Cu2−xS@MnS CSNPs with an independent core and shell ratio are synthesized by a controllable hot‐injection method, resulting in an optimal photothermal (PT) effect with a PT conversion efficiency of up to 47.9%. An enhanced photodynamic (PD) effect also occurs in an H2O2 environment. More significantly, in vivo experiments demonstrate that Cu2−xS@MnS CSNPs can mediate tumor shrinkage in both HeLa tumor cell line‐derived xenograft (CDX) and head and neck squamous cell carcinoma (HNSCC) patient‐derived xenograft (PDX) models, with the capability of being used as a T1‐enhanced magnetic resonance (MR) contrast agent. These results suggest the great potential of as‐prepared Cu2−xS@MnS CSNPs as photo/H2O2‐responsive therapeutic‐agents against tumors, even in a complicated and heterogeneous environment, thus promoting the clinical translation of nanomedicine.Stimuli‐responsive nanomedicines have become a recent research focus as a candidate for cancer treatment because of their effectiveness, sensibility, and minimal invasiveness. In this work, a novel nanosystem is developed based on Cu 2−xS@MnS core–shell nanoparticlesCu are developed for cancer theranostics. The Cu2−xS core serves as a photosensitizer to generate hyperthermia and reactive oxygen species, and the MnS shell is used in H2O2‐responsive O2 production, therefore mediating tumor shrinkage in both HeLa tumor cell line‐derived xenograft and head and neck squamous cell carcinoma patient‐derived xenograft models, with the capability of being used as a T1‐enhanced magnetic resonance contrast agent. However, most reported nanotheranostics are manganese‐supporting nanostructures with organic photosensitizers loaded and might not be ideal for realizing precisely controlled release of therapeutic payloads or supply of a sufficiently effective antitumor effect with mono‐therapy.Phototherapy has been heavily researched as a promising treatment option for cancer treatment due to its minimal invasiveness, high selectivity, and lower systemic toxicity.2−xS), is a promising PT agent due to its stoichiometry‐dependent near‐infrared region (NIR) localized surface plasmon resonance (LSPR) absorption derived from the copper vacancy.2−xS and magnetic manganese compounds into a single unit could be advantageous for efficient PT applications and a hyperthermia/O2‐enhanced PD effect, as well as avoidance of photobleaching and uncontrolled release of organic photosensitizers.Similar to PD therapy, photothermal (PT) therapy is also a novel modality that operates upon appropriate laser irradiation, with negligible attenuation into biological tissues and minimal photodamage to cells of adjacent tissues.Even though with the goal of improving tumor oxygenation and enhancing cancer treatment, performance evaluation of these proposed nanomaterials is mostly based on tumor cell line‐derived xenograft (CDX) mouse models.2−xS@MnS core–shell nanoparticles (CSNPs) as a photo/H2O2‐responsive platform for enhanced cancer theranostics. The Cu2−xS@MnS CSNPs were synthesized in a one‐pot hot‐injection method and were subsequently surface modified. The incorporation of Mn in the CSNP was found to vary in distribution and transfer of charge carriers, resulting in tunable absorption spectra. The intense optical absorption from the CSNPs in the NIR led to the perfect PT conversion and IR thermal imaging property. At the same time, the MnS shell endows the Cu2−xS@MnS CSNPs with the ability to be used as a T1‐MR contrast agent. Furthermore, the Cu2−xS@MnS CSNPs possess a catalase‐like activity to function as an H2O2‐responsive platform for enhancing the PD effect in which the Cu2−xS serves as a photosensitizer. Above all, the catalase‐like Cu2−xS@MnS CSNPs integrate PT therapy, enhanced PD therapy and MRI as a whole, thus demonstrating their potential against tumors in both CDX and PDX models and promoting further applications of nanomedicines based on these nanostructures.In the current work, we prepared multifunctional Cu22.12−xS@MnS CSNPs were synthesized via a one‐pot hot‐injection method in an oleyamine environment under an N2 atmosphere. Figure3COO)2) was selected as the copper source to create the Cu2−xS nanoseeds with the aid of 1‐dodecanethiol (C12H25SH).2−xS@MnS CSNPs were conducted by injecting an oleylamine dispersion of manganese acetate (Mn(CH3COO)2·4H2O) into the vessel and reacting at 200–220 °C for 20 min.Cu2−xS (JCPDS No. 23‐0959) and hexagonal MnS (JCPDS No. 03‐1062). In parallel, the high‐resolution transmission electron microscopy (HRTEM) image confirmed the lattice fringes from the core corresponding to the (804) and (226) planes of the Cu2−xS crystal, with spacings of 2.34 and 2.14 Å, respectively. The lattice spacing of 3.22 Å in the shell is associated with the (002) plane of the MnS crystal. In the XPS data 2·4H2O in oleylamine was injected, a portion of the Cu in the Cu2−xS nanoseeds was replaced by Mn, and the Cu2−xS@MnS CSNPs with various shell thickness formed at different rates of temperature increase in the reaction process. As shown in Figure −1, a thin layer of MnS shell was coated outside the Cu2−xS core (thin‐shell Cu2−xS@MnS). With the rates increased up to 0.55 and 1.00 °C min−1, intermediate‐shell Cu2−xS@MnS . Moreover, as the cation exchange proceeded, a void emerged between the MnS shell and the Cu2−xS nucleus. It is believed that this unique phenomenon can be attributed to the unequal ion diffusion between Cu+ and Mn2+, where the more rapidly exported Cu+ diffusion process induced the nanoscale Kirkendall effect.The high chalcocite Cu2.22−xS@MnS NPs with different MnS shell thicknesses were examined. The pure Cu2−xS NPs have an optical absorption band centered at 1700 nm , consistent with the high chalcocite Cu2−xS material. After partial transformation to a MnS shell, the absorption band gradually blueshifted with an obvious intensification and moved to 950 nm for the thick‐shell Cu2−xS@MnS CSNPs, indicating the strong interaction between the Cu2−xS core and the MnS shell.El‐Sayed and coworkers reported that an optimal PT conversion effect could be reached when the LSPR from the plasmonic NPs resonates with the incident light, which means that a stronger absorption of the incident light leads to a higher PT activity.2−xS@MnS samples were first transferred from the organic phase into water using an amphiphilic poly‐based polymer as a protecting group for the hydrophilic carboxylic acid groups and hydrophobic oleylamine sidechains. The Fourier transform infrared spectroscopy (FTIR) patterns  showed that selected new peaks  obviously appeared after material modification, representing the characteristic absorption peaks of C=O and C—O, which indicated successful hydrophilic modification. The PT conversion performances of these Cu2−xS@MnS NPs were measured in aqueous dispersion under irradiation with an 808 nm laser for 5 min at an intensity of 0.72 W cm−2. Figure2−xS NPs suspension increased from 28.1 to 38.1 °C within 5 min. The thin‐shell Cu2−xS@MnS and intermediate‐shell Cu2−xS@MnS CSNPs suspensions had final temperatures of 44 and 47.9 °C, respectively, under the same conditions. The highest temperature of 49.4 °C after irradiation was stimulated by the thick‐shell Cu2−xS@MnS CSNPs aqueous dispersion. In other words, the thick‐shell Cu2−xS@MnS CSNPs obtained an optimal PT conversion efficiency upon photo‐excitation at 808 nm, and therefore, they were selected as the object for the following study.Before comparison of their PT properties, the four as‐synthesized Cu2−xS@MnS CSNPs, aqueous dispersions with various concentrations  were excited by an 808 nm laser. The temperature elevation of these dispersions was found to be proportional to the Cu2−xS@MnS concentration  maintained a low dissolved oxygen level. After addition of H2O2 (28 × 10−3m) to the Cu2−xS@MnS CSNPs suspension, a large amount of bubbles was observed, and the dissolved oxygen concentration increased from 7.6 to 13.7 mg L−1 within 12.5 min. Moreover, the dissolved oxygen concentration increased more rapidly and appeared to be higher post‐H2O2 addition with comparatively more H2O2 , a fluorescence indicator for 1O2. As shown in Figure 2−xS@MnS CSNPs incubation under irradiation using an 808 nm laser. A larger concentration of the Cu2−xS@MnS CSNPs suspension resulted in an obvious increase in the fluorescence intensity of SOSG. Furthermore, considering the dependence of ROS generation on O2, the generation of ROS upon H2O2 treatment was also assessed  via covalent bonds. Cells were stained by 4,6‐diamidino‐2‐phenylindole (DAPI) and imaged with a confocal laser scanning microscope (CLSM) after incubation with the Cu2−xS@MnS CSNPs. As shown in Figure S7 in the Supporting Information, a green signal appeared around the domains covered by DAPI after 20 min, suggesting that the CSNPs were taken up by cellular endocytosis and delivered to the cytoplasm. Moreover, we used ICP‐AES to quantize the NPs in the cells after incubation with Cu2−xS@MnS CSNPs at different concentrations. With the increasing of CSNPs' concentration  and propidium iodide . As shown in Figure 2−xS@MnS CSNPs were greater when the suspension concentration increased from 100 to 200 ppm, confirming the photodamage of the Cu2−xS@MnS CSNPs on cancer cells under 808 nm laser irradiation.The CCK‐8 assay was performed to evaluate the potential cytotoxicity of the Cuh Figure b. Withou2−xS@MnS CSNPs could mediate an efficient PT therapy effect under laser irradiation and generate O2 and ROS in response to H2O2. Moreover, it is known that the cancer cells inside solid tumors are able to constitutively produce H2O2 (from ≈50 × 10−6 to 100 × 10−6m) by relying on the decreased antioxidant enzyme level. Therefore, Cu2−xS@MnS CSNPs are anticipated to guide an enhanced phototherapy effect in response to H2O2 by generating O2 and more ROS after internalization by tumor cells. To confirm this, the in vitro cell cytotoxicity of Cu2−xS@MnS CSNPs in an H2O2 environment was also evaluated via the standard CCK‐8 assay. HeLa cells were first incubated with Cu2−xS@MnS CSNPs suspension for 24 h and subsequently treated with exogenous H2O2 (100 × 10−3m). H2O2 did not influence cell viability under laser irradiation when the added CSNPs concentration was 0  lower than that in normoxic conditions at a CSNPs concentration of 200 ppm. These results indicated that extra H2O2 was helpful in enhancing the phototriggered cancer cell‐ablation efficiency of the Cu2−xS@MnS CSNPs. Taken together, these results showed that Cu2−xS@MnS CSNPs could mediate an improved therapeutic effect in response to H2O2 and photo‐irradiation.As verified above, the nanoplatforms based on Cu2.52−xS@MnS CSNPs as an MR contrast agent. First, we recorded the MR images and relaxation times of aqueous dispersions containing Cu2−xS@MnS CSNPs using a 0.5 T MR animal scanner (MesoMR23‐060H‐I) at varied Mn concentrations. As shown in Figurer1, effectiveness as a contrast agent) of these CSNPs was calculated to be 1.243 mM−1 s−1, suggesting that our material is suitable for use as a contrast agent in MRI.Considering that manganese‐based nanomaterials are one of the most current T1‐enhanced MR contrast agents,2−xS@MnS CSNPs dispersed in a saline solution. The tumor region has a contrast‐enhanced signal after CSNPs injection compared with that before, and the relative MR signal intensity in the tumor region shows an increase from 23 046 to 36 516 . This result suggests the potential application of thick‐shell Cu2−xS@MnS CSNPs as a promising MRI agent for tumor diagnosis.The contrast‐enhancing effect was evaluated in nude mice bearing HeLa tumors. Figure 2.62−xS@MnS CSNPs upon laser irradiation, we assessed the in vivo phototherapy effect. CDX mice bearing HeLa tumors were first intratumorally injected with CSNPs suspension  and subsequently laser irradiated . The control groups were treated with PBS injection (100 µL) and the same laser exposure. A thermal infrared camera was used to record the full‐body IR thermal images and monitor the temperature changes in the mice, revealing massive contrast differences in tumor sites between the two groups . We subsequently measured the body weight and tumor sizes of the mice during a 16 day period, starting from the laser irradiation on the first day. The results showed no significant difference in body weights between the treatment group (CSNPs + laser) and the other three control groups , implying that the thick‐shell Cu2−xS@MnS nanomaterials and laser irradiation showed no overall side effects in mice. Relative tumor volumes were calculated based on the size measured for each mouse. As shown in Figure To investigate the potential antitumor efficacy of our thick‐shell Cu2.72−xS@MnS CSNPs, a PDX model was constructed based on head and neck squamous cell carcinoma (HNSCC), which belongs to superficial tumor that is preferable for photo‐illumination and photo‐penetration. The 4th generation of PDX animals was used in this work due to its stabilization . Similar to the experiments on the HeLa tumour CDX model , mice inoculated with the PDX model were intratumorally injected with the CSNPs suspension  followed by laser irradiation  . Controls in these experiments included mice lacking CSNPs injection , mice lacking laser irradiation , and mice lacking both treatments .The PDX model not only preserves the intratumoral heterogeneity and histology of the primary human cancer tissues but also preserves the regenerative TME, such as blood vessels, tumor stroma, fibroblasts, macrophages, and extracellular matrices, thus making it more significant for preclinical assessments of nanomedicines compared with CDX.Figure2−xS@MnS CSNPs induced the highest cell apoptosis rate in tumors upon laser excitation. A decrease in the number of Ki‐67‐positive proliferating cells was observed in tumors treated with CSNPs + laser (10% positive) compared with the other three groups . These results suggested that Cu2−xS@MnS CSNPs had an inhibitory effect on tumor cell proliferation and pro‐apoptosis. The decrease in positive proliferating cells in the CSNPs control might be due to the Fenton effect induced by the Cu2−xS@MnS material within the special TME with a high H2O2 level. In addition, the tumor growth curves  proved the safety of phototherapy stimulated by CSNPs, thus reinforcing that our Cu2−xS@MnS CSNPs can be used as an efficient therapeutic agent for cancer therapies.Over these treatments, we evaluated the therapeutic efficacy using haematoxylin and eosin (H&E) staining Figure8a. Lit2.82−xS@MnS CSNPs suspension. Blood and tissue samples were collected and analyzed at different time points. The results showed that Cu2−xS@MnS material treatment did not affect the normal range of blood biochemistry indicators  including alanine aminotransferase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), creatinine (CRE), and uric acid (UA), and the physiological morphology of tissues including heart, liver, spleen, lung, and kidney, observed from H&E‐stained images in Figure S14 in the Supporting Information. Moreover, tissue samples were also digested with HNO3/HCl to determine the Cu contents via ICP‐AES measurements. As shown in Figure S15 in the Supporting Information, the Cu2−xS@MnS material distribution reached a high level in liver and spleen at first, which could be possibly correlated with reticuloendothelial system (RES) uptake.2−xS@MnS CSNPs at the tested dose. However, additional systematic studies of pharmacokinetics, pharmacodynamics and pharmaco‐immunology are still necessary for future clinical translation of such a material.The in vivo biosafety of nanomedicines is always a considerable concern for application in cancer theranostics. To test biosafety, nude mice were used as models and intravenously injected with the Cu32−xS@MnS CSNPs was developed for cancer theranostics using a controllable hot‐injection method. The external MnS shell mediates O2 production to overcome hypoxia and regulates TME in a special tumor environment with excessive H2O2. The Cu2−xS core is excited by a NIR laser, leading to an enhanced PD effect with an O2 self‐supplement condition and an improved PT effect via ROS and heat generation, resulting in successful eradication of subcutaneous tumors in CDX and PDX models. Moreover, the MR effect induced by the Mn‐based nanomaterial can be used to noninvasively locate tumors and guide treatment. This multifunctional nanoplatform demonstrated effective tumor ablation and imaging, promoting clinical translation of nanomedicine.In conclusion, a novel nanosystem based on Cu4Materials and Reagents: Copper (II) acetate (Cu(CH3COO)2), manganese (II) acetate (Mn(CH3COO)2·4H2O), sodium carbonate (Na2CO3), cyclohexane (C6H12), and chloroform (CHCl3) were obtained from Sinopharm Chemical Reagent Co. Ltd, and 1‐dodecanethiol (CH3(CH2)11SH) and oleylamine (OM) were sourced from Aladdin Industrial Corporation. Tetrahydrofuran (C4H8O) was purchased from Shanghai Boer Chemical Reagent Co. Ltd. Poly was obtained from Sigma‐Aldrich Chemie GmbH. Fluorescein isothiocyanate was obtained from Shanghai Macklin Biochemical Co. Ltd. All chemicals were of at least analytical reagent grade and used without further purification.2−xS@MnS CSNPsSynthesis of Cu: The Cu2−xS@MnS CSNPs were synthesized using a one‐pot hot‐injection method. In brief, 0.6 mmol Cu(CH3COO)2 was dissolved in 20 mL of oleylamine under vigorous stirring, and 8 mL of 1‐dodecanethiol was added. The mixture was heated to 220 °C in an N2 atmosphere and maintained for 15 min. Subsequently, 8 mL of oleylamine containing 1 mmol Mn(CH3COO)2·4H2O was injected into the hot solution, and the temperature of the solution was changed to 195 °C. The reactions continued for another 20 min with different rates of temperature increase . Finally, solid black products were collected by centrifugation after cooling to room temperature.2−xS NPs were prepared using the procedures above but without the addition of Mn(CH3COO)2·4H2O and following another 20 min reaction.Pure Cu2−xS@MnS CSNPsSurface Modification of Hydrophobic Cu: The amphiphilic polymer composed of poly and oleylamine was synthesized based on a protocol in the literature.2−xS@MnS CSNPs, 150 µL of amphiphilic polymer stock solution (0.8 m in CHCl3) and 10 mL of hydrophobic Cu2−xS@MnS CSNPs (500 ppm in CHCl3) were combined and stirred for 4 h at room temperature. The solvent was removed by rotary evaporation, and 25 mL sodium carbonate solution (0.05 g mL−1) was added to the flask and stirred for 15 min at room temperature to disperse the hydrophilic Cu2−xS@MnS CSNPs. The solution was centrifuged and dispersed into PBS for further use.2−xS@MnS‐FITC CSNPs, 5 mg of FITC was added into an amphiphilic polymer stock solution (20 mg) and stirred for 24 h in the dark until CHCl3 was volatilized. The hydrophobic Cu2−xS@MnS CSNPs were modified with this production using the above procedures.To prepare the hydrophilic Cu2−xS@MnS CSNPsIn Vitro PT Performances of Cu: To measure the PT effect of Cu2−xS@MnS CSNPs with different MnS shell thicknesses, 100 µL of aqueous dispersions containing pure Cu2−xS NPs, thin‐shell Cu2−xS@MnS CSNPs, intermediate‐shell Cu2−xS@MnS CSNPs, and thick‐shell Cu2−xS@MnS CSNPs were each irradiated by an 808 nm laser  at a power density of 0.72 W cm−2. A thermal imaging camera  was used to record the temperature change.2−xS@MnS CSNPs, 100 µL of aqueous dispersions with different concentrations (0–500 ppm) were irradiated using the 808 nm laser for 5 min. In the photostability test, the sample was laser irradiated for 5 min and cooled naturally. The on–off process was repeated six times.To further investigate the PT effect of the selected thick‐shell Cu2 Generation of Cu2−xS@MnS CSNPs in Response to H2O2O: A portable dissolved oxygen meter was used to measure O2 generation. First, the Cu2−xS@MnS CSNPs were dispersed in 7 mL of aqueous solution with a concentration of 71 ppm, and different amounts of H2O2 were added. The dissolved oxygen concentration in the aqueous dispersions was recorded every 30 s after H2O2 was added. For investigation of the effect of the Cu2−xS@MnS CSNPs suspension concentration on O2 release, the amount of H2O2 was added and maintained at 71 × 10−3m, and the Cu2−xS@MnS CSNPs concentration was changed from 0 to 143 ppm.ROS Production: For ROS detection, the commercial chemical probe SOSG was used to detect its fluorescence intensity at 525 nm via fluorescence emission spectroscopy excited at 488 nm. SOSG (2.5 × 10−6m) was added to 1 mL of Cu2−xS@MnS CSNPs suspension (0–16.67 ppm), and the mixture was kept in the dark and irradiated with an 808 nm laser (0.72 W cm−2) for 60 min. Fluorescence emission spectroscopy was recorded. To test ROS production in response to H2O2, laser irradiation was conducted after addition of H2O2 (71 × 10−3m).Cell Uptake: HeLa cells were seeded into a 96‐well plate at 1 × 104 cells per well at 37 °C in the presence of 5% CO2 for 24 h. The Cu2−xS@MnS CSNPs suspensions were added to these wells at various concentrations . After incubation for 6 and 12 h, the medium was removed, each well was washed three times with PBS, and HeLa cells were digested with HNO3/HCl to determine the Cu uptakes via ICP‐AES measurements.2−xS@MnS‐FITC CSNPs. The medium was removed, and each well was washed three times with PBS. The cell nuclei were stained with DAPI for 20 min. Finally, the cells were observed by CLSM. The Cu2−xS@MnS‐FITC CSNPs and DAPI were excited separately using laser excitation at 494 and 358 nm and captured by emission at 525 and 461 nm.For visualization, HeLa cells were seeded into a 6‐well plate and cocultured with Cu2−xS@MnS CSNPsBiocompatibility of Cu: The in vitro cytotoxicity was evaluated using a CCK assay. HeLa cells were seeded into a 96‐well plate at 1 × 104 cells per well at 37 °C in the presence of 5% CO2. After incubation for 24 h, the Cu2−xS@MnS CSNPs suspensions were added to the wells at various concentrations and incubated for another 24 h. An amount of 200 µL complete medium with 10% (volume) CCK‐8 dispersion was added to each well of the microtiter plate and incubated in the CO2 incubator for another 2 h. The optical density (OD) was measured at 490 nm using a microplate reader. All experiments were independently repeated three times.In Vitro Phototherapy Effect: HeLa cells were seeded into a 96‐well plate at 1 × 104 cells per well at 37 °C in the presence of 5% CO2 for 24 h. The Cu2−xS@MnS CSNPs suspensions were added to the wells at various concentrations. After 24 h, cells were irradiated with an 808 nm laser at 0.72 W cm−2 for 7 min. After further incubation for another 2 h, the cell viability was detected using a CCK assay and observed on a CLSM after costaining with calcein AM and PI for 20 min.2O2 on phototherapy, H2O2 (100 × 10−3m) was added to the wells after Cu2−xS@MnS CSNPs suspension addition, and the cell viability was detected using a CCK assay after laser irradiation.To study the effect of HAnimals and Tumor Model: All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine. For the CDX model, cancer cells from a HeLa tumor cell line were subcutaneously injected into BALB/c nude mice (6–8 weeks old).3, the mice were sacrificed, and the tumor tissues were sampled and implanted into another group of mice with the same protocol. The 4th generation of animals was used in in vivo experiments in this study.For the PDX model, tumor samples were obtained from Shanghai Ninth People's Hospital with written informed consent from each patient and research ethics board approval in accordance with the Declaration of Helsinki. Herein, a tumor tissue sample from an HNSCC patient after surgical treatment was cut into ≈5 mm pieces and implanted into the BALB/c nude mice. After the tumor volume reached ≈1000 mmMR Measurement: For in vitro MR measurement, Cu2−xS@MnS CSNPs suspensions with different Cu concentrations were scanned under a 0.5 T small animal MR scanner (MesoMR23‐060H‐I) at room temperature. For MRI in vivo, HeLa tumor‐bearing mice were scanned with the same MR scanner before and after intratumour injection with Cu2−xS@MnS CSNPs suspension . During the experiments, all mice were first anaesthetized using chloral hydrate (8%).In Vivo Therapy Experiment: Tumors in mice were allowed to grow to a size of 5–10 mm in diameter and the mice were randomly assigned to four groups: Group I, untreated control; Group II, PBS + laser; Group III, CSNPs only; and Group IV, CSNPs + laser. For Groups III and IV, the Cu2−xS@MnS CSNPs suspensions  were injected intratumorally. Mice in Groups II and IV were subsequently irradiated by an 808 nm laser at a power density of 0.72 W cm−2 for 10 min, and an infrared camera was applied to monitor the temperature changes. The tumor sizes were measured after treatments using a Vernier caliper. The tumor volume was calculated according to the following formula: volume = 0.5 × length × width2.For tumor sectioning and staining experiments, the tumors from different groups were harvested 4 h after treatment, embedded in paraffin, and cryo‐sectioned into 4 µm slices using a conventional microtome. The slides were stained with H&E, TUNEL, and Ki‐67 and subsequently observed.In Vivo Systemic Toxicity Study: Tumor‐bearing mice were intravenously injected with the Cu2−xS@MnS CSNPs suspension . The control group was untreated. At different time points, major tissues including heart, liver, spleen, lung, and kidney were collected and digested with HNO3/HCl to determine the Cu contents by ICP‐AES measurements. Blood was collected for blood biochemistry analysis. Moreover, these organs were observed with H&E staining for comparison with untreated healthy mice.The authors declare no conflict of interest.SupplementaryClick here for additional data file.


The phenotype of HDC and functional relevance of CX3CR1 was assessed in mice following necro-inflammatory liver injury induced by the hepatotoxic agent carbon tetrachloride (CCl4) and in steatohepatitis caused by a methionine/choline-deficient (MCD) diet. In both the experimental models, hepatic inflammation was associated with a massive expansion of CD11c+/MHCIIhigh/CD11b+ myeloid HDCs. These cells also expressed the monocyte markers Ly6C, chemokine (C-C Motif) receptor 2 (CCR2), F4/80 and CD88, along with CX3CR1, allowing their tentative identification as moDCs. Mice defective in CX3CR1 showed a reduction in liver-moDC recruitment following CCl4 poisoning in parallel with a defective maturation of monocytes into moDCs. The lack of CX3CR1 also affected moDC differentiation from bone marrow myeloid cells induced by granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) in vitro. In wild-type mice, treatment with the CX3CR1 antagonist CX3-AT  24 h after CCl4 administration reduced liver moDCS and significantly ameliorated hepatic injury and inflammation. Altogether, these results highlight the possible involvement of moDCs in promoting hepatic inflammation following liver injury and indicated a novel role of CX3CL1/CX3CR1 dyad in driving the differentiation of hepatic moDCs.Recent evidence suggests that hepatic dendritic cells (HDCs) contribute to the evolution of chronic liver diseases. However, the HDC subsets involved and the mechanisms driving these responses are still poorly understood. In this study, we have investigated the role of the fractalkine receptor CX In recent years, growing attention has been paid to the importance of myeloid cells in modulating the evolution of both acute and chronic liver injury ,2. Altho+/CD11b− in mice; CD141+/CD14− in humans) and type-2  cells [During homeostasis, hepatic dendritic cells (HDCs) represent a small sub-fraction (less than 1%) of total liver myeloid cells. HDCs are mainly localized in the portal areas, although a recent study in mice has identified them also under the liver capsule . Based os) cells . In heals) cells ,8. Howevs) cells ,10,11. + type-1 myeloid HDCs are lowered in patients with advanced chronic liver diseases [+ type-1 myeloid HDCs display a hepatoprotective action in mice, and their depletion in Batf3-deficient mice favors the onset of steatohepatitis [3CR1 and producing IL-10 are present in the liver during homeostasis and counteract acute hepatic inflammation [3CR1 and an increased TNF-α production contribute to the evolution of steatohepatitis [The actual role of HDCs in the pathogenesis of liver diseases is still a matter of debate, due to contradictory data obtained in experiments in which HDCs have been either artificially expanded or depleted ,14,15,16diseases . Furtherepatitis . 3CR1 in directing moDC differentiation within the liver.A further element of complexity is given by the fact that—in response to inflammatory stimuli—monocytes can differentiate to a distinct dendritic cell subset called monocyte-derived dendritic cells (moDCs) or inflammatory dendritic cells. MoDCs have several surface markers and functional properties in common with type-2 myeloid dendritic cells, although they develop independently from the transcription factors driving myeloid dendritic cell differentiation ,22,23. F3CR1gfp/+ and CX3CR1gfp/gfp mice were housed in pathogen-free conditions and fed ad libitum with a standard chow diet and water. Liver injury was induced by injecting intra-peritoneally eight-week-old male mice with CCl4 (0.6 mL/kg in olive oil). Control animals received an injection with olive oil alone. Non-alcoholic streatohepatitis (NASH) was induced by feeding wild-type C57BL/6 mice with a choline/methionine-deficient diet , as previously reported [3CR1 antagonist CX3-AT (European Patent EP2913060A1) [4. Control animals received a similar amount of saline alone. All animals were euthanized 36 h after CCl4 administration. The animal experiments were performed at RWTH University Hospital Aachen and comply with ethical guidelines for animal experimentation. The protocols were approved (AZ 84-02.04.2013.A054 and AZ 84-02.04.2014.A165) by the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Recklinghausen (Germany) according to the European legal requirements. C57BL/6 wild-type, CXreported . Control13060A1) ,25 was kLivers were rapidly removed and, after rinsing in ice-cold saline, cut in pieces. Aliquots were immediately frozen in liquid nitrogen and kept at −80 °C until analysis. Two portions of each liver were fixed in 10% formalin for histology. Plasma alanine aminotransferase (ALT) and aspartate amino transferase (AST) were determined by a spectrometric kit supplied by Radim S.p.A. . The extension of liver injury was assessed morphologically in hematoxylin/eosin-stained liver sections.3CR1, Ly6C and Ly6G (Biolegend). After surface staining, cells were fixed using 2% formalin and permeabilized using 0.5% saponin . Total cell numbers were determined by adding fixed numbers of Calibrite APC beads  before measurement as an internal reference [3CR1low/− and CX3CR1high CD11b+ type-2 myeloid HDCs obtained from the livers of CCl4-treated mice were cell sorted using a FACS Aria-II cytometer (BD Bioscience). Sorted cells  were analyzed using the Nanostring Immunology gene array kit covering 561 genes , according to the manufacturer’s instructions. Differential gene expression was calculated using the R package DESeq2. A log2 fold change threshold greater than 2 and an adjusted P value of <0.01 were used for comparison.Livers were digested by type-IV collagenase , and intrahepatic leukocytes were isolated by multiple differential centrifugation steps according to . The celeference . Sample −ΔCt, was expressed as a fold increase over the control samples.mRNA was extracted from snap-frozen liver fragments using the peqGOLD  reagent. cDNA was generated from 1 µg of RNA using the Transcriptor first-strand cDNA synthesis kit . The quantitative real-time polymerase chain reaction (PCR) was performed using SYBR Green Reagent  and a QuantStudio 6 Flex Real-Time PCR System . All samples were run in duplicate and the relative gene expression, calculated as 23CR1gfp/+ and CX3CR1gfp/gfp mice according to [Myeloid cells were isolated from the tibia and femur bone marrow of CXrding to . Red bloStatistical analyses were performed by SPSS statistical software  using a one-way ANOVA test with Tukey’s correction for multiple comparisons or a Kruskal–Wallis test for non-parametric values. Significance was taken at the 5% level. Normality distribution was assessed by the Kolmogorov–Smirnov algorithm.4)  A–C. Thisoid HDCs D. Comparoid HDCs D. + HDCs expanding in response to hepatic inflammation were characterized by a high expression of CX3CR1 (3CR1low type-2 myeloid HDCs present at homeostasis [+/Ly6C+/CD88+/CD26− monocyte-derived dendritic cells (moDCs) from CD88−/CD26+ type-1 and type-2 myeloid dendritic cells. In our hands, CD11b+/Ly6C+/CX3CR1+ HDCs detectable in the livers of CCl4-treated mice were largely CD88+ and did not express CD26 , toll-like receptors , chemokines , immunoglobulin Fc receptors , CD14, macrophage scavenger receptor 1, urokinase and urokinase receptor  induced by feeding mice with a methionine/choline-deficient (MCD) diet ,14. Sinc of NASH , we inve3CL1) was upregulated during hepatic inflammation in parallel with the expansion of CX3CR1+ moDCs  inserted in the CX3CR1 gene (CX3CR1gfp/gfp), showing that the expansion of myeloid dendritic cells associated with CCl4 poisoning were strongly reduced in CX3CR1gfp/gfp mice compared with CX3CR1-proficient (CX3CR1+/gfp) animals. Such an effect specifically involved a fraction of liver CD11c+/MHCIIhigh/CX3CR1+/CD11b+/CD88+ moDCs . In this setting, the lack of CX3CR1 specifically hampered MHCII expression via CD11b+/CD11c+ pre-dendritic cells (p < 0.05), indicating a CX3CR1 action in the processes leading to full moDC development. To further evaluate such a possibility, we investigated CX3CR1 effects on the mRNA levels of the transcription factors Zbtb46, interferon responsive factor-4 (IRF-4) and interferon responsive factor-8 (IRF-8) that have been implicated in moDC maturation induced by GM-CSF/IL-4 [3CR1 significantly reduced Zbtb46 and IRF-8 mRNAs, while IRF-4 mRNA was unaffected (3CR1 in monocyte differentiation to moDCs. MoDCs can be readily obtained in vitro by culturing bone marrow myeloid cells with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) . We obsed CX3CR1 A. Thus, ic cells . ConversCSF/IL-4 ,30,31,32affected C, furthe3CR1 affects the anti-inflammatory properties of resident CX3CR1+ type-2 myeloid HDCs [3CR1gfp/gfp mice were unsuitable to address the possible role of CX3CR1+ moDCs in sustaining inflammatory responses associated to hepatic damage. To overcome such a limitation, we took advantage of CX3-AT, a NH2-terminal CX3CL1-derived peptide that has been previously reported to block CX3CR1 signaling in lymphocytes [4, and the effects on hepatic inflammation were monitored over the following 12 h. At this dosage, CX3-AT was not hepatotoxic and did not affect the liver distribution of monocyte/macrophages and CX3CR1+ type-2 myeloid HDCs , while they are negative for the dipeptidyl peptidase-4 (CD26), two surface markers that discriminate CD11b+ monocyte-derived DCs (CD88+/CD26−) from type-1 and type-2 classical DCs (CD88−/CD26+) [+/CX3CR1+/CD88+ moDCs also account for HDC expansion occurring at the onset of experimental NASH, confirming the previous implication of moDC in sustaining liver injury and inflammation in experimental models of chronic steatohepatitis [+ moDCs contribute to the formation of intrahepatic myeloid-cell aggregates for T-cell expansion (iMATEs), which are responsible for CD8+ T-lymphocyte stimulation during viral infection. On these bases, we propose that the differentiation of dendritic cells from liver-infiltrating monocytes might represent a mechanism to rapidly expand the pool of HDCs in response to pro-inflammatory stimuli. Nonetheless, we are well aware that the above surface markers do not allow a definitive differentiation of moDCs from liver-infiltrating inflammatory monocyte-derived macrophages that are also Ly6Chigh/CX3CR1+ and can express CD11c, MHCII and CD88 to varying extents [Growing evidence indicates that HDCs play an important role in modulating hepatic immune and inflammatory responses both at homeostasis and in liver diseases ,6. Alongeostasis ,16. Howe−/CD26+) . In lineepatitis  have rep extents .3CR1-deficient mice showed that the expansion of moDCs in response to liver injury requires CX3CR1. It is noteworthy that, despite CX3CR1 being widely present in liver myeloid cells (including inflammatory monocyte-derived macrophages), such an effect is specific to moDCs. In fact, in line with a previous report [3CR1 in CCl4-treated mice did not affect the pool of liver-infiltrating monocyte-derived macrophages, further indicating that CX3CR1+ moDCs might represent a different cell subset. At present, CX3CR1 function in moDCs is still poorly characterized. David et al. have shown that CX3CR1 contributes to liver dendritic cell replenishment after selective depletion by directing bone marrow-derived precursors [3CR1/CX3CL1 interaction is critical for monocyte adhesion to endothelial cells and their migration into atherosclerotic plaques [3CR1-deficient mice receiving CCl4 do not involve the hepatic recruitment of monocytes. This is consistent with the recent observation that chemokine (C-C Motif) receptor 2 (CCR2) is the main responsible for monocyte attraction within injured livers [3CR1 deficiency impairs the capacity of dendritic cell precursors to fully express MHCII. Moreover, lack of CX3CR1 reduces in vitro moDC maturation from bone marrow myeloid cells incubated with MG-CSF/IL-4. Altogether these data suggest that CX3CR1-mediated signals are required for moDC differentiation. Supporting this view, we have observed that in MG-CSF/IL-4-treated myeloid cells CX3CR1 influences the expression of Zbtb46 and IRF-8, two transcription factors implicated in driving monocyte differentiation to moDC [3CR1 might have additional effects on moDCs, as this receptor is required for the survival of liver-infiltrating monocyte-derived macrophages [The experiment using CXs report , the lac plaques . Howeverd livers . On the  to moDC ,31. Howerophages .3CR1+ moDC differentiation using the CX3CR1-blocker CX3-AT ameliorates lobular inflammation and parenchymal damage following acute CCl4 poisoning. These findings are consistent with previous data showing that the H2S donor NaHS lowers hepatic TNFα levels and ameliorates parenchymal injury in an experimental model of chronic steatohepatitis by selectively affecting the development of CX3CR1-expressing HDCs [3CR1+ moDCs can contribute to sustaining hepatic injury and inflammation through the production of pro-inflammatory mediators. Such an interpretation is in line with the data of Connolly et al. [It is well established that moDCs are not only effective in antigen presentation to lymphocytes, but also produce pro-inflammatory mediators contributing to sustaining inflammatory processes ,22,23. Ring HDCs . Taken ty et al.  showing 3CR1-expressing HDCs might have different functional capabilities in relation to their origins and/or exposure to different environmental stimuli. In fact, during homeostasis liver CX3CR1+/CD11b+ type-2 myeloid HDCs produce IL-10 and have anti-inflammatory capabilities [3CR1+ moDCs, in response to inflammatory stimuli, is responsible for the development of HDCs with pro-inflammatory and immune-stimulating activities. These opposite functions of dendritic cells expressing similar surface markers might help to explain the inconsistent results so far obtained in assessing the role of HDCs in the evolution of chronic liver diseases [The results of this work also provide evidence that CXbilities , therebydiseases ,14,15,163CR1/CX3CL1 dyad in modulating moDC differentiation within the liver.In conclusion, the present results add functional data regarding the complex role of dendritic cells in the mechanisms of liver injury, indicating that the rapid expansion of HCDs in response to hepatic injury involve monocyte differentiation to inflammatory moDCs. Furthermore, our data point to the importance of CX


Astragali Radix, in ameliorating refractory cutaneous wound healing in vitro, which may provide a new insight into therapeutic strategy for diabetic wounds.  Refractory wound healing is a severe complication of diabetes with a significant socioeconomic burden. Whereas current therapies are insufficient to accelerate repair, stem cell-based therapy is increasingly recognized as an alternative that improves healing outcomes. The aim of the present study is to explore the role of cycloastragenol (CAG), a naturally occurring compound in β-catenin knockdown cell line. Cell counting kit-8, scratch wound healing, and transwell migration assay were used to determine the effects of CAG in cell growth and migration. The activation of TERT, β-catenin, and c-Myc was determined using real-time qPCR and western blot analysis. Chromatin immunoprecipitation (ChIP) was performed to evaluate the associations among CAG, TERT, and Wnt/β-catenin signals.  Human epidermal stem cells (EpSCs) obtained from nine patients were exposed to CAG, with or without DKK1 . A lentiviral short hairpin RNA (shRNA) system was used to establish the telomerase reverse transcriptase (TERT) and β-catenin, c-Myc. These effects of CAG were most pronounced at a dose of 0.3 μM. Notably, the CAG-promoted proliferative and migratory abilities of EpSCs were abrogated in TERT and β-catenin-silenced cells. In addition, the ChIP results strongly suggested that CAG-modulated TERT was closely associated with the activation of Wnt/β-catenin signaling.  CAG not only promoted the proliferation and migration ability of EpSCs but also increased the expression levels of TERT, β-catenin signaling. Our data indicate that CAG is a TERT activator of EpSCs and is associated with their proliferation and migration, a role it may play through the activation of Wnt/ In recent years, wound repair, especially for chronic nonhealing wounds, has emerged as a global public health issue that concerns physical and mental health in both type 1 and type 2 diabetes .Wound healing is a complex process that requires an inflammatory reaction, wound clotting, re-epithelialization, remodeling regulation, and control of stem cells , 5. The β-catenin signaling, which could be improved with the administration of Wnt activator [β-catenin signaling. However, despite this progress, current therapies and research into the relevant mechanisms remain inadequate.In terms of mechanism, there is an accumulation of evidence that telomere is critically important for wound repair and that the dysfunction of telomere has a negative effect on wound healing by impairing the ability of EpSCs to proliferate or migrate . The Winctivator . The datAstragali Radix, has been confirmed to have a variety of pharmacological functions, including antiaging, anti-inflammation, antifibrosis, and protection of the liver and endothelium [β-catenin pathway in EpSCs, in order to provide new insight into therapeutic strategy for diabetic wounds.Cycloastragenol (CAG), a triterpenoid saponin compound and a hydrolysis product of othelium , 13. Resothelium . Howeverβ1-integrin  were, respectively, performed to identify the phenotype of our EpSCs. Finally, isolated cells were cultured in a Keratinocyte medium  supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin, in a humidified atmosphere of 95% air and 5% CO2 at 37°C. All the participants wrote the informed consent, and the experimental procedures were approved by the Ethical Review Board at the First Affiliated Hospital of Zhejiang Chinese Medicine University (No. 2017-KL-024-01).Human foreskin samples were obtained from seven healthy donors. Primary EpSCs were isolated using a standard protocol adopted from Yang et al. , which aβ-catenin were manufactured by GenScript . The sequence of short-hairpin RNA (shRNA) for target genes was provided by the authors. The plasmids were constructed using vector pRNAi-U6.2 (+) as the backbone and Hpa I and Xho I as the cloning site. The primer of shRNAs were synthesized by Genepharma Corp. , and the sequences used were as follows: shTERT #1, 5′-GCTGCTTTATTCTCCCATTGA-3′ and 5′- TCGACCTGCTGGAATCTCGTG-3′; shTERT ##2, 5′-GGTTAATAA-GGCTGCAGTTAT-3′ and 5′- TCGACCTGCTGGAATCTCGTG-3′; shTERT ###3, 5′-GCTTATGGCAACCAAGAAAGC-3′ and 5′-TCGACCTGCTGGAATCTCGTG-3′; shβ-catenin #1, 5′-GCTCGTGGAGACCATCTTTCT-3′ and 5′-TCGACCTGCTGGAATCTCGTG-3′; shβ-catenin ##2, 5′-GGAAGACAGTGGTGAACTTCC-3′ and 5′-TCGACCT-GCTGGAATCTCGTG-3′; shβ-catenin ###3, 5′-GGAAGAGTGTCTGGAGCAAGT-3′ and 5′-TCGACCTGCTGGAATCTCGTG-3′. For stable transfection, T293 cells were infected with TERT and β-catenin shRNA lentiviral particles and control lentiviruses, according to the manufacturer's instructions  and using Lipofectamine® 3000 . The transfection efficiencies were detected using qRT-PCR and western blot, respectively.Plasmid constructs of TERT and 3 cells per well; three replicates per group) were seeded into a 96-well plate and incubated at 37°C. At the indicated time , the absorbance of cells cultured in different concentrations  of GCA was measured at 450 nm with 10 μL of CCK-8 reagent  for approximately 1.5 h. The assay was conducted in six replicate wells for each sample.Cells (2 × 105 and incubated with GCA (0.3 μM) or with an equal volume of dimethyl sulfoxide (DMSO) as a control for a further period of 24 h. Migrated cells were determined using crystal violet staining. Images of the crystal violet stain were captured using a light microscope . The assay was conducted on three replicates per group, and three parallel experiments were performed.The cells were distributed on the transwell inserts  at a density of 1 × 105 cells per well. After the cells had attached, the monolayer was scratched with a P1000 pipette tip and washed three times with PBS to remove the floating cells. The cells were then exposed to GCA from different groups or an equal volume of DMSO. EpSCs were photographed at 0 h and 24 h after wounding, and the distance between the scratches was taken to represent the proliferative ability of EpSCs in wound healing.Cells were seeded in six-well plates at 5 × 10−ΔΔCT method [β-catenin forward: 5′-CATCTACACAGTTTGATGCTGCT-3′ and reverse: 5′-GCAGTTTTGTCAGTTCAGGGA-3′; c-Myc forward: 5′-GGCTCCTGGCAAAAGGTCA-3′ and reverse: 5′-CTGCGTAGTTGTGCTGATGT-3′; GAPDH forward: 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse: 5′- GGCTGTTGTCATACTTCTCATGG-3′.Total RNAs of fresh cells were extraction by using the TRIzol reagent  and then synthesized to complementary DNA according to the instruction of manufacturer . Real-time PCR reaction  was conducted in a Bio-Rad system for 40 cycles, with each cycle consisting of denaturation at 95°C for 5 s and annealing at 60°C for 60 s; the melt curve was 95°C for 60 s and 65°C for 60 s. GAPDH was used as an endogenous control for normalization, and the relative quantitation of target gene expression was quantified using the 2β-catenin , c-Myc , and GAPDH . Next day, proteins were incubated with horseradish peroxidase-conjugated secondary antibody and visualized by using the enhanced chemiluminescence method. The intensity of the bands was quantified using the image lab 6.0 system , and the data were normalized to the GAPDH as loading controls. All western immunoblot analyses were performed three times.Western blot was performed as described elsewhere. Briefly, the fresh cells were harvested, washed with PBS, and lysed in lysis buffer. After being boiled, the proteins were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and then electrotransferred onto polyvinylidene fluoride (PVDF) membranes. Primary antibodies used were as follows: anti-TERT , β-catenin , and a normal rabbit IgG was used as a control. All experiments were performed in triplicate and repeated a minimum of three times.The total protein was extracted and processed according to the Total Protein Extraction Kit for Animal Cultured Cells and Tissue User Manual v.4. Immunoprecipitation was performed with antibodies against TERT  and t test, and P value <0.05 was considered as statistically significant.All data were analyzed using SPSS  and presented as mean ± standard deviation (SD). The Kolmogorov–Smirnov method was used to test whether the data were distributed normally. Statistical analysis was performed by the paired two-sample Student's β1-integrin were examined using flow cytometry, and K19, K15, CD34, and K14 were detected using IF staining [β1-integrin and K19, K15, and CD34 were detected in our EpSCs. In contrast, there was almost no expression of K14 in these cells, which suggests that successful human primary EpSCs were achieved in our culture conditions . Cells in each group clearly displayed a similar, normal epithelial-like appearance, with close cell-to-cell contacts, up to 72 h. Neither low nor high concentrations of CAG were associated with any morphologic alterations in EpSCs under a phase-contrast microscope. Our CCK-8 assay demonstrated that there were no concentration- or time-dependent cytotoxic effects of CAG on these human primary EpSCs over the indicated time spans . More importantly, the cell viability of EpSCs increased significantly in co-culture with CAG at days 3, 5, and 7 compared with normal cells, and the changes were especially pronounced at a concentration of 0.3 μM. In other settings, there were no detectable differences in the cell viability of each group . However, in comparisons between CAG-treated and CAG-treated TERT-silenced EpSCs, the latter group persistently showed a detectable decrease in cell viability compared with the former group (Telomerase reverse transcriptase (TERT) is a catalytic component of human telomerase, undetectable in normal somatic cells but upregulated in cancers and stem cells, where telomere length is maintained by telomerase . In the AG EpSCs . Most imAG EpSCs . As shower group . Collectβ-catenin signaling is one of the key regulators for cell proliferation and tissue regeneration [β-catenin signaling in EpSCs. As expected, the results of real-time qPCR indicated that the relative expression levels of β-catenin and c-Myc were significantly increased in EpSCs treated with CAG compared with those without CAG treatment  TERT and Wnt/β-catenin signaling was dramatically activated and co-expressed in EpSCs after CAG treatment. These observations suggest that CAG effectively contributes to the proliferation and migration of EpSCs, and this may be related to the increased expression of TERT and the activation of Wnt/β-catenin signaling. Our data provide new insight into this mechanism and its potential therapeutic use in experimental diabetic wound repair.In this study, we first investigated the role of CAG in the proliferation and migration of EpSCs and the mechanism involved. Our data demonstrated that (1) CAG promotes the proliferation and migration of primary human EpSCs; (2) CAG effectively increases the expression of TERT in primary human EpSCs; (3) there was a significant activation of Wnt/Skin acts as a barrier to foreign pathogens, regulates body temperature, provides sensation, and prevents dehydration of the body . With tyCAG is an aglycone of astragaloside IV that has various pharmacological actions , and it μM as the most effective concentration in terms of contribution to cell proliferation and migration.First, as expected, we found that CAG promoted the proliferation and migration of primary human EpSCs. Notably, our data also demonstrated that, during the indicated time spans, CAG was not associated with any morphologic alterations in EpSCs, and there were no concentration- or time-dependent cytotoxic effects of CAG on these primary human EpSCs. Most importantly, we identified 0.3 Next, we explored whether CAG had a protective role on TERT in primary human EpSCs. TERT, a reverse transcriptase of telomerase has been shown to be involved in certain types of injury through the activation of specific signals, such as NF-kappa B and autophagy , and it β-catenin signaling. The Wnt/β-catenin signaling pathway is known to be crucial for proper organ development. Classically, Wnt signaling has been separated into canonical and noncanonical signaling, with Wnt signaling that relies on the activation of the transcriptional coactivator β-catenin designated as canonical [β-catenin signaling is of great importance for stem cell growth, maintenance, and turnover of epithelium during homeostasis and in response to injury, there is still some controversy as to the details. For example, Sun et al. proposed that Wnt signaling is a potent regulatory molecule for stem cell turnover and skin regeneration but that it is not well activated in diabetic wounds [β-catenin pathway in wound repair [β-catenin contributes to the expression of TERT, eventually promoting cell proliferation and migration. In combination, these results provide useful data for research into the mechanism of diabetic wound healing.Most importantly, we further confirmed that TERT may play this role though Wnt/anonical . Althougc wounds . Houschyc wounds . By contd repair . NotablyThe present study has some limitations. It should be acknowledged that our data propose a general role for CAG in promoting the proliferation and migration of EpSCs, rather than a mechanism involved specifically in the evolution of diabetic wound healing. The in vitro experiment may therefore not be fully representative of the conditions in clinical settings with diabetic patients. Further research using classic animal models or target gene-deficient animals is required to confirm our findings. Furthermore, CAG is an aglycone derivative of astragaloside IV that has various pharmacological actions and regulatory signals , and thuβ-catenin signaling.In conclusion, our data indicate that CAG can promote the proliferation and migration of EpSCs, and that this is associated with increased expression of TERT in EpSCs. CAG may play this role through activation of Wnt/


The reported data deal with the derivation of the equation of isostatic line of compression (ILC) and splitting force in a bottle-shaped strut with different height-to-width ratios. The final data show that the splitting force in a bottle-shaped strut is not only related to the height-to-width ratio (h/b), but also related to the load area ratio. Specifications Table•The data provide the detailed derivation process of the equation of isostatic line of compression.•The data provide a formula to calculate the magnitude of the splitting force•The data provide a formula to determine the location of the resultant splitting force.•The data serves as a methodological benchmark for further attempts to improve the formula of the splitting force subjected to different geometric and boundary conditions.•Considering the different assumptions based on the previous researches, A mathematic and explicit describing the equation of ILCs is established.•Splitting force formulae for the struts with different load area ratios are obtained.This datea article refers to the research paper Splitting force of Bottle-shaped Struts with Different Height to Width Ratios  To calculate the transverse stresses, the CDM should be defined as the mathematical model of principal compressive-stress trajectories. Five geometric and physical boundary conditions of the ILCs of the CDM are given as follows.(1) The equations of ILCs are assumed to have the polynomials form, given byThe first and second derivative of Eq. For For For For For b− (6):(4) × (8) − (7):b2 − (9) × 2:(7) × By Substituting Eq. By Substituting Eqs. By Substituting Eqs. By Substituting A–E into Eq. Based on the above results, we get the second derivative of the Eq. Let thatBased on the relationship between the curvature of the ILCs and the transverse stresses, we get that ,3(18)σTxBy integrating the transverse tensile stresses along the axis of the struts, the total transverse force below the axis of the struts can be determined bybd can be expressed as follows:The location of the splitting force By Substituting Eqs. None.


Hepatoblastoma is a malignant tumor in the liver of children that generally occurs at the age of 2–3 years. There have been ample evidence from the preclinical as well as clinical studies suggesting the activation of Wnt/β-catenin signaling in hepatoblastoma, which is mainly attributed to the somatic mutations in the exon 3 of β-catenin gene. There is increased translocation of β-catenin protein from the cell surface to cytoplasm and nucleus and intracellular accumulation is directly linked to the severity of the cancer. Accordingly, the alterations in β-catenin and its target genes may be used as markers in the diagnosis and prognosis of pediatric live tumors. Furthermore, scientists have reported the therapeutic usefulness of inhibition of Wnt/β-catenin signaling in hepatoblastoma and this inhibition of signaling has been done using different methods including short interfering RNA (siRNA), miRNA and pharmacological agents. Wnt/β-catenin works in association with other signaling pathways to induce the development of hepatoblastoma including Yes-associated protein (YAP)1 (YAP-1), mammalian target of rapamycin (mTOR) 1 (mTOR-1), SLC38A1, glypican 3 (GPC3), nuclear factor κ-light-chain-enhancer of activated B cells (NF-kB), epidermal growth factor receptor, ERK1/2, tumor necrosis factor-α (TNF-α), regenerating islet-derived 1 and 3 α (REG1A and 3A), substance P (SP)/neurokinin-1 receptor and PARP-1. The present review describes the key role of Wnt/β-catenin signaling in the development of hepatoblastoma. Moreover, the role of other signaling pathways in hepatoblastoma in association with Wnt/β-catenin has also been described. Hepatoblastoma is a malignant tumor in the liver of children that generally occurs at the age of 2–3 years . HepatobThe Wnt/β-catenin signaling pathway is crucial in controlling hepatic homeostasis, maintaining adherens junctions, metabolic zonation and regeneration suggesting its role in almost every aspect related to liver functioning. However, its aberrant activation is associated with the development of various hepatic diseases including hepatoblastoma . Indeed,Since β-catenin expression has been detected in all the heterologous elements such as squamous, osteoid and chrondroid tissues, along with the mesenchymal derived cells, therefore, scientists have employed the β-catenin staining to differentiate different histological variants of hepatoblastoma . StudiesThe studies in late 1990s reported that the mutations in the β-catenin genes lead to accumulation of β-catenin protein in the cytoplasm and nucleus of the cells of hepatoblastoma. Bläker et al.  reported2-terminal domain of β-catenin gene in epithelial and mixed hepatoblastoma patients revealed the presence of missense mutations or interstitial deletions in the GSK-3β phosphorylation motif . The study performed by Takayasu et al. [Later, scientists verified these initial findings and reported that somatic mutations in β-catenin gene are responsible for its accumulation inside the tumor cells, a crucial event in tumorigenesis ,24–27. Iu et al.  in 68 pru et al. .Apart from the clinical studies, scientists have explored the role of mutations of β-catenin in different animal models of hepatoblastoma. In 19-anthraquinone-induced and 8-oxazepam-induced hepatoblastoma in B6C3F1 mice, mutations have been reported in the β-catenin gene. Moreover, the nuclear localization of β-catenin protein was identified in hepatoblastoma in comparison with normal liver cells, in which β-catenin was detected mainly on the plasma membrane . In anotAs described in above studies, scientists have directly measured the expression of β-catenin in the cytoplasm or nucleus in tumor cells to delineate its role in hepatoblastoma. Apart from it, the expression of other related proteins has also been used as an indirect method to identify the activation of Wnt signaling. It is well reported that four and a half of LIM-only protein 2 (FHL2) is a novel β-catenin-interacting protein and it acts as a co-activator of β-catenin. In hepatoblastoma patients, an increase in the expression of FHL2 has been detected, which suggests the activation of Wnt pathway. An increase in the expression of FHL2 along with β-catenin mutation suggests that FHL2 may be possibly involved in transactivating the β-catenin signaling in cancer cells . StudiesIt is shown that Wnt/β-catenin works in association with other signaling pathways to induce the development of hepatoblastoma .Increasing number of studies has shown the cooperation between β-catenin and Yes-associated protein 1 (YAP1) in inducing the development of hepatoblastoma. In a clinical study, the nuclear co-localization of β-catenin and YAP1 was identified selectively in approximately 80% of the cases of hepatoblastoma, but not in hepatocellular carcinoma and cholangiocarcinoma . AnotherRaptor (the unique subunit of mTORC1) was shown to inhibit cell growth in hepatoblastoma cells and delay YAP/β-catenin-induced development of hepatoblastoma in mice, respectively. Moreover at the molecular level, there was a parallel increase in the expression of amino acid transporter (SLC38A1) and mTORC1 in the hepatoblastoma tissues of mice, which was significantly reduced during mTOR1 inhibition. Silencing of YAP led to decrease in the SLC38A1 expression and inhibition of mTORC1 suggesting that the activation of YAP-β catenin may induce activation of mTORC1 to promote the development of hepatoblastoma by inducing the expression of SLC38A1 [In Yap1-β-catenin mice model, treatment with rapamycin  inhibitor) was shown to attenuate the tumor growth over a period of time along with inhibition of mTORC1 suggesting that mTCOR1 is a potential target in YAP-1-β-catenin signaling cascade in hepatoblastoma . A previ SLC38A1 .Glypican 3 (GPC3), a heparan sulfate proteoglycan, is a cell surface oncofetal protein, which is highly expressed in number of pediatric tumors including hepatoblastomas. Immunostaining has revealed the co-expression of GPC3 and β-catenin in the tissues of hepatoblastoma . It is sNuclear factor κ-light-chain-enhancer of activated B cells (NF-kB) is an important gene, which plays a crucial role in the regulation of physiological and pathological processes, including cancer development . To undeThe close relationship between epidermal growth factor receptors and β-catenin signaling has been explored in the development of hepatoblastoma. In transgenic mice with mutated β-catenin gene, an increase in hepatocyte proliferation along with increase in cytoplasmic and nuclear accumulation of β-catenin was demonstrated. Along with it, there was also a parallel increase in the mRNA and protein levels of epidermal growth factor receptors in the transgenic livers. The inhibition of epidermal growth factor receptors led to decrease in the liver size in these transgenic mice. More analysis confirmed the activation of epidermal growth factor receptor in response to Wnt-3A treatment, which was attenuated by, in the presence of Wnt antagonist, a frizzled related protein 1. Accordingly, it may be suggested that the activation of Wnt/β-catenin signaling increases the expression of epidermal growth factor receptors on the liver to increase proliferation of hepatocytes in hepatoblastoma .Epithelial to mesenchymal transition (EMT) is a critical process in the progression of epithelial tumors including hepatoblastoma . MutatioThere have been other molecules that participate in Wnt/β-catenin triggered signaling pathway in hepatoblastoma including tumor necrosis factor-α (TNF-α), regenerating islet-derived 1 and 3 α (REG1A and 3A), substance P (SP)/neurokinin-1 receptor and PARP-1. Using immortalized murine hepatocyte cell line (AML12) that stably expressed mutant β-catenin, it was shown that stabilization of β-catenin enhanced proliferation of hepatocytes, suppressed TNF-α-induced apoptosis, induced anchorage-independent cell growth and increased the expression of c-myc and cyclin D1. It suggests that increase in β-catenin may produce its cellular proliferative effects in hepatoblastoma due to inhibition of TNF-α-induced apoptosis . REG1A a50 ranging from 4.87 to 32 μmol/l [SFRP1), which normally down-regulates Wnt signaling and is transcriptionally silenced in hepatoblastoma cells [CTNNB1 expression. Among the different mimetics, miR-624-5p has been shown to trigger cell senescence in in vitro as well as in vivo studies involving experimental hepatoblastoma growth along with inactivation of Wnt/β-catenin signaling [Considering Wnt/β-catenin as a potential therapeutic target, scientists have attempted to explore the usefulness of suppression of Wnt/β-catenin signaling in the preclinical studies related to hepatoblastoma. In hepatoblastoma cell lines (HuH-6 and HepG2) transfection of siRNAs or sequence-specific antisense morpholino oligonucleotides leads to degradation of overexpressed β-catenin in the nucleus and reduces the levels of c-myc. It suggests that β-catenin may be potential target of gene therapy to manage pediatric hepatic tumors, with mutations and overexpression of β-catenin gene ,55. Tran2 μmol/l . Adminisma cells . Co-admima cells . The sciignaling . TherefoBased on the number of studies, it has been proposed that Wnt/β-catenin signaling pathway may be an important target to modulate the proliferation of cancer cells including cancers of female reproductive system  and breaThere has been an exploration of new targets in the management of hepatoblastoma including signal transducer and activator of transcription signaling 3 (STAT3) ,72; NOTCApart from the association of Wnt/β-catenin signaling with the induction of hepatoblastoma, studies have shown that the activation of this signaling also regulates hallmarks of tumor progression including degree of differentiation, invasiveness, metastasis . The dowSomatic mutations in the exon 3 of β-catenin gene (CTNNB1) may lead to activation of Wnt/β-catenin signaling in hepatoblastoma, which is characterized by increased cytoplasmic/nuclear accumulation of β-catenin. The accumulation may result either directly or indirectly through other signaling pathways involving activation of GPC3, NF-kB, GSK-3β, ERK1/2 and FHL2, which serves as a co-activator of β-catenin. The activation of β-catenin signaling may trigger the activation of other signaling pathways to induce the development of hepatoblastoma, which may include inhibition of TNF-α-dependent apoptosis, increase in the secretion of epithelial growth factor and formation of complex of with YAP-1 protein. The interaction between β-catenin and YAP-1 protein may activate mTOR pathway to increase the expression of amino acid transporter, i.e. SLC38A1, which may be critical in the development of hepatoblastoma. The inhibition of Wnt/β-catenin signaling using siRNA, miRNA or pharmacological agents (specific or non-specific) may be potentially employed for the attenuating the progression of hepatoblastoma.


OBJECTIVES/SPECIFIC AIMS: Intestinal stem cells (ISC) primarily act in the repair of ulcerated epithelium, and their proliferative capacity relies on Wnt/β-catenin signaling. However, the role of GCs on basal epithelial cell signaling has not been fully characterized. The objective of this study was to interrogate a mechanism by which steroids may limit ISC activation. GCs inhibit NFκB signaling, which has been shown to play a role in nuclear β-catenin activation in epithelial cells. We hypothesized that GCs limit Wnt/β-catenin signaling required for ISC activation and epithelial restitution by inhibiting NFκB activation in epithelial cells. METHODS/STUDY POPULATION: To examine the effects of GCs on intestinal epithelial cells, we treated a nontransformed human colonic epithelial cell line (NCM460) with dexamethasone and observed the effects on NFκB and Wnt/β-catenin signaling events. We isolated mouse epithelial cells from the distal colon for stem cell culture as 3D “organoids.” We obtained pure epithelial cell preparations from mucosal biopsies isolated from patients treated at GI clinics at the University of Kentucky Chandler Hospital and VA Medical Center, Lexington. Steroid treated patients with equivalent levels of inflammation, but no mucosal ulceration were used as controls. RESULTS/ANTICIPATED RESULTS: In steroid-treated NCM460 cells, we saw an increase in steroid-responsive genes GILZ and SGK1. We saw a significant decrease in transcripts for Wnt target genes, including Axin2 and cmyc; NFκB target genes, including IFNG and IL6; and the shared NFκB and Wnt pathway co-activator CREBBP, despite unchanged transcript levels for β-catenin (CTNNB1). This data was corroborated in 3D stem cell cultures from cells isolated from mouse colon tissue, which had significant decreases in transcripts for stem cell markers Lgr5 and Ascl2, proliferative markers KI67 and PCNA, and Wnt target Axin2. NCM460s transfected with a lentivirus carrying a TCF/LEF luciferase construct showed a 2.5-fold decrease in TNF-stimulated luciferase activity with dexamethasone treatment. Interestingly, this effect can be rescued by glucocorticoid receptor (GR) blockade with RU-486. Intestinal epithelial cells from patient biopsies showed significant decreases in colitis-induced Axin2, p-LRP6  and nuclear β-catenin, which correlated with decreased p-p65 protein levels. DISCUSSION/SIGNIFICANCE OF IMPACT: Together, these data suggest that steroid therapy inhibits Wnt/β-catenin signaling at multiple levels, and effects stem cell proliferation in pure stem cell cultures. Decreases in TCF/LEF transcriptional activation (nuclear β-catenin’s DNA binding target) can be reversed with steroid receptor blockade with RU-486, suggesting that a receptor level interaction may be occurring. Interestingly, the required co-activator CBP, shared between NFκB and Wnt pathways, has decreased transcription following steroid treatment, which may provide a mechanism for limited Wnt activation following steroid therapy. Although steroids play a significant role in regulating the amount of inflammatory damage that occurs during IBD treatment, our data suggest that they may be limiting pathways required for effective healing as well.


Increased individual resources (condition) can be correlated with either increased or decreased longevity. While variation in resource acquisition and allocation can account for some of this variation, the general conditions that select for either pattern remain unclear. Previous models suggest that nonlinearity of payoffs from investment in reproduction  can select for high‐condition individuals that sacrifice longevity to increase reproductive opportunity. However, it remains unclear what mating systems or patterns of sexual competition might select for such life‐history strategies. We used a model of condition‐dependent investment to explore how expected payoffs from increased expression of secondary sexual traits affect optimal investment in lifespan. We find that nonlinearity of these payoffs results in a negative relationship between condition and lifespan under two general conditions: first, when there are accelerating marginal benefits from increasing investment; second, when individuals that invest minimally in secondary sexual trait expression can still achieve matings. In the second scenario, the negative relationship occurs due to selection on low‐condition individuals to extend lifespan at the cost of secondary sexual trait expression. Our findings clarify the potential role of sexual selection in shaping patterns of condition‐dependent ageing, and highlight the importance of considering the strategies of both low‐ and high‐condition individuals when investigating patterns of condition‐dependent ageing. In some species, when access to resources is increased, for example through higher quality diet, expected lifespan increases. Yet, in other species expected lifespan is decreased with increasing resources. Why do these different patterns exist? We developed a new theory to explore the conditions under which either pattern might occur. We uncovered two important factors related to investment in sexual traits  that might explain these different patterns of ageing. First, how mating success is determined by sexual trait expression is important. For example, if the highest quality males with the most elaborate sexual traits are able to effectively monopolize access to females, then the best strategy is to increase investment in those traits at the sacrifice of lifespan. On the other hand, if this type of monopolization does not occur then the best strategy for high quality males is to increase lifetime mating success by extending lifespan instead. Second, the ability to obtain matings without bearing elaborate traits is important. For example, a poor quality male may never have the resources to compete with high quality males. So, the best strategy may be to extend lifespan and rely on sneak matings to increase lifetime reproductive success. But this strategy can only evolve where sneak matings are possible. Both these factors can independently contribute to patterns ageing in response to individual access to resources. Our results show how the interplay between resources and mating systems could drive the evolution of different ageing strategies. This can shed light on broad patterns of ageing observed in the animal kingdom.Ageing, the deterioration in performance with age, is an almost universal trait with important fitness consequences  in individuals of varying condition. Kokko  exploredWhile our results support the findings of Kokko , we alsoAs we are primarily interested in how secondary sexual trait expression affects mating success and ultimately fitness, our model focuses on investment decisions by males. Thus, in the context of the model description, “individuals” refers to males within a population.T, to invest in traits relating to fitness. T therefore represents individual condition , so that:q is the proportion of T that is invested in somatic maintenance, then:Therefore, if b, is determined by investment in the secondary sexual trait, and lifespan, E, is determined by investment in somatic maintenance, so that b is a function of γ, E is a function of ω, and hence both are functions of q. Individual fitness, W, is the product of mating success, b, and lifespan, E.Mating success, q, denoted *q, which is the allocation to somatic maintenance that maximizes W for each value of condition over the range 0 < T < 1. This allows us to investigate the relationship between T and optimal lifespan, E*, the lifespan that maximizes W.Hence, fitness is a function of secondary sexual trait expression and somatic maintenance, and individuals should invest more in whichever trait yields the higher marginal fitness returns on that investment. Below, we first find optimal As mating success depends largely on external factors, including behaviors of other individuals, the relationship between secondary sexual trait expression and mating success can be expected to vary between populations, and take on a variety of function forms. As such, we investigate four potential relationships: Linear, decelerating power, accelerating power, and logistic Fig. . We alsop < 1. This curve passes through the points  and  meaning that an individual that invests nothing in somatic maintenance has zero lifespan, and the maximum possible lifespan is 1. A linear relationship between investment in somatic maintenance and lifespan is also plausible, but using this function form leads to qualitatively similar results .On the other hand, the relationship between investment in somatic maintenance and lifespan is likely to be much less variable. Because return on investment in somatic maintenance in terms of increased lifespan is associated with internal physiological processes such as increasing efficiency of cellular maintenance, we expect a function with diminishing returns. Therefore, we assume a function form representing diminishing lifespan returns on investment in somatic maintenance:n = 1; for the decelerating power model, n = 1/3; and for the accelerating power model, n = 3. For the tan function, m and r are set at 1.5 and 14.101, respectively so that this function is the same shape as the one used by Kokko  and , meaning that b is zero if males invest nothing in the secondary sexual trait, and the maximum value of b is 1 for values 0 < T < 1 . Therefore, for the values of T that we are interested in, the b(γ) functions only differ in shape, but do not differ in the minimum or maximum expected mating success . Numerically solving each W equation for *q yields a function q*(T), which represents how *q changes with condition over the range 0 < T < 1. This allows us to find the optimal lifespan, *E, over the same values of T using equation Functions for mating success and fitness used in our models are outlined in Table by Kokko , but is T, and optimal allocation to somatic maintenance (q*), and optimal lifespan (*E) for each of the fitness functions representing different relationships between secondary sexual trait expression and mating success , but this is probably not the case in many mating systems. Low‐condition males of many species are able to achieve matings in ways that do not involve the use of the signal or weapon traits employed by high‐condition males  becomes b = s for each model. For the following analyses s = 0.1. Varying s over a wide range of values does not qualitatively change patterns of optimal *q and *E, except in some cases at very high values of s that select for decreased investment in secondary sexual traits  and optimal secondary sexual trait expression (*γ) for the models shown in Table s = 0.1. With the accelerating power, tan, and logistic functions, low‐condition individuals are selected to allocate all of their resources to somatic maintenance, and invest nothing in the secondary sexual trait. In the tan function, this results in a similar overall relationship as where s = 0: there is a positive relationship between condition and lifespan at low values of condition, but a negative relationship at high values of condition. However, in the accelerating power and logistic models, the patterns of optimal allocation indicate that there are two distinct optimal life‐history strategies within the population. While low‐condition individuals invest in extending their lifespan, high‐condition individuals are selected to increase investment in the secondary sexual trait. This results in a negative correlation between condition and lifespan across the specified range of condition. On the other hand, when payoffs from investing in a secondary sexual trait increase linearly, or have a saturating response (decelerating power function), optimal allocation patterns do not change qualitatively with changing values of s: there is always a positive correlation between condition and lifespan.Figure s = 0, increasing nonlinearity in both the accelerating power function model , the optimal allocation patterns for low‐condition individuals change. In the high‐condition individuals, increasing nonlinearity still selects for increased investment in the secondary sexual trait, resulting in a similar investment pattern for this region of condition as in models where s = 0. However, the marginal benefits of investing in a secondary sexual trait decrease for low‐condition individuals. The overall effect is that increasing nonlinearity of mating success selects for high‐condition individuals to increase investment in γ, but for low‐condition individuals to decrease their investment in γ to zero and instead extend their lifespan. This results in greater discontinuity in patterns of optimal lifespan and secondary sexual trait expression, as low and high‐condition males gain higher fitness with increasingly different investment strategies. As a result, as n increases, the relationship between condition and optimal lifespan becomes more negative across the range of condition. The patterns in Figs. By contrast, when minimum mating success is greater than zero , and when minimum expected mating success is greater than zero. In these cases, low and high‐condition males pursue different allocation strategies, which results in a discontinuous allocation pattern . The population might therefore consist of longer lived sneaker males, and shorter lived males with large secondary sexual traits. Interestingly, the patterns of secondary sexual trait expression obtained with these models approximate observed patterns of male secondary sexual trait (horn) expression in some species of beetles that show sigmoidal trait scaling and condition‐dependent male mating strategies, including alternative/sneak tactics in low‐condition males , or higher for small trait sizes (decelerating power model), positive associations between condition and lifespan are predicted regardless of whether matings can be achieved without expressing the secondary sexual trait. These findings are consistent with previous theoretical studies including Nur and Hasson . Data frOur findings reflect the key assumption that the payoffs from investment in lifespan do not increase steeply in the way that payoffs from investment in reproduction do. We also assume that the mating success function is highly variable between populations, and can take on multiple forms, while the relationship between investment in somatic maintenance and lifespan is less variable between populations. We believe that this is a reasonable assumption because of the nature of the physiological systems that determine ageing rate, and the fact that these systems are unlikely to vary between species to the same extent as the traits that determine mating success. We also assumed that the costs of secondary sexual traits arise only through resource allocation trade‐offs. However, secondary sexual traits can also impose other types of costs. For example, increased secondary sexual trait expression can result in increased predation pressure , optimal investment strategies may change with age or in response to somatic wear‐and‐tear. Such facultative adjustment is also likely to characterize investment in costly sexual behavior. Such systems and traits may be characterized by feedback between secondary sexual trait expression and resulting somatic wear‐and‐tear, such that individuals might be selected to dynamically adjust their investment in secondary sexual traits and in somatic maintenance so as to maximize expected lifetime reproductive success. Understanding the evolution of condition‐dependent ageing in such systems may require the development of more complex models.s = 0 , there is only a very weak positive relationship between condition and lifespan at low levels of condition, but a positive relationship at higher levels of condition. Additionally, with the logistic model where s > 0, our analysis predicts that individuals with intermediate values of T will have the shortest lifespans, because those intermediate individuals have the most to gain from sacrificing investment in lifespan for the sake of enhancing secondary sexual traits. These patterns suggest that an empirical study of condition‐dependent ageing could find any correlation between condition and lifespan – positive, negative, or neutral – depending on how the range of conditions investigated relates to the curves that define mating success and lifespan. Hence, future empirical studies of condition‐dependent ageing would benefit from including more than just two condition values or treatments in their analyses , whereas females are selected to pursue strategies involving less risky strategies with delayed payoff  , and s = 0.1 (B). The value of m for each function is shown in the legend.Click here for additional data file.


Casein kinase 2 interacting protein-1 (CKIP-1) has exhibited multiple functions in regulating cell proliferation, apoptosis, differentiation, and cytoskeleton. CKIP-1 also plays an important role as a critical regulator in tumorigenesis. The aim of this study is to further examine the function of CKIP-1 in glioma cells.β/β-catenin pathway was detected by western blot analysis. The expression level of CKIP-1 protein was determined in gliomas tissues and cell lines by immunohistochemistry stain and western blotting while the association of CKIP-1 expression with prognosis was analyzed by Kaplan-Meier method and compared by log-rank test. CKIP-1 was overexpressed or silenced in gliomas cell lines. CCK-8, colony formation assay, and BrdU incorporation assay were used to determine cell proliferation and DNA synthesis. Cell cycle and apoptosis rate were determined with fluorescence-activated cell sorting (FACS) method. Then, expression of key members in AKT/GSK3β at Ser9, and promoted β-catenin degradation. In the present study, we reported new evidence that CKIP-1 was reversely associated with the proliferation of glioma cells and survival in glioma patients. Additionally, the overexpressed CKIP-1 significantly inhibited glioma cell proliferation. Further experiments revealed that CKIP-1 functioned through its antiproliferative and proapoptotic activity in glioma cells. Importantly, mechanistic investigations suggested that CKIP-1 sharply suppressed the activity of AKT by inhibiting the phosphorylation, markedly downregulated the phosphorylated GSK3 Overall, our results provided new insights into the clinical significance and molecular mechanism of CKIP-1 in glioma, which indicated CKIP1 might function as a therapeutic target for clinical treatment of glioma. In the central nervous system, gliomas are the most common type of aggressive primary solid tumor, accounting for ~80% of primary malignant brain tumors. The incidence of gliomas has increased in recent years and is still with significant mortality . Malignaα subunit  in U251 cells that transfected with CKIP-1 but was expressed at relatively higher levels in U251-Ctrl and parental cells. This result demonstrated that CKIP-1 induced the downregulation of p-Akt (T308), (S473), and p-GSK3β (S9). In addition, there was no accumulation of β-catenin, but the level of phosphorylated-β-catenin at Ser33/37 [p-β-catenin (S33/37)] was significantly upregulated. Conversely, the levels of c-myc, cyclin D1, and Smurf1 appeared to decrease by CKIP-1 overexpression. CKIP-1 promotes the self-ubiquitylation and autodegradation of Smurf1. CKIP-1 functions as an inhibitor of Smurf1. We also observed a significant decrease in Smurf1 when CKIP-1 was overexpressed. These results indicated that CKIP-1 may suppress glioma cell proliferation and induce apoptosis through inhibiting the AKT/GSK3β/β-catenin pathway.We next investigated underlying mechanism through which CKIP-1 inhibited glioma tumorigenesis. For this, we analyzed the association between CKIP-1 and candidate signaling pathway proteins by Western analysis Figures . CKIP-1 igration . Phosphoradation , and itsradation . In thisβ/β-catenin proteins in normal brain tissue and patient glioma tissues. From the score we can see that the expression level of CKIP-1 in glioma tissues is lower than that in normal brain tissues, and the activation level of AKT/GSK3β/β-catenin signaling pathway is significantly higher than that in normal brain tissues. This also indicates that CKIP-1 is inhibited in glioma tissues, while the AKT/GSK3β/β-catenin signaling pathway is significantly activated  which is the most common and also the most aggressive form of gliomas. Currently, the standard treatment for glioma is surgical resection followed by combination radiotherapy and chemotherapy. It is difficult to completely remove glioma tumor owing to no obvious boundaries from normal brain tissues. This leads to the high recurrence rate of glioma. Therefore, exploring and evaluating new gene targets against glioma cell invasion is a promising approach for treatment of glioma. CKIP-1 was originally identified as a specific interacting protein of casein kinase 2 (CK2) ude mice . HistoriIt has been widely demonstrated that CKIP-1 acts as a suppressor in several types of human cancer cells including human osteosarcoma SaOS-2, human epithelial carcinoma A431, gastric cancer cell, colon cancer HCT116, and SW480 cells , 21. Howβ-catenin pathway is intensively studied in multiple cancers, but its role in glioma has only started to emerge [β [β signaling upon M-CSF treatment [β at Ser9 and upregulated the phosphorylated β-catenin (Ser33/37) which leads to the ubiquitylation and proteasomal degradation of β-catenin [β/β-catenin signaling pathway in glioma cell. These results suggested an essential role for CKIP-1 in regulation of biological behavior of glioma cell.The Wnt/o emerge –24. It hmerge [β . This inmerge [β . Similarreatment  while ov-catenin . In thisβ/β-catenin signaling pathway. These findings suggested that CKIP-1 might be a potential therapeutic target in the treatment of glioma.In summary, we demonstrated that the CKIP-1 is commonly downregulated in glioma tissues. Lower expressed CKIP-1 promotes glioma cell proliferation and patients with low level of CKIP-1 are associated with a poor prognosis. Our results also showed that upregulation of CKIP-1 inhibits the proliferation of human glioma cell line via suppression of AKT/GSK3