resins represent the most common class of thermosetting materials. Their highly crosslinked microstructures endow them with many useful properties for industrial applications, for instance, high strength, excellent thermal stability, and good adhesion. Nevertheless, their resistance to crack initiation and propagation is poor due to their inherent brittleness, which affects their durability and strongly constrains their design parameters ,2,3,4. TBlock copolymers (BCs) have attracted considerable attention through the toughening of epoxy polymers via the ordered micro/nanostructures since Hillmyer et al. first reported the ordered nanostructures within thermosets modified with BCs in 1977 . BCs canTg) and mechanical strength, even at the lower contents of additives of 2 and 5 wt %. The intramolecular and intermolecular interactions between the related components arising from the significant hydrogen bonding are proposed to be the main driving force for the ordered microphase separation and prominent toughness improvement of modified epoxy polymers with the tetra-carbamates.However, little has been published on introducing carbamates of –NHCOO– as the molecular segments of additives to mediate the interaction and miscibility of modifiers with the epoxy matrix to achieve the ordered micro/nanostructures. Herein, the tetra-carbamates with four hydrogen bond motifs separated by one poly(tetra-methylene glycols) (PTMG) segment and having two terminal poly(propylene glycols) (PPG) chains were firstly synthesized through the facile addition reaction of the commercial materials of HDI, PTMG, and PPG. The average molecular weights of up to 2000 g/mol were preferred for both polyether glycols considering the processability of modified epoxy with the prepared tetra-carbamates . The curN,N-dimethylformamide  and acetone (AR) were purchased from Aladdin Industrial Corporation and dried by 3A molecular sieves for one week. Organic tin (Thermolite 890) was obtained from PMC Group  and used as received. Bisphenol-A epoxy resin whose epoxide equivalent is about 189g/mol (DER331), was purchased from DOW  and used as received.Hexamethylene diisocyanate  and triethylenetetramine (TETA) were purchased from Aladdin Industrial Corporation  and used as received. PTMG , PPG , 2 atmosphere at 80 °C. Then a weighed amount of PTMG dissolved in DMF was added dropwise into the reactor within 3 h under N2 atmosphere at 80 °C. The molar ratio of NCO to OH was fixed at 2.05/1.00. A transparent DMF solution containing NCO–terminated PTMG was obtained for the next synthesis reactions. Secondly, a certain amount of DMF, PPG400, and Thermolite 890 was added into another reactor and stirred by a mechanical stirrer for 15 min under N2 atmosphere at 80 °C. A weighed amount of DMF solution containing NCO–terminated PTMG was added dropwise into the reactor within 3 h under N2 atmosphere at 80 °C. The molar ratio of NCO to OH was fixed at 1.00/2.05. The reaction concentration of 20 wt % was employed for all synthesis reactions, and Thermolite 890 was controlled at 0.005 wt % for all synthesis reactions. The synthetic pathway is shown in The preparation of the tetra-carbamates was performed in a 250 mL three-necked glass reactor. Firstly, a certain amount of DMF, HDI, and Thermolite 890 was added into the reactor and stirred by a mechanical stirrer for 15 min under NThe tetra-carbamates sample was introduced into DER331 at different mass percentages under constant stirring for 15 min at 60 °C in a vacuum condition until a homogeneous blend was achieved. The obtained modified epoxy samples were cooled down to 25 °C. Subsequently, the modified epoxy resins were mixed with TETA through centrifugal mixing for 2 min in a vacuum condition at the mass ratio of DER331/TETA = 100:15. The final epoxy ternary mixtures obtained were cast into stainless molds in a vacuum, and were subjected to the following curing procedures: firstly cured at 60 °C for 2 h, and then post-cured at 130 °C for 2 h for the complete curing. Finally, the cured modified epoxy samples were naturally cooled down to room temperature.−1. The spectra were obtained as percentage of transmittance vs. wavenumber. Thermal properties of samples were analyzed by a differential scanning calorimeter  at a temperature ramping rate of 5 °C/min in a nitrogen atmosphere. The rheological properties of various uncured epoxy ternary mixtures were investigated in oscillation modes by a hybrid rheometer  with parallel plates 25 mm in diameter. A small amplitude oscillatory shear test was carried out in a fixed strain of 3.125% with a constant frequency of 1 rad/s at a heating rate of 5 °C/min. The flexural strength of unmodified epoxy and cured modified epoxy thermosets were tested on a universal testing machine  with specimens (80 mm × 10 mm × 4 mm) at a loading rate of 2 mm/min according to ISO178. The critical stress concentration factor (KIC) of the single edge notch bend (SENB) specimens (40 mm × 8 mm × 4 mm) was measured according to ASTM D5045-14 with a loading rate of 10 mm/min at the temperature of 23 °C. The no-notch impact strength of the cured epoxy specimens (80 mm × 10 mm × 4 mm) was determined by an impact tester  according to ISO179. The glass translation temperature (Tg) and flexural modulus of the cured epoxy specimens (35 mm × 7 mm × 2 mm) were analyzed under a bending mode (1 Hz) with an amplitude of 5 mm and a heating rate of 10 °C /min over a temperature range of 25–200 °C on a dynamic mechanical analysis  according to ASTM D4092 and D4085. The fracture surfaces of the cured epoxy composites after impact tests were observed with a scanning electron microscope . An FT-IR Spectrophotometer  was employed to record the infrared spectra of samples. The KBr tablet with the sample was scanned from 4000 to 300 cm−1 in the FT-IR spectra of the HT2P4 sample. This indicates that the additional reactions of –N=C=O from HDI and hydroxy groups (–OH) from PTMG2000 and PPG400 were fully completed [−1 corresponding to the stretching vibration of N–H and C=O, respectively, indicated that there exist carbamate groups of –NHCOO– in the prepared HT2P4 molecules [−1 corresponding to the stretching vibration of –OH and C–O, respectively, showed the existence of the hydroxyl groups and ether bonds in the molecular structure of prepared HT2P4 [−1 but at 1721 cm−1. Many researches have shown that the absorbance position of C=O stretching is always at 1683~1707 cm−1 due to the strong hydrogen bonding with –N–H composed in reported carbamates and polyurethane [−1) in −1 in the IR spectra of the crystalized PTMG2000 to 1111 cm−1 in the IR spectra of the melted PTMG2000 as shown in The molecular structure of tetra-carbamates is shown in ompleted . The strolecules ,19,20. Murethane ,23. This−1 via a log-log scale. This is a distinct advantage of these additives when compared to the commercial toughening additives, which always result in the significant increase in viscosity of modified epoxy system, such as CTBN and BCs [The effect of different tetra-carbamates additives on the viscosities of DER331 resin systems at 25 °C is shown in  and BCs ,12,13,14 and BCs ,25,26. T and BCs ,28,29,30The effect of different tetra-carbamates additives on the curing profile of the DER331/TETA system at 60 °C is shown in The cured properties of modified epoxy/TETA systems with HT1P4 and HT2P4 were characterized to study the effect of tetra-carbamates on the cured epoxy thermosets. Less than 10 wt % additives were employed in the DER331/TETA system considering the balance of cost and toughness improvement in practical applications. All modified epoxy systems showed as transparent liquids before the curing reaction. However, the cured aspects were observed differently according to the employed tetra-carbamates in epoxy systems. The modified epoxy systems with HT1P4 often showed the cured semitransparent aspects, which can be ascribed to the good miscibility of HT1P4 in epoxy systems. Microphase separation induced by curing reaction was limited in the epoxy system containing HT1P4, whereas phase separation was executed fully in the modified epoxy system with HT2P4, which resulted in the cured opaque aspects as shown in Tg, flexural strength, and modulus of epoxy systems modified with HT1P4 decreased with the increase in the content of additives from 0 to 5 wt %, which can be ascribed to the internal plasticization of miscible HT1P4. The impact strengths and KIC of the modified epoxy systems showed a slight increase accordingly. However, it is exciting to find that the flexural strength and modulus of epoxy systems modified with 10 wt % HT1P4 increased with little further reduction in Tg. Compared to the unmodified epoxy system, they declined approximately 14% and 5% with a 15 °C reduction in Tg, respectively. Meanwhile, it was observed that the modified epoxy systems exhibited significantly higher impact strength and KIC than the unmodified epoxy systems. Impact strength increased 84.8% (16.5–31.0 kJ/m2) and KIC increased 107% (1.5–3.1 MPa·m1/2). The reduction in Tg by HT1P4 was much lower than that by the commercial plasticizers such as PPG400 at high additive contents. It is speculated that phase separation of HT1P4 in an epoxy matrix reduced the residual HT1P4 in a cured epoxy matrix. The effect of HT1P4 additive on the cured properties of modified epoxy systems is shown in .8% 16.5–.0 kJ/m2 KIC than the unmodified epoxy polymers. Impact strength of the modified epoxy system with 5 wt % HT2P4 increased 195.8% (16.5–48.8 kJ/m2), and accordingly KIC increased 226.7% (1.5–4.9 MPa·m1/2). This is a particular advantage of the tetra-carbamates when compared to the commercial additives such as CTBN. It was reported that the toughness was improved by 60% at a level of 10 wt % of CTBN [Tg and flexural strength values of the modified epoxy systems remained nearly invariant irrespective of the content of HT2P4. This indicates that molecules of HT2P4 were hardly retained in the epoxy matrix during the curing process. The flexural modulus values of the modified epoxy systems slightly decreased with the increase in the mass percentage of HT2P4, which could be ascribed to the deformation of the epoxy matrix around the particle cavitation [Tg of the modified epoxy system with 5 wt % commercial CTBN was reported to be more than 15 °C [The effect of HT2P4 additives on the cured properties of modified epoxy systems is shown in  of CTBN . The tou of CTBN ,35,38. Tvitation , whereasan 15 °C .KIC of the modified epoxy systems with the relatively low concentration of prepared tetra-carbamates composed of PTMG segments with bigger molecular weight were significantly higher than those of unmodified epoxy polymer without sacrificing Tg and flexural strength. The toughening mechanism is supposed as a synergism combination among the phase separation mechanism, an in situ homogeneous toughening mechanism, and a particle cavitation mechanism. In conclusion, the prepared tetra-carbamates have excellent potential for use in the practical applications of epoxy resin considering the cost, processability, and desired toughness improvement.PPG-terminated tetra-carbamates were synthesized as a new toughening additive for epoxy thermoset polymers. The modified epoxy systems with these additives exhibited lower steady viscosities than the unmodified epoxy resin, which indicates that they had a positive effect on the processing viscosity. The impact strength and


Human parathyroid hormone (1-34) (PTH) has been widely used as the subcutaneous injection formulation for the treatment of osteoporosis. In the present study, we developed an efficient transdermal delivery system of PTH by using dissolving microneedle arrays (MNs) composed of hyaluronic acid (HA) for the treatment of osteoporosis. PTH-loaded MNs, with needle length 800 µm, were fabricated via a micro-molding method. The stability of PTH in MNs was found to be 6-fold higher than that of PTH solution when stored at room temperature (15–20 °C) for one month. Micron-scale pores were clearly visible in rat skin following application of PTH-loaded MNs. PTH-loaded MNs were completely dissolved by 60 min following application to rat skin. The bioavailability (BA) of PTH relative to subcutaneous injection was 100 ± 4% following application of PTH-loaded MNs in rats. In addition, PTH-loaded MNs were found to effectively suppress decreases in bone density in a rat model of osteoporosis. Furthermore, no skin irritation was observed at the site of application in rats. These findings indicate that our dissolving MNs have a potential use in formulations for the transdermal delivery of PTH and for the treatment of osteoporosis. Human recombinant parathyroid hormone 1-34) (PTH) is a peptide that consists of 34 amino acids. It is widely used for the treatment of osteoporosis. PTH is unique in that intermittent administration of this peptide leads to an increase in bone formation and bone mass. This is a result of its anabolic effect, which is not a characteristic of other osteoporosis therapeutics that are currently available [-34 (PTH)Transdermal drug delivery has several advantages. It is painless and allows ease of application. Therefore, transdermal delivery is considered to be an attractive method for the delivery of PTH. However, the stratum corneum, the outermost layer of the skin, restricts the permeation of hydrophilic and macromolecular drugs ,6. In orOf the various strategies available, MNs are considered to be an efficient approach for the transdermal delivery of PTH. MNs are composed of micron-scale needles arrayed onto a transdermal patch. MNs enhance the permeation of macromolecules and hydrophilic compounds by creating micron-sized pores in the skin ,13,14,15®, a PTH-coated titanium microneedle patch system, with needle length 190 µm, was developed for the treatment of osteoporosis [ZP-PTHoporosis . Howeveroporosis ,18,19. Toporosis . FurtherIn the present study, we designed and developed dissolving MNs composed of HA as the base material for the efficient transdermal delivery of PTH. The stability of PTH in the MNs and the process by which MNs dissolve in the skin were evaluated. Pharmacokinetic analyses were performed in order to evaluate the transdermal absorption of PTH following application of PTH-loaded MNs. In addition, the therapeutic potential of MNs to treat post-menopausal osteoporosis following the application of PTH-loaded MNs was assessed in a rat osteoporosis model. The risk of skin irritation following application of PTH-loaded MNs was also assessed.® O.C.T. Compound was purchased from Sakura Finetek Japan Co., Ltd. . Paraformaldehyde, ethylenediaminetetraacetic acid (EDTA), and aprotinin were purchased from Wako Pure Chemical Industries, Ltd. . All other chemicals were obtained commercially as reagent-grade products.HA was purchased from Kikkoman Biochemifa Company . PTH was supplied from CosMED Pharmaceutical Co., Ltd. . The SEP-Column containing 200 mg C18 was purchased from Phoenix Pharmaceuticals, Inc. . Trypan blue was purchased from Nacalai Tesque, Inc. . Tissue-TeKMale Wistar rats (250–270 g) and female Sprague-Dawley rats (180–200 g) were purchased from Japan SLC, Inc. . All animal experiments were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The protocols for animal experiments were approved by the Animal Experimentation Committee of the Kyoto Pharmaceutical University.The MNs were fabricated by using micromolding technologies with HA as a base material ,21, and In order to confirm the PTH content in the needle part of MNs, the needles were separated from the back layers of MNs and dissolved in 5 mL of phosphate buffered saline  in a vial. The solution was then stirred for 1 h at 72 rpm at 32 °C . ConcentPTH-loaded MNs were packaged into an aluminum sachet purged with nitrogen and stored at −20 °C, 4 °C, or at room temperature (15–20 °C) . PTH sol®; KEYENCE, Osaka, Japan) [The abdominal skin of Wistar rats was shaved while the animals were placed under pentobarbital anesthesia . PTH-loaded MNs were then applied to the abdominal skin of rats. Following a 1 h application, the MNs were removed and the application sites were stained with 0.4% trypan blue in water for 10 min. Following the removal of the trypan blue solution with tissue paper, the surface of the skin with applied MNs was observed. The areas of skin to which MNs had been applied were then excised from euthanized rats and embedded in optimal cutting temperature (OCT) compound. The embedded skin samples were frozen in liquid nitrogen and sectioned into 10 µm slices using a cryomicrotome . The sections were then visualized under a microscope  ,24.The abdominal skin of Wistar rats was shaved while the animals were placed under pentobarbital anesthesia . PTH-loaded MNs were then applied to the abdominal skin of rats. Following a 1 h application, the MNs were removed. The TEWL values of rat skin were measured using a Tewameter . This was done by applying a probe to the skin until a stable reading was obtained. TEWL values were determined before and directly following the removal of MNs at indicated time intervals over a period of 48 h. Separately, a group of rats were treated by tape stripping 20 times each as a positive control. Untreated rats were used as a negative control. TEWL values were also determined over similar time intervals for both the positive and negative control groups .The abdominal skin of Wistar rats was shaved while the animals were placed under pentobarbital anesthesia . PTH-loaded MNs were then applied to the abdomen of rats. At pre-determined times, the MNs were removed and visualized using a digital microscope  .max) and time to reach Cmax (Tmax) were determined directly from the plasma concentration–time profiles. Area under the plasma concentration-time curve (AUC) after transdermal administration or subcutaneous injection was calculated by the trapezoidal formula and extrapolation to infinite time based on monoexponential equation [The abdominal skin of Wistar rats was shaved while the animals were placed under pentobarbital anesthesia . PTH-loaded MNs (20 µg/rat) were then applied to the abdominal skin of rats. The dosage of PTH in MNs was calculated based on the content of PTH in the needle part of MNs. Separately, PTH solution (20 µg/rat) was subcutaneously injected into the backs of rats. At pre-determined intervals, 0.3 mL of blood was withdrawn from the jugular veins while the animals were placed under pentobarbital anesthesia  and collected in tubes containing 0.3 mg of EDTA and 150 KIU of aprotinin. The collected blood was then centrifuged for 15 min at 4000 rpm to obtain plasma. The plasma samples were then stored at −80 °C until required for determination of PTH concentration. The peak PTH plasma concentration (Cequation .The PTH in the plasma sample was extracted and purified using SEP-columns. The solution obtained was lyophilized and dissolved in EIA buffer. Concentrations of PTH were determined using the PTH (1-34) ELISA kit (PTH (1-34), Human, EIA Kit, High Sensitivity, Peninsula Laboratories International, Inc. San Carlos, CA, USA).PTH-loaded MNs were applied to the abdomen of rats (20 µg/rat). The MNs were then removed 1 h after their application. Skin irritation was assessed by observation of the application site of rats at pre-determined times ,24.The post-menopausal rat model of osteoporosis was established by performing ovariectomies (OVX) of female Sprague-Dawley rats . Four we2/h. The TEWL significantly increased and reached a level of 75.30 ± 3.61 g/m2/h immediately following the tape stripping as a positive control. Although the mean TEWL value transiently increased and reached a level of 52.30 ± 3.16 g/m2/h immediately following the 1 h application of MNs, the mean TEWL value gradually recovered to its initial level 24 h following the removal of MNs.To evaluate the piercing ability of MNs and the recovery of the skin barrier following the application of MNs to the skin, the TEWL values of rat skin were measured as an indicator of the barrier function of the stratum corneum. max) following application of PTH-loaded MNs was slightly delayed compared with that observed after subcutaneous injection, the plasma concentrations of PTH rapidly increased following application of PTH-loaded MNs in a manner similar to that observed following subcutaneous injection. The AUC0–∞ values of PTH were 1354 ± 484 and 1355 ± 54 ng·mL/min after the application of MNs and after subcutaneous injection, respectively. The bioavailability (BA) of PTH relative to subcutaneous injection was 100 ± 4% after application of PTH-loaded MNs.In the present study, we developed dissolving MNs fabricated from HA for the efficient transdermal delivery of PTH. HA has been widely used in the manufacturing of drug delivery and cosmetic products ,28,29 anTo evaluate the MNs in terms of their potential clinical use, a dosage of PTH equivalent to the clinical dose (20 µg) was enclosed in the MNs in the present study. Although it has previously been reported that needle strength decreases as the amount of enclosed drug is increased ,33, our ®) was 37.4% following application of the PTH-coated titanium microneedle patch system (ZP-PTH®) [® would not be able to reach the upper dermis where blood vessels are present. It was reported that MNs with longer needles (>600 µm length) showed effective transdermal drug delivery [max after application of MNs was slightly delayed compared with that after subcutaneous injection of PTH. This was probably caused by the dissolution process of MNs in the skin following application [Daddona et al. reported that the bioavailability of PTH relative to subcutaneous injection (FORTEOZP-PTH®) . This loZP-PTH®) ,39. Therdelivery ,41. Therlication .It has previously been reported that PTH has pharmacological activities for both bone formation and resorption. A previous study reported that bone formation was observed following intermittent treatment of rats with PTH ,42. In tWe also demonstrated that PTH-loaded MNs were relatively safe for application to the rat skin. The results of the skin irritation study, together with the results of TEWL, indicate that PTH-loaded MNs transiently created a pathway for the transdermal delivery of PTH and caused minimal skin irritation. HA is known to be both an endogenous and highly biocompatible compound and hardly caused irritation to the rat skin. Although further studies are needed to evaluate the safety of PTH-loaded MNs in detail for potential clinical use, PTH-loaded MNs show promise as formulations for inclusion in a safe and efficient transdermal delivery system.In conclusion, we developed dissolving MNs fabricated from HA as the base material for the transdermal delivery of PTH. The storage stability of PTH in MNs was much higher than that of PTH solution kept at room temperature (15–20 °C) for one month. Efficient transdermal delivery of PTH and treatment of osteoporosis were successfully achieved by application of PTH-loaded MNs without the development of any skin irritation. These findings indicate that our dissolving MNs are promising formulations for the transdermal delivery of PTH and the treatment of osteoporosis.


Xiphophorus (platyfish and swordtails) puberty onset is genetically determined by a single locus, which encodes the mc4r. Wild populations of Xiphophorus are polymorphic for early and late-maturing individuals. Copy number variation of different mc4r alleles is responsible for the difference in puberty onset. To answer whether this is a special adaptation of the Mc4r signaling system in the lineage of Xiphophorus or a more widely conserved mechanism in teleosts, we studied the role of Mc4r in reproductive biology of medaka (Oryzias latipes), a close relative to Xiphophorus and a well-established model to study gonadal development. To understand the potential role of Mc4r in medaka, we characterized the major features of the Mc4r signaling system . In medaka, all these genes are expressed before hatching. In adults, they are mainly expressed in the brain. The transcript of the receptor accessory protein mrap2 co-localizes with mc4r in the hypothalamus in adult brains indicating a conserved function of modulating Mc4r signaling. Comparing growth and puberty between wild-type and mc4r knockout medaka revealed that absence of Mc4r does not change puberty timing but significantly delays hatching. Embryonic development of knockout animals is retarded compared to wild-types. In conclusion, the Mc4r system in medaka is involved in regulation of growth rather than puberty.In mammals the melanocortin 4 receptor (Mc4r) signaling system has been mainly associated with the regulation of appetite and energy homeostasis. In fish of the genus  Xiphophorus (platyfish and swordtails) show polymorphisms in puberty onset timing, body length and reproduction tactics (P (Puberty) locus encoding the melanocortin 4 receptor (Mc4r) is critically involved in establishing these polymorphisms  and is a member of the melanocortin receptor family, which has five members, Mc1r to Mc5r . These rMc4r has been reported to be involved in many physiological processes in several fish species. The most prominent physiological processes affected are food intake and energy balance, as reported in goldfish  and rainMC4R are connected to early-onset obesity (Mc4r knockout causes hyperphagic obesity (Agrp overexpression mice (Avy)  . Mc4r wace (Avy) . Leptin ce (Avy) . Due to Xiphophorus provide a useful tool to study the genetic basis of puberty regulation and the molecular factors involved in this process (Oryzias latipes) is a phylogenetically closely related species to Xiphophorus. For both species, high-quality genomes are available  were maintained under a standard light/dark cycle of 14/10 h at 26°C in the fish facility of Biocenter at the University of Wuerzburg. Eggs of medaka fish were collected and cultured in Danieau's medium 2 0.18 mM, HEPES 1.5 mM, methylene blue 0.0001%) until hatching.All wild-type medaka used in the study were from the Carbio strain. The knockout tant see . Adult mAll animals were kept and sampled in accordance with the applicable EU and national German legislation governing animal experimentation, in particular, all experimental protocols were approved through an authorization  of the Veterinary Office of the District Government of Lower Franconia, Germany, in accordance with the German Animal Protection Law (TierSchG).The annotated Mc4r, Mrap2, Pomc, Agrp1 sequences from human and 14 fish species were retrieved from either National Center for Biotechnology Information nucleotide sequences database (NCBI)Poecilia formosa), cave fish (Astyanax mexicanus), cod (Gadus morhua), fugu (Takifugu rubripres), medaka (Oryzias latipes), Southern platyfish (Xiphophorus maculatus), spotted gar (Lepisosteus oculatus), stickleback (Gasterosteus aculeatus), tetraodon (Tetraodon nigroviridis), tilapia (Oreochromis niloticus), zebrafish (Danio rerio); one Sarcopterygian (lobe-finned fish), coelacanth ; one Chondrichthyan (cartilaginous fish), elephant shark ; and one Agnathan (jawless fish), lamprey (Petromyzon marinus) were used in the study. Common carp (Cyprinus carpio) was additionally used in Mrap2 phylogeny. These were compared with human (Homo sapiens). Accession numbers are listed in Eleven Actinopterygians (ray-finned fish) including Amazon molly . The exact amounts of transcripts could not be determined in 0 dpf sample due to lack of proper normalization factors. To determine the tissue expression of Mc4r signaling pathway genes, three pools of male or female adult fish tissues (n = 3–4) of brain, eye, gill, kidney, liver, gonad, skin, and muscle were analyzed. Total RNA was isolated using TRIZOL reagent . After DNase treatment, a reverse transcriptase with random hexamer primers was used to synthesize first strand cDNA using RevertAid First Strand cDNA Synthesis Kit  according to manufacturer's manual. Primers for mc4r, mrap2, pomca, pomcb, agrp1, and housekeeping gene ef1a are listed in 2 Eppendorf machine Eppendorf . Data were analyzed by the ΔΔCT method and normalized to housekeeping gene ef1a. The values were presented as mean ± standard deviation (SD).To determine the time course of expression of genes from the Mc4r signaling system, three pools of medaka embryos and larvae from 0, 1, 2, 3, 4, 5, 6, 8, 10, 15, 20 days post fertilization (dpf) were collected  with digoxigenin or fluorescein RNA labeling mix (Roche). Whole mount in situ hybridization was performed on dissected intact brains fixed in 4% PFA and dehydrated in methanol for storage. Before hybridization brains were rehydrated with PBST and digested with 10 μg/ml proteinase K for 45 min. Samples were hybridized with riboprobes overnight at 68°C in a humidified chamber and then were subjected to stringent washes of SSC series (0.05X SSC used for washes of high stringency) and by PBST. The samples were embedded in 3% agarose and 100 μm section were cut by TPI Vibratome Series 1000 Sectioning System . After blocking with 5% sheep serum, anti-digoxigenin-AP  or anti-fluorescein-AP  were applied to the sections overnight at 4°C. NBT/BCIP solution (Roche) or FastRed Tablets  were used to develop the signals by AP reaction according to manufacturer's instruction. Double in situ hybridization was performed by first visualizing fluorescein probes and thereafter digoxigenin probes. Sections were mounted in 80% glycerol for microscopy . Fluorescence pictures were taken with a Confocal microscope . The transcript localization was determined according to the medaka brain atlas  fish (Carbio strain) and Mc4r knockout (KO) −2/+3 TALEN-KO strain   tests. GraphPad Prism 6 was used to display the results.Statistical treatment of the data was done with GraphPad Prism 6 software. Two-way mc4r of Southern platyfish and blind cavefish have longer branches indicating faster evolution toward specialized functions in these lineages. Different from all other species, only zebrafish has two copies of mrap2 (Cyprinus carpio) genome reveals also two copies of mrap2, mrap2a, and mrap2b  21, which is the sex chromosome. However, in medaka, mc4r is on linkage group LG 20 and not on the sex chromosome (LG 1). None of the other Mc4r signaling system genes are sex-linked, neither in Xiphophorus nor in medaka  expression in the female brain  and ovary (2.9-fold)  and agrp1 is intriguing and warrants further studies to elucidate a sex-related function.Differential expression among tissues and between males and females were analyzed using RT-qPCR. Brains of both sexes highly express nd mrap2 . High exanalysis . The pomth sexes . The agrle brain . Besidespression . The mra.9-fold) . No othe muscles . Testis  tissues . In summmrap2 has to be co-expressed with mc4r in the same cells. To investigate the localization of mc4r and mrap2 expression, whole mount in situ hybridization was performed using intact brains from adult females and males.Mrap2 has been described as Mc4r accessory protein of important function . To exermc4r was expressed in the preoptic region and hypothalamus region, mrap2 was only expressed in the hypothalamus  and compared to wild-type medaka. In medaka, puberty onset can be determined from the development of secondary sexual characteristics in males , which can be easily observed under a stereomicroscope. In females, the first egg-laying indicates puberty completion  probably arose during the 3rd round of whole genome duplication of the teleost, since only one copy of pomc is present in other vertebrates. However, two sequences of pomca (pomca1 and pomca2) were found in most of Percomorpha teleost, indicating a lineage-specific duplication of this gene. The agrp gene has been found in some species to exist in two versions, agrp1 and agrp2. agrp1  has been related to obesity (agrp2  is s asip2b  and pigms asip2b . Taken tmc4r and high levels for mrap2. The mc4r and mrap2 genes, which code for membrane proteins, are expressed early in development, concurrent with the late neurula stage and early brain development. Thus, mc4r and mrap2 genes are expressed already in early and late embryonic stages, when the brain and integrated functional proteins in the neurons are developing. The pomc genes, precursors of Mc4r agonists, start expression 2 days after mc4r and mrap2. At the hatching stage, all genes are expressed, indicating that the whole system is in place for full function when the fish start feeding. Intriguingly, agrp1 showed a dramatic upregulation after hatching, coinciding with first feeding. This may be explained by the important role of agrp1 in appetite regulation and implies the role of the Mc4r system in energy metabolism regulation from the very first feeding event (In the next step toward a physiological characterization of the Mc4r signaling system of medaka, temporal expression analysis showed mRNA maternal contribution at low levels for ng event , 46.mc4r signaling pathway genes are expressed highly in brains of both sexes consistent with the center of food uptake regulation in the hypothalamus and neighboring regions of the brain, e.g., the preoptic area (mc4r and mrap2 were identified in the hypothalamus, and the two genes showed colocalization in certain areas. This indicates interaction of Mc4r and Mrap2 in those cells. Mc4r and Mrap2 are both membrane proteins, their interaction could tune signal strength and intracellular signal transduction as previously proposed for the zebrafish homologs (mc4r is expressed, mrap2 is not present, implying that in such regions mc4r may be able to transmit signals without its co-factor mrap2. Nevertheless, mrap2 expression was observed only in regions where the mc4r signal was present, supporting the specific role of Mrap2 in interacting with Mc4r in the brain (Spatial expression analysis revealed that the tic area . The exphomologs . At somehe brain .mrap2 in ovary could be explained by the very high maternal RNA contribution. Mature eggs contain high levels of mrap2 inferred from maternal contribution level, and this is reflected in the high mRNA levels in the ovary. High expression of mrap2 in the male kidney has been shown before. In zebrafish, mrap2 is highly expressed in the head kidney (mrap2 is expressed mainly in brain and adrenal gland in human (mrap2 might be similarly expressed in the interrenal gland on the kidney. A male-specific function of mrap2 in kidney still needs to be investigated.The high transcript levels of d kidney . Moreovein human  and widein human . Fish mrXiphophorus fish. In Xiphophorus, mc4r is expressed highest in the brain (mc4r on puberty timing was not noted. Our results indicate that Mc4r may not be the critical puberty signal in medaka fish. Alternatively, the lack of a puberty phenotype in our TALEN KO fish might be due to the fact that other genes can compensate for the loss of Mc4r function (The expression data did not exclude that the Mc4r signaling system in medaka could have the same function in regulating puberty timing as in he brain . We findhe brain . Howeverhe brain , an effefunction .Xiphophorus species, where Mc4r is involved in male size but has no noticeable effect on female growth and adult size. In addition, KO fish are slightly shorter than WT, indicating that possibly growth in general is retarded in Mc4r KO fish. In zebrafish, mc4r KO led to increased body length at 42 dpf (mc4r KO in medaka surprisingly show retarded growth suggesting that eventually other receptors like Mc3r, which is also related to energy balance and reacts with Pomc and Agrp1, could compensate for the function of compromised Mc4r by responding to Pomc signals and thus reducing growth.Although the timing of puberty onset is not altered in KO fish, female body length at puberty in KO fish is significantly shorter than WT. WT Females are significantly larger than WT males and KO males. KO females and males do not show a difference in body length at puberty. Since puberty is normally related to animal size/weight, earlier puberty and shorter length at puberty in KO females implies an advanced pubertal process in KO females. This suggests that the Mc4r function is relevant for female growth in medaka, rather than male growth. This is unlike the situation in Southern platyfish and other t 42 dpf . Overexpt 42 dpf , 54. mc4mc4r expression. This effect becomes even more obvious from day three on. It could be speculated that Mc4r has a role in medaka development by influencing growth. Zebrafish Mc4r KO, however, does not show changes in linear growth during embryonic and larval stages. An increased length of Mc4r KO zebrafish was first seen only at 42 dpf (Xiphophorus fish (mc4r led to increased body length at 42 dpf (Intriguingly, we observed that the hatching time of Mc4r KO medaka was affected. The Mc4r KO fish show delayed development from day one coinciding with the onset of t 42 dpf . The embt 42 dpf .Xiphophorus, but rather in growth and development. Our findings suggest that from an evolutionary perspective the role of mc4r as the critical P locus gene is a specific innovation in the Xiphophorus lineage.In summary, the regulatory network of Mc4r signaling system in medaka appears not to be involved in puberty regulation as in in situ hybridizations, KO adult and embryos functional analysis, and drafted the manuscript. MK established and provided the KO medaka line. MA supervised the experiments and helped to review the manuscript. MS defined and designed the study, coordinated all steps of the research and reviewed all versions of the manuscript.RL performed the phylogeny, sampling, RT-qPCR, 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.


The effects of the ultrathin Al2O3 layer and the thickness of Nd:IZO layer in the nanolaminate structure on the improvement of electrical performance and stability of thin film transistors (TFTs) were systematically studied. Besides the carrier movement confined along the near-channel region, driven by the Al2O3 layer under an electrical field, the high performance of the TFT is also attributed to the high quality of the 8-nm-thick Nd:IZO layer and the corresponding optimal Nd:IZO/Al2O3 interface, which reduce the scattering effect and charge trapping with strong M–O bonds in bulk and the back-channel surface of Nd:IZO, according to the X-ray reflectivity (XRR), X-ray photoelectron spectroscopy (XPS), and micro-wave photo conductivity decay (μ-PCD) results. As a result, the Nd:IZO/Al2O3 TFT exhibits an outstanding performance, with a high μsat of 32.7 cm2·V−1·s−1, an Ion/Ioff of 1.9 × 108, and a low subthreshold swing (SS) value of 0.33 V·dec−1, which shows great potential for the room temperature fabrication of TFTs in high-resolution or high-frame-rate displays by a scalable, simple, and feasible approach.In this work, a high-performance thin film transistor with an neodymium-doped indium zinc oxide (Nd:IZO) semiconductor via a room temperature approach and adopting the Nd:IZO/Al However, it is difficult to have a higher breakthrough on device performance for the a-IGZO TFT, which is still not sufficient for the high-resolution or high-framerate displays requiring mobility of over 30 cm2·V−1·s−1 [2·V−1·s−1) with an Nd:IZO semiconductor processed by a room temperature approach, and investigate the effects of a sputtered Nd:IZO/Al2O3 nanolaminate structure. Moreover, the thickness of the Nd:IZO layer in Nd:IZO/Al2O3 structure also showed a great impact on the device performance, due to the nature of film growth during the sputtering process.Great interest in metal oxide semiconductors (MOS) for thin film transistors (TFTs) has grown dramatically in recent years, due to their visible-light transparency, good uniformity, compatibility with different substrates, fewer limitations on processing temperature, and the high mobility to drive flexible Active Matrix Organic Light Emitting Diode (AMOLEDs) or high-resolution displays ,2. As th·V−1·s−1 ,4, is li·V−1·s−1 ,6, we ha·V−1·s−1 . Therefo·V−1·s−1 , the bon·V−1·s−1  is much 2O3 TFT, respectively. Besides the insertion of an ultrathin Al2O3 layer with a thickness of 3 nm for the Nd:IZO/Al2O3 TFT, the processes for the fabrication of both devices are consistent. Firstly, a 300-nm-thick Nd-doped aluminum was deposited as a gate metal on glass by direct-current (DC) magnetron sputtering and patterned by photolithography. Then, a 200-nm-thick Nd:AlOx was formed on the surface of the gate metal by anodic oxidation. The introduction of the Nd element was also reported to contribute to a smooth surface of the Al electrode and high dielectric properties of the AlOx gate insulator [2 = 20:1) using the Nd:IZO target with an Nd2O3:In2O3:ZnO ratio of 1:62.5:36.5 wt.%. For the Nd:IZO/Al2O3 TFT, the 3-nm ultrathin Al2O3 layer was then prepared by RF magnetron sputtering, with a power of 120 W and a pure Ar pressure of 1 mTorr onto the surface of the Nd:IZO layer for 120 s. Finally, the 150-nm-thick Al source/drain (S/D) electrodes were prepared by DC magnetron sputtering and patterned by a shadow mask. The width/length ratio (W/L) of all devices was 500/100 μm·μm−1. Besides the baking process for the photoresist, carried out on thermal platform under 150 °C for 1 min, no further thermal treatment was adopted during the whole process. The electrical characteristics of the TFTs were measured by a semiconductor parameter analyzer  under ambient conditions. X-ray photoelectron spectroscopy (XPS) analysis was carried out to investigate the chemical changes in the oxide films by using a THERMO ESCALAB250Xi  with an Al Ka (hν = 1486.6 eV) 15 kW beam spot source. The thickness (±0.1 nm), roughness and density of the Nd:IZO films were determined by X-ray reflectivity measurements  using a Cu-Ka X-ray source at 40 kV and 40 mA. Microwave photo conductivity decay  was used to characterize the relative carrier concentrations of the Nd:IZO/Al2O3 films and their relationship with the electrical performance of the TFT devices.nsulator . The Nd:2O3 TFT—therein, the thicknesses of Nd:IZO layers are both 8 nm. The output characteristics of TFTs were obtained with VG = 0~20 V in steps of 5 V, and the transfer curves were measured with VD = 20.1 V (saturation regime). The single Nd:IZO TFT could already exhibit an electrical performance with a saturation mobility (μsat) of 4.1 cm2·V−1·s−1 and an on-to-off current ratio (Ion/Ioff) of 1.0 × 107, which implies an acceptable concentration of carriers for the room-temperature-prepared Nd:IZO layer. However, the mobility and on-state current (Ion) is still low, due to the high density of defects in the as-deposited oxide thin film, especially near the back-channel region. By inserting an ultrathin Al2O3 film upon the Nd:IZO layer, as shown in on can be observed, the corresponding saturation mobility of the Nd:IZO/Al2O3 TFT can be remarkably improved to 32.7 cm2·V−1·s−1, and the Ion/Ioff reaches 1.9 × 108. Moreover, the hysteretic phenomenon reduces in the transfer curves of Nd:IZO/Al2O3 TFT. According to our previous study [2O3 acts as an electrical controller layer, inducing high-flux electron movement in the bulk and near-channel regions of the semiconductor layer by the carrier confinement effect under an electrical field, which avoids the scatting and trapping effect by the back-channel defects, and hence efficiently improves the device’s mobility. Meanwhile, in the Nd:IZO/Al2O3 system, the Al2O3 layer should have a specific role in the modification of the Nd:IZO semiconductor layer, which will be further discussed in this paper.us study  and otheus study ,13, the 2O3 on Nd:IZO layer, X-ray photoelectron spectra (XPS) measurements on the Nd:IZO/Al2O3 (8 nm/3 nm) multilayered films and a single Nd:IZO film (8 nm) were carried out. As shown in 2O3 layer. Herein, it is difficult to judge the difference between oxygen vacancies in the Nd:IZO by the O 1s core level spectra, because of the existence of Al2O3. Therefore, the XPS results for In 3d5/2, Zn 2p3/2, and Nd 3d5/2 core level spectra of the both samples were acquired as shown in 2O3 exhibit the positive shifts towards higher energy direction, indicating that the introduction of the Al2O3 layer can prevent oxygen decomposing from the metal–oxygen (M–O) bonds near the back-channel surface of the Nd:IZO film, which reduces the density of defects and thus enhances carrier mobility. Moreover, the decomposition of the Nd 3d5/2 lines through nearly Gaussian fitting shows that the Nd:IZO film covered by Al2O3 layer has a lower content of the Nd 3d5/24f4 configuration, which is considered to be associated with scattering centers or charge traps [To further investigate the effect of ultrathin Alge traps ,15.2O3 TFT under negative/positive bias stress (NBS/PBS) in 1 h, respectively. Both of the TFTs exhibit favorable stabilities under NBS after the first 900 s, with slight shifts of Von, indicating that the introduction of the Nd element can actually prevent the ionized oxygen vacancies from migrating to the semiconductor–insulator interface under the negative bias field [on for the single Nd:IZO TFT shows that the diffusion of absorbed water or oxygen molecules can efficiently affect the device stability [on can reach 3.2 V after 1 h under PBS. Therefore, it is essential to introduce an ultrathin Al2O3 film, which also acts as a good passivation layer. As a result, the Nd:IZO/Al2O3 TFT exhibits an outstanding stability under PBS, so that no obvious change is observed on the device transfer characteristics.as field ,17. Howetability , due to 2O3 TFTs with different thickness  of the Nd:IZO layers, and the corresponding electrical parameters are summarized in 2O3 structure shows a great impact on the device performance. A significant trend can be found that the device performance increased at first, but then degraded as the Nd:IZO thickness increased from 3 to 10 nm. As a result, the TFT with an Nd:IZO thickness of 8 nm exhibits the best electrical properties, with an Ion of 4.5 × 10−4 A, a μsat of 32.7 cm2·V−1·s−1, an Ion/Ioff of 1.9 × 108, and an SS value of 0.33 V·dec−1. The XRR measurements on the 3, 5, 8, and 10-nm-thick Nd:IZO films were adopted, and the information for film density and roughness was acquired, as shown in −3, with the optimal thickness of 8 nm, which contributes to fewer structural defects and consequently a higher device performance. Meanwhile, as the thickness increases, the roughness of the Nd:IZO increases continuously, and reaches 1.2 nm when the thickness is 10 nm, which is due to the nature of film growth during the sputtering process. The Nd:IZO/Al2O3 interfaces are sensitive to the surface roughness of over 1 nm for the Nd:IZO layers, since the thickness of upper Al2O3 layers are only 3 nm. Therefore, the rough back-channel surface of the Nd:IZO layer would enhance the carrier scattering effect and deteriorate the interface between Nd:IZO and Al2O3, consequently weakening the electrical properties of the Nd:IZO/Al2O3 TFTs.2O3 stacks on glass with a size of 10 × 10 mm2, in order to further investigate the effect of Nd:IZO thickness on the Nd:IZO/Al2O3 interfaces, as shown in 2O3 stacks with an Nd:IZO thickness of 8 nm exhibit the highest peak mean value of the μ-PCD signal, which indicates the highest relative concentration and mobility of carriers, as well as the lowest density of shallow localized defects [2O3 TFT. Associated with the XRR results, the growth of the Nd:IZO film during sputtering process can be simply demonstrated by 2O3 interface, according to the μ-PCD results, reducing the mobility of the carriers by scattering and trapping effects.Microwave photoconductivity decay (μ-PCD) mapping scan measurements were carried out for the above-mentioned Nd:IZO/Al defects ,20,21 th2O3 stacks can also unveil the effect of varying Nd:IZO thicknesses. As shown in p peak and the ratio of (Nd/(Nd + In + Zn) at.%) implies that the higher concentration of Nd can also contribute to a stronger formation of Al–O bonds (reflected by the positive shift of the Al 2p peak) [2O3. The variation in Nd concentrations of the Nd:IZO films with varying thickness can be attributed to the different depositing rate of Nd/In/Zn atoms during the sputtering process. As the sputtering proceeds, the atomic ratio of each element should first gradually change, and then become stable as soon as the film reaches a certain thickness. According to the XRR/μ-PCD/XPS results above, the thickness of 8 nm is optimal for the high quality of the Nd:IZO film and a favorable Nd:IZO/Al2O3 interface, leading to the high carrier mobility and consequently, the high electrical performance of Nd:IZO/Al2O3 TFT.Furthermore, the XPS measurements on the Nd:IZO/Al2p peak) , forming2O3 nanolaminate structure. The Al2O3 layer is considered to induce high-flux electron movement in the bulk and near-channel region of the Nd:IZO layer, prevent oxygen decomposing from the M–O bonds near the back-channel surface of Nd:IZO, and act as a good passivation layer, so that the Nd:IZO/Al2O3 TFT remains highly stable under both NBS and PBS. Meanwhile, the thickness of the Nd:IZO layer has a great impact on the electrical performance of Nd:IZO/Al2O3 TFTs, due to the nature of the sputtering method. The film quality and carrier concentration are optimal when the thickness of the Nd:IZO layer reaches 8 nm, and the high concentration of Nd also benefits the formation of a tight Nd:IZO/Al2O3 interface with strong Al–O bonds, reducing the scattering centers and charge traps near the back-channel surface. As a result, the Nd:IZO/Al2O3 TFT exhibits an outstanding performance with a high μsat of 32.7 cm2·V−1·s−1, an Ion/Ioff of 1.9 × 108, and a low SS value of 0.33 V·dec−1, which shows great potential for the room temperature fabrication of TFTs in high-resolution or high-framerate displays by a scalable, simple, and feasible approach.In conclusion, we demonstrated a high-performance TFT with an Nd:IZO semiconductor via a room temperature approach, by adopting an Nd:IZO/Al