Type 2 diabetes (T2D) is an independent risk factor for Alzheimer's disease (AD). Exendin-4 (Ex-4), a widely used glucagon-like peptide-1 receptor agonist drug in the treatment of T2D, has been demonstrated the therapeutic effects on diabetic encephalopathy (DE). Especially, the Ex-4 ameliorates the tau hyperphosphorylation and cognitive impairment in DE. And these crucial alterations are also important bridge between T2D and AD. However, its unique mechanism is unclear.Ins2. Moreover, the chromatin immunoprecipitation coupled with quantitative polymerase chain reaction (ChIP-qPCR) and luciferase reporter assays were performed. The db/db mice, high-fat-diet (HFD) / streptozotocin (STZ)—induced diabetic (HF-diabetic) mice, and high-glucose-damaged (HGD) HT-22 hippocampal cells were enrolled to examine the effects of Ex-4 on AD-like changes in T2D. The Novel object recognition test (NORT) and Morris water maze test (MWMT) were conducted to evaluate the cognitive impairment. The Dickkopf-1 (DKK1) was employed to weaken the activation of the Wnt/β-catenin pathway to explore the mechanism of Ex-4 in protecting the brain functions. The JASPAR was based to predict the interaction between NeuroD1 and the promoter region of Ins2. And this interaction could be promoted by Ex-4. Ex-4 alleviated the tau hyperphosphorylation, increased the brain-derived insulin, and improved the PI3K/AKT/GSK3-β signalling in db/db mice, HF-diabetic mice, and HGD HT-22 hippocampal neuronal cells. The NORT and MWMT indicated that Ex-4 alleviated the learning and memory deficits in HF-diabetic mice. The inhibitor Dickkopf-1 (DKK1) of the Wnt/β-catenin pathway significantly blocked the protective effects of Ex-4. Regarding further molecular mechanisms, NeuroD1 was affected by Ex-4 in vivo and in vitro, and the knockdown or overexpression of NeuroD1 suggested its crucial role in promoting the brain insulin by Ex-4. Meanwhile, the ChIP‒qPCR and luciferase reporter assays confirmed the combination between NeuroD1 and the promoter region of the insulin-encoding gene Ins2-derived brain insulin through the Wnt/β-catenin/NeuroD1 signaling in T2D. And its also show new lights on part of the progress and mechanism on treatment targets for the DE in T2D.Our study proposes that Ex-4 alleviates tau hyperphosphorylation and cognitive dysfunction by increasing The online version contains supplementary material available at 10.1186/s10020-023-00718-2. Metabolic diseases often cause systemic metabolic disorders, unbalancing the homeostasis and damaging various organs and tissues. Type 2 diabetes (T2D), a chronic metabolic disease characterized by glucose metabolism disorder and insulin resistance, has become an important healthcare burden in China and worldwide (Magliano and Boyko Exendin-4 (Ex-4), a glucagon-like peptide-1 (GLP-1) analogue in treating T2D, is highly affinitive and specifically actives the GLP-1 receptor (GLP-1R)  / streptozotocin (STZ) -induced diabetic (HF-diabetic) mice and high-glucose-damaged (HGD) neuron cell model to evaluate the definitely therapeutic effects of Ex-4, and explored the underlying mechanisms which may closely related with DE, bran-derived insulin and Wnt/β-catenin signaling pathway.The 8-week-old male C57BL/6 mice and diabetic db/db (C57BLKS/J-LepRdb/LepRdb) mice were purchased from the HFK Bioscience Co., Ltd. (HFK) (China). All mice were maintained at room temperature (25 ± 2 °C) under a standard 12-h/12-h light–dark cycle with free access to water and food. All operation procedures related to the animals were approved by the Animal Care Committee of the Tongji Hospital, Huazhong University of Science and Technology. And complied with the standards described in the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.Sixty male C57BL/6 mice were randomly assigned to the HFD/STZ induced T2D mice  and the control mice . They were fed a HFD  or a normal control diet (NCD) . After 12 weeks feeding, the mice fed with HFD were injected intraperitoneally with STZ  at 30 mg/kg for three consecutive days, while the CTL mice were injected with the sodium citrate buffer. Weight and the whole blood glucose (WBG) were monitored 3 days after the last injection and once a week thereafter. Mice with the random WBG levels higher than 16.7 mmol/L were set for the experimental T2D, and the others were excluded from this work. Finally, the CTL mice were regarded as the CTL group . The HF-diabetic mice were randomly divided into five groups, including the HF-diabetic group , the Ex-4 group , the Ex-4 + DKK1 group , the insulin-S group , and the insulin-I group .Twenty-five male db/db mice were fed a NCD and randomly assigned to 5 groups (n = 5). The groups included the db/db group , the Ex-4 group , Ex-4 + DKK1 group , Insulin-S group , and Insulin-I group . Weight and WBG were measured weekly during the administration.Specifically, each mouse was treated daily with both subcutaneous injection and nasal drip for 4 weeks. 2 IU insulin  for morphological studies. Besides, the hippocampus was dissected from the left hemisphere and stored at -80 °C for biochemical analyses.2 and grown to 70–90% confluence were collected for analyses unless otherwise noted.The mouse hippocampal neuronal cell line HT22 and human embryonic kidney (HEK) 293 T cells were purchased from the American Type Culture Collection . And they were cultured in the Dulbecco's modified eagle medium (DMEM) basic  supplemented with 10% foetal bovine serum , 100 IU/ml penicillin, and 100 μg/ml streptomycin. Cells maintained in a 37 °C incubator with 5% COTo establish an in vitro model of chronic high glucose damage and explore the effect of Ex-4. Because the optimal growth and survival rate of HT-22 cells requires 25 mM basic glucose, high glucose DMEM containing 25 mM glucose was used as the control glucose (CON) group. An additional 25 mM glucose was added to DMEM  as the high glucose (HG) group  were constructed by lentivirus containing short hairpin RNA (shRNA) targeting Ins2 . Then, they were co-cultured with its negative control (V-CON) at 50 mM for 48 h. Next, they were again cultured with or without the Ex-4 (HG + Ex-4 or HG) for another 48 h to determine whether the effects of Ex-4 on HT22 cells were attributed to increased insulin synthesis due to Ins2 transcription.The The Wnt/β-catenin signaling inhibitor was treated as follows. The HT22 cells under high glucose (50 mM) were treated with DMSO (HG) or Ex-4  or 100 ng/μl DKK1 for 2 h .NeuroD1 overexpressed (NeuroD1-KI), NeuroD1 knockdown (NeuroD1-KD), and negative control (NC) HT22 cells were constructed by the lentivirus transfection to clarify the role of NeuroD1 when the Ex-4 affected the HGD HT22 cells. Lentivirus production and transient transfections were performed by Lipofectamine 3000  according to the manufacturer’s instructions. The target sequence 5’-3’ for the lentivirus containing shRNA targeting Hippocampus or HT22 cells were homogenized in radioimmunoprecipitation assay (RIPA) buffer  containing complete protease inhibitor cocktail and phosphatase inhibitor cocktail I and II . Western blotting was performed as previously described  and analysed using a Roche RT-PCR System as previously described  for 24 h, and the brain was dehydrated in 30% sucrose-PBS for 48 h. According to a previous method . The chromatin was incubated overnight at 4 °C with the anti-NeuroD1 antibody , and a normal immunoglobulin G (IgG) antibody  was selected as the NC. The RT-PCR analysis were performed as described previously, and the primers were designed by Sangon Biotech Co., Ltd. (China).Ins2-WT, pGL3-Ins2-MUT1, and pGL3-Ins2-MUT2 luciferase reporters, respectively. Ins2 transcriptional activity was assessed using the Dual Luciferase Report Assay Kit DL101-01 on a TD-20/20 Luminometer .During transfection of HT22 cells with the Lipofectamine 3000, the overexpressed plasmid (or vector) of the NeuroD1 was transfected with the pGL3-P < 0.05.All the experiments above were performed in triplicate independently at least. The GraphPad Prism (version 8.3.0) was employed for statistical analysis, and all data were presented as means ± standard deviations (M ± SD). A two-tailed Student’s t test was applied to compare the data of the two groups with a normal distribution, and the one-way ANOVA with Tukey’s post hoc analysis was employed for the multiple comparison. The data difference was significant when The datasets, and adenoviruses and plasmids are available by a reasonable request from the corresponding authors.Tau hyperphosphorylation of several AD-associated has been reported in the brains of T2D patients  among those mainly insulin-related transcriptional regulators in the HT22 cells were detected under the high-glucose conditions with and without Ex-4. Meanwhile, the NeuroD1 was selected for the further study, as illustrated in Additional file Ins2 transcription  targeting the two binding sites, and a NeuroD1 overexpression plasmid were constructed, based on which the double luciferase experiment was carried out. Ex-4 treatment resulted in a 1.53-fold (WT-INS2), 0.84-fold (MUT1-INS2), and 0.40-fold (MUT2-INS2) in Ins2 promoter activity compared with the HG group . Table S3. Predicted binding sites for NeuroD1 on Ins2 promoter. Figure S1. The damage of HT22 cells induced by high glucose is not caused by increased osmotic. Figure S2. The blood glucose and body weight changes during experiments in HF-diabetic mice and db/db mice. Figure S3. The effect of knocking down the Ins2 in HT22 cells. Figure S4. NeuroD1 played a crucial role in increasing the Ins2-induced brain-derived insulin and reduced tau hyperphosphorylation.


This work demonstrates that β-catenin–mediated suppression of p21 is essential for endocardial cushion formation, suggesting a potential role of β-catenin in the etiology of congenital heart defects. β-cateninDM allele in which the transcriptional function of β-catenin is selectively disrupted, we further reveal that β-catenin regulated cell proliferation and migration through its transcriptional and non-transcriptional function, respectively. At the molecular level, loss of β-catenin resulted in increased expression of cell cycle inhibitor p21 in cushion endocardial and mesenchymal cells in vivo. In vitro rescue experiments with HUVECs and pig aortic valve interstitial cells confirmed that β-catenin promoted cell proliferation by suppressing p21. In addition, one savvy negative observation is that β-catenin was dispensable for endocardial-to-mesenchymal fate change. Taken together, our findings demonstrate that β-catenin is essential for cell proliferation and migration but dispensable for endocardial cells to gain mesenchymal fate during endocardial cushion formation. Mechanistically, β-catenin promotes cell proliferation by suppressing p21. These findings inform the potential role of β-catenin in the etiology of congenital heart defects.Endocardial cushion formation is essential for heart valve development and heart chamber separation. Abnormal endocardial cushion formation often causes congenital heart defects. β-Catenin is known to be essential for endocardial cushion formation; however, the underlying cellular and molecular mechanisms remain incompletely understood. Here, we show that endothelial-specific deletion of β-catenin in mice resulted in formation of hypoplastic endocardial cushions due to reduced cell proliferation and impaired cell migration. By using a  Endocardial cushions give rise to heart valves and the septa that separate the cardiac chambers  and mainCtnnb1 gene and conducts two distinct functions at cell membrane and nucleus, respectively . Immunostaining confirmed that β-catenin was expressed in both endocardium and myocardium of E9.5 control (β-cateninf/f or β-cateninf/+) embryos, but its expression in endocardium was selectively disrupted in their littermate β-cateKO embryos  in which endocardial cells and their mesenchymal progenies completely lost the transcriptional function of β-catenin, whereas maintained one copy of β-catenin with the cell adhesive function. We then analyzed the AVC cushion development in control, β-cateKO/eKO  and β-cateKO/DM embryos. HE staining showed that both β-cateKO/eKO and β-cateKO/DM embryos had hypocellular AVC cushions . The qRT-PCR results showed that the expression of Notch1, Hey1, Hey2, Bmp2, Tgfbr2, Bmpr1a, and Bmpr2, which are involved in the NOTCH and BMP signals essential for EndoMT , known for their functions in EndoMT  and found their expression was not altered in either mutant  . Down-ree groups . This re embryos . On the d PDGFRβ . SNAIL, d PDGFRβ . Togethep21 expression and supports cell migration via mediating filopodia formation , proliferation, and migration. WNT/β-catenin signaling is essential for endocardial cushion formation in zebrafish and mice. Consistent with previous reports, we found that endothelial-specific deletion of β-catenin in mice resulted in hypoplastic endocardial cushions. In zebrafish, truncated adenomatous polyposis coli–mediated activation of WNT/β-catenin signaling led to excessive endocardial cushions associated with increased cell proliferation . In addiebrafish . These febrafish . In thisebrafish . In addiebrafish . Togetheβ-cateninDM allele which has the cell adhesive function but not the transcriptional activity of β-catenin, we demonstrate that β-catenin regulates cell proliferation and migration via its transcriptional function and non-transcriptional function, respectively.β-Catenin interacts with VE-cadherin at cell membrane and mediates cell–cell adhesion. It can also shuttle to nucleus and activate target gene expression by forming a transcriptional complex with transcriptional factor TCF/LEF upon stimulation of WNT ligands . By usinSnail is known to reduce VE-cadherin expression transcriptionally and triggers the EndoMT process . In line with the qRT-PCR results, immunostaining revealed that the level of active NOTCH1 protein in cushion endocardial cells was comparable between control and β-catenin mutant embryos. These results suggest that Notch1 is not a downstream target regulated by WNT/β-catenin signaling during endocardial cushion formation in mice. This discrepancy between previous and our study may reflect a species-specific mechanism underlying endocardial cushion formation.We used a candidate gene approach to characterize the molecular mediators through which β-catenin regulates endocardial cushion formation. NOTCH and TGFβ/BMP are two well-known signaling pathways essential for this process. ia Snail . In zebrcushions . In our Crosstalk between TGFβ/BMP and WNT/β-catenin signaling pathways has been implicated in both heart valve development and myxomatous valve disease . We haveIn conclusion, our findings demonstrate that β-catenin is dispensable for endocardial cells to gain mesenchymal fate but essential for later cell migration and proliferation. The underlying mechanisms may involve β-catenin–mediated filopodia formation and p21 suppression. Our findings provide a new insight into the cellular and molecular mechanisms by which β-catenin controls endocardial cushion formation during early heart valve development.Tie2Cre mice  staining was performed for histology by using a standard protocol. The HE-stained tissue sections were examined and photographed using a Zeiss Axio Observer Z1 inverted microscope. Serial sections across the entire AVC cushion region were used for quantification of the number of mesenchymal cells. To analyze the distribution of mesenchymal cells in the endocardial cushions, cells located underneath the endocardium (sub-endocardium) and far away from the endocardium and underneath the myocardium (sub-myocardium) were counted, respectively. The percentage of mesenchymal cell in the sub-endocardium or the sub-myocardium was calculated and the data were presented as mean ± SD. Data from 4–6 embryos for each genotype were used for statistical calculation.Tissue sections prepared as described above were boiled for 10 min in sodium citrate  (Vector Laboratories) to retrieve antigens and then blocked with 5% normal horse serum in PBS before being incubated with primary and secondary antibodies (Table S1). The signal was amplified using the TSA Plus Cyanine 3 System (Perkin Elmer) as noted for some antibodies. The stained tissue sections were photographed using a Leica confocal microscope.List of antibodies.Table S1. Cell proliferation was determined by the EdU assay. 2 h before collecting embryos, pregnant female mice at E9.5 were administrated with EdU (Life Technology) through intraperitoneal injection at a concentration of 100 mg/kg. After the 2 h chasing, embryos were fixed in 4% PFA at 4°C for 1 h, soaked in 15% and 30% sucrose sequentially, and embedded in OCT compounds in the sagittal orientation. The tissues were cut at 8 μm thickness and mounted on the positive charged slides. Tissue sections were then fixed in cold ethanol and acetone (1:1) solution for 5 min and stored at −80°C. Before staining, tissue sections were air dried for 45 min. Serial sections crossing the entire cushion region were first stained with PECAM1 antibody followed by EdU staining using an EdU imaging Kit (Life Technology) and counterstained with DAPI. The stained sections were photographed using a Leica confocal microscope. The positive EdU labeling was quantified for the endocardium, mesenchyme, or myocardium using ImageJ software. At least three embryos were analyzed for each genotype.Gapdh) and calculated using the 2−ΔΔCT method. Three samples per genotype were analyzed, and the mean relative expression of each gene between groups was used for statistical analysis.The AVC cushion tissues were microdissected from E9.5 embryos and subjected to isolation of total RNAs using TRIzol (Invitrogen). Tissues from 2–3 embryos were pooled as one sample. First-strand cDNA was synthesized using the Superscript II Reverse Transcriptase Kit (Invitrogen). qRT-PCR was carried out using the Power SYBR Green PCR Master Mix (ABI). Gene-specific primers were used for genes of interest (Table S2). The relative level of gene expression was normalized to an internal control (level of List of PCR primers.Table S2. E9.5 embryos were collected and processed for frozen sections. mRNA expression pattern of genes was detected by using an RNAscope 2.5 HD Reagent Red Kit (ACD BIO) according to the manufacturer’s instructions. The images were collected using a Leica confocal microscope.EndoMT assay was performed as described previously . AVC tisCtnnb1 (β-catenin) and Cdkn1A (p21), the cells were transfected with gene-specific siRNA, whereas the scramble siRNA were introduced simultaneously as controls. The knockdown efficiency was evaluated using qRT-PCR. EdU was added into the culture media with a final concentration of 10 μM. 2 h later, the cells were processed for EdU staining as described above. Immunostaining was performed to detect the expression level of p21 protein.Primary HUVECs and PAVIC were cultured in endocardial cell medium (ScienCell Research Laboratories) and DMEM, respectively. To block the canonical WNT signaling, XAV939 were added to the culture media at a final concentration of 1 μM for HUVEC or 2.5 μM for PAVIC. PAVIC was treated with 2.5 μM of UC2288 to inhibit p21. To knockdown the expression of t test was used for comparisons between two groups. One-way ANOVA was used for comparisons among three groups. Probability value < 0.05 was considered as significant.Statistical analysis was performed using GraphPad Prism. All data were presented as mean ± SEM.


The development of executive function (EF) in children, particularly with respect to self-regulation skills, has been linked to long-term benefits in terms of social and health outcomes. One such skill is the ability to deal with frustrations when waiting for a delayed, preferred reward. Although robots have increasingly been utilized in educational situations that involve teaching psychosocial skills to children, including various aspects related to self-control, the utility of robots in increasing the likelihood of self-imposed delay of gratification remains to be explored. Using a single-case experimental design, the present study exposed 24 preschoolers to three experimental conditions where a choice was provided between an immediately available reward and a delayed but larger reward. The likelihood of waiting increased over sessions when children were simply asked to wait, but waiting times did not increase further during a condition where teachers offered activities as a distraction. However, when children were exposed to robots and given the opportunity to interact with them, waiting times for the majority of children increased with medium to large effect sizes. Given the positive implications of strong executive function, how it might be increased in children in which it is lacking, limited, or in the process of developing, is of considerable import. This study highlights the effectiveness of robots as a distractor during waiting times and outlines a potential new application of robots in educational contexts. Among self-regulation skills, delay of gratification (DG) is considered as one crucial to be fostered during child development  and inteversus two marshmallows , these projects have implemented procedures that are very dissimilar to the classic Marshmallow Test. For example, Although the procedure of The experiment took place at a preschool near the university of the first author. Participants were recruited using cluster sampling to achieve an approximately even distribution by sex and age and to ensure that participation was spread around the various preschool classes, enabling the study to be conducted within the space and time constraints outlined by the preschool teachers. Inclusion criteria required participants to be able to understand and respond to instructions in English and not to have any diagnosis for an intellectual disability. Among the 29 participants initially recruited, data from 5 children were eventually excluded due to non-participation in more than one session in any of the three experimental conditions or due to parents’ request to exclude students from the study. Of the 24 participants, 11 were female and 13 male . None ofThe research was advertised on school notice boards where the study was conducted. A session with parents was organised by the school management to introduce the research objectives. An information sheet detailing the objectives of the study was also given to the parents, who all gave signed consent for their child’s participation to the school principal. No financial remuneration was provided. Prior to commencing the study, information was obtained about each child’s preferred sweet or snack.The participants were grouped into three teams using simple random sampling. All sessions were conducted between 10 and 11a.m. at their classroom. The participants were seated at a table. Each participant’s favorite snack was placed in a bowl in front of them and they were told that they could wait for 5 min to get double the reward amount or choose to consume the smaller reward without waiting. Three teachers working at the school used a stopwatch to time the latency between the start of each session and when the children indicated, by tapping the table, that they would like to consume a reward. Three research assistants assisted the teachers with the procedure, recording the results on notepads.The study was conducted over a period of 3 weeks with three sessions a week. Each block of three sessions was part of a different experimental condition. In Condition 1, no intervention was provided. Here, the participants were simply informed that they would need to wait for 5 min for the larger reward. Alternatively, they could choose to tap the table at any time and collect the reward available in front of them. In the first session of this condition, the teachers left the room and monitored the activity remotely. In the second session, one teacher was present in the room but had no interaction with children. For the final session of Condition 1, two teachers were present in the room and again had no interaction with the children. Participants who waited for 5 min were rewarded double the amount of snack and also received applause from other participants. In Condition 2, the teachers helped children to potentially increase their wait time by telling stories (Session 1), enabling access to the children’s preferred toys (Session 2), and saying nursery rhymes (Session 3). In Condition 3, children were exposed to a humanoid robot, Nao . This tenon-overlap of all pairs (NAP) and TAU-U are considered superior among those . The number of times that a score in the second condition was higher than that of a data point in the first condition was calculated . A tied score was counted as 0.50. NAP was then calculated as the sum of A) the instances that the latency increased and 2) the scores for ties, divided by the total number of comparisons. For example, if there were six instances where a score in the second condition was higher than that in the first condition, and if there was one tie, NAP was 6.50/9.00. In cases of missing data, the measure was still calculated but only with the maximum number of comparisons that was possible. All calculations were conducted using Microsoft Excel.novlap. The latter is calculated in a similar way as NAP. However, instead of adding tied scores, TAUnovlap is calculated by subtracting instances of score decreases from the increases, yielding a score S. This S score is then divided by the total number of comparisons . For example, if a latency score increased five times but decreased four times, S was 1, and TAUnovlap was thus 1/9. TAU-U has the advantage that trends in the baseline phase  are accounted for. The S score is thus adjusted by subtracting from it a S score obtained from separate trend analysis of the first phase. TAU-U does not require this trend to be linear  = 5.56, p < .01) but not for Condition 2 (F(2) = 0.14, p > .05). For Condition 3, scores again increased significantly across sessions (F(2) = 10.60, p < .01).The individual participant latency results by session and experimental conditions are shown in novlap and TAU-U. novlap that it adjusts for baseline trend, only TAU-U is shown here. For the comparison of Condition 1 with 2, therefore, TAU-U can adjust for the fact that there was a significant positive trend in Condition 1. The number of participants in medium and large NAP effect size categories was substantially higher when comparing Condition 2 with 3 than for Condition 1 versus 2  increased across sessions indicates that the novelty of the robots is unlikely to have been the sole contributor to the comparatively high number of medium and large effect sizes, although the increasing levels of possible child-robot interactions across session may have played a role. Given logistical constraints and the need to restrict the length of the study at the school setting, we were unable to arrange a larger number of sessions in each condition and explore at what stage latency scores in the robot intervention condition were reaching a stable level. A further limitation is the lack of follow-up to explore to what extent the intervention effects were long lasting; that is, if children would continue to delay gratification even in the subsequent absence of the robots.reverse ego-depletion indicates that the exertion of willpower may have an energizing effect in cultures such as India where religious and philosophical traditions tend to emphasize the application of mental self-control to everyday life. To what extent this also applies to the values embedded within Indian educational contexts for young children needs to be explored in future research. Other factors to be explored include to what extent DG in preschoolers in India tend to be specific to types of reward used: Additionally, our delay time was only a third of that in the original Mischel studies e.g., . AlthougFurther limitations of the present study need to be noted. Given time constraints, Condition 1 was not run until stability had been reached, and possibly the addition of the teachers across sessions affected waiting time . In Condition 3, the intensity of the intervention (extent of possible interaction with robots) increased across sessions, which, as we note above, may have been partly the reason for the positive trend in that condition. The trend during Condition 1 did not continue into Condition 2, and effect size calculations were able to adjust for trend using the measure TAU-U. For Condition 3, effect sizes may have been larger if the condition had contained more sessions. Future studies thus need to replicate the study with longer conditions. Of course, increasing the number of sessions will eventually create experimental fatigue or a ceiling effect if sufficient DGskills have already been acquired by the participants. A further limitation of the present study was that the rewards used varied to a very high extent, which may have resulted in variability of waiting time results. Previous studies have limited the choice of rewards to two items . HoweverTo summarize, the present study has demonstrated the possible utility of robots as a distraction when studying DG in preschoolers. We emphasize that our goal was merely to investigate the feasibility of a robotic intervention in principle, as a starting point for future studies. We do note, however, that compared to no intervention or standard teaching, exposure to robots during the self-imposed waiting period increased the likelihood of participants waiting for the delayed, more preferred reward as opposed to consuming a smaller, immediate reward. The medium and large effect sizes for the robot intervention indicate that this approach has merit in the development of future, more automized robotic interventions.


Dendritic arborization is essential for proper neuronal connectivity and function. Conversely, abnormal dendrite morphology is associated with several neurological pathologies like Alzheimer’s disease and schizophrenia. Among major intrinsic mechanisms that determine the extent of the dendritic arbor is cytoskeletal remodeling. Here, we characterize and compare the impact of the four proteins involved in cytoskeletal remodeling–vertebrate members of the p120-catenin subfamily–on neuronal dendrite morphology. In relation to each of their own distributions, we find that p120-catenin and delta-catenin are expressed at relatively higher proportions in growth cones compared to ARVCF-catenin and p0071-catenin; ARVCF-catenin is expressed at relatively high proportions in the nucleus; and all catenins are expressed in dendritic processes and the soma. Through altering the expression of each p120-subfamily catenin in neurons, we find that exogenous expression of either p120-catenin or delta-catenin correlates with increased dendritic length and branching, whereas their respective depletion decreases dendritic length and branching. While increasing ARVCF-catenin expression also increases dendritic length and branching, decreasing expression has no grossly observable morphological effect. Finally, increasing p0071-catenin expression increases dendritic branching, but not length, while decreasing expression decreases dendritic length and branching. These distinct localization patterns and morphological effects during neuron development suggest that these catenins have both shared and distinct roles in the context of dendrite morphogenesis. Dendrites were discovered over 100 years ago, and perhaps their most notable feature is their elaborate range of morphologies that make up the dendritic arbor. Through precise extension, branching, elongation, retraction, and pruning, dendritic arbors allow neurons to connect with each other to form neural circuits . ConversThe vertebrate p120-catenin subfamily is made up of four proteins: p120-catenin (gene name CTNND1), delta-catenin (gene name CTNND2), ARVCF-catenin (gene name ARVCF), and p0071-catenin (gene name PKP4). This subfamily of proteins is characterized by a central series of 9 or 10 Armadillo (ARM) repeats that are located between each catenin’s N- and C-terminal tails. Of these four proteins, all except p120-catenin contains a PDZ-binding motif at the C-terminus, which enables them to selectively bind to partner proteins that contain appropriate PDZ domains . The domWhile p120-catenin, ARVCF-catenin, and p0071-catenin are widely distributed throughout mammalian tissues, including the central nervous system, delta-catenin expression is primarily restricted to the central nervous system . Furtherp120-catenin and delta-catenin have been found to regulate dendrite morphology in neurons through the cadherin-catenin complex and by modulating small Rho GTPase activity . Depletiin vitro) and 5 DIV. The timepoint of 1 DIV was chosen to best visualize growth cones, while 5 DIV was chosen to better visualize dendritic processes. Using general criteria , antibodies specifically directed against the endogenous catenins were used to visualize and quantify the localization pattern of each p120-subfamily catenin  promoter so that we only scored neurons . FurtherWe determined that increasing p120-catenin levels significantly increases dendritic length and branching, while decreasing p120-catenin levels decreases dendritic length and branching . Like p1While we observe that each catenin is present in neuronal dendritic growth cones to some degree, a relatively larger proportion of p120-catenin and delta-catenin locate to growth cones compared to ARVCF-catenin and p0071-catenin. As growth cones are sites of neurite outgrowth, p120-catenin and delta-catenin may play a relatively more substantial role in dendritic morphogenesis than ARVCF-catenin and p0071-catenin . All fouin vitro . This is likely due to the inclusion of multiple types of neurons in the cultures. These primary cultures originating from the rat hippocampus contained neurons from the CA1, CA2, CA3 regions, the dentate gyrus, and the subiculum complex . TherefoOverall, the relative localization patterns of the p120-catenin subfamily are consistent with the possibility that they each play roles in dendritic arborization and maintenance, with p120-catenin and delta-catenin potentially playing a relatively stronger role in dendrite morphogenesis than ARVCF-catenin and p0071-catenin based on their relative abundance in growth cones. Furthermore, our morphologic analysis suggests that these catenins contain some shared and some distinct effects on dendrite shaping during neuronal development. The effects of the p120-subfamily catenins on dendrite morphology are likely due in part to their interactions with small Rho GTPases. However, further studies are needed to determine if the effects of each catenin are primarily caused by modulating small Rho GTPase activity, other effector molecules, or a combination of both. Finally, as abnormal dendrite morphology is seen in multiple neurological disorders like Alzheimer’s disease, schizophrenia, and autism spectrum disorder, determining the mechanisms of how each catenin modulates dendrite morphology may help point to new therapeutic approaches or considerations for these diseases. Interestingly, p120-subfamily catenins, most prominently delta, have associations with pathological states involving neurons and brain functions .5 cells per well. The cultures were maintained in Neurobasal Medium supplemented with B-27, GlutaMAX, and penicillin-streptomycin. Transfections in neurons were done at 3 DIV using Lipofectamine 2000 according to the manufacturer’s protocol.Primary hippocampal neurons were isolated from rat embryos at E18 as previously described . All proNeurons were fixed with 4% paraformaldehyde for 10 min at room temperature, incubated for 5 min in PBS supplemented with 100 mM glycine, washed with PBS for 30 min, then permeabilized with 0.5% Triton X-100 for 10 min at room temperature. The neurons were then blocked with PBS containing 1% bovine serum albumin overnight at 4°C. After blocking, the neurons were incubated with primary antibodies in blocking solution overnight at 4°C. The neurons were washed with PBS 3 times, then incubated with secondary antibodies for 1 h at room temperature. Following a second washing step of 3 washes at 5 min each, coverslips were mounted onto slides with 2 μL of VECTASHIELD Antifade Mounting Medium.Neurons were imaged using a Nikon T2i Inverted Confocal Microscope with a 20× Plan Achromat 0.75 NA lens for morphology studies and an Apo-Plan 60 × 1.4 NA oil objective for endogenous localization studies. Images were captured with a Nikon A1-DUG GaAsP hybrid four-channel multi-detector and Nikon NIS-Elements software. All z-series images were acquired at a pixel size of 100 nm and a step size of 1.1 μm at 20× and 0.2 μm at 60×. Excitation was done with a 488 nm laser line and emission was collected with a BP 525/50 m filter. The pinhole was set at 1 airy unit for all images. For imaging endogenous localization of catenins, the laser intensity, offset, and high voltage settings on the microscope were kept consistent between images of the same condition. All images were acquired below the threshold for pixel saturation. For morphological analysis of images acquired at 20×, montages of at least 5 × 5 tiles were captured and stitched together to visualize larger fields of view.p120-catenin antibody  was used at 1:250 dilution. Delta-catenin antibody  was used at 1:250 dilution. ARVCF-catenin antibody  was used at 1:100 dilution. p0071-catenin antibody  was used at 1:250 dilution. For secondary antibodies, Alexa Fluor 488  was used at 1:1,000 dilution, Alexa Fluor 555 (Cat. #A32732) was used at 1:1,000 dilution, and Alexa Fluor 568 (Cat. #A-11004) was used at 1:1,000 dilution.All cDNA transfections for overexpression analyses utilized an AAV vector, an eGFP reporter, an hSyn promoter, and a P2A ribosomal skipping sequence to separate the exogenous catenin from the eGFP reporter to visualize the neurons’ full morphology. All shRNA-mediated knockdowns utilized a pSUPER vector with an eGFP reporter. The GFP controls contained only the vectors and eGFP. Overexpression plasmids were transfected with Lipofectamine 2000 according to the manufacturer’s protocol at 0.5 μg/well, and knockdown plasmids were transfected at 0.25 μg/well. The human forms of each catenin were used for exogenous expression plasmids and are as follows: . The sequences for the shRNA targeting each catenin are as follows: p120-catenin, 5′-CGATATTGGTAGGATTGTT-3′; delta-catenin, 5′-TAGAAGTCGACATAGTTGC-3′; ARVCF-catenin, 5′-CGAGTTAGCTATTTACTGA-3′; p0071-catenin, 5′-GGACTGTTCATGACATGGA-3′.After capturing confocal images in the manner described above, several analyses were performed on the neurons. Endogenous localization was quantified using ImageJ by measuring the relative average fluorescent intensity of each catenin compared to itself in the nucleus, soma, processes, and growth cones. These measurements were obtained by going through each slice of the image, creating regions of interest for the four different subcellular regions, and using ImageJ to measure the average fluorescent intensity of each region. This process was repeated until all slices of the image were measured. The values presented for each cell are the average value for each slice to account for differences in thickness. Cells were co-stained with DAPI and phalloidin to determine subcellular regions. Measurements were averaged from 15 neurons for each condition from three separate cultures for endogenous localization studies. Fluorescent intensity is to be compared between subcellular localizations for the same catenin but is not to be compared between different catenins. For morphological analysis, cells were transfected at 3 DIV and analyzed at 7 DIV. Imaris Filament Tracer was used to create a skeleton of each neuron. From this tracing, the software automatically measures dendrite length, dendrite branching, and performs Sholl analysis. The radii interval for sholl analysis was set at 5 μm. Laser intensity and detector settings were kept the same between samples while collecting images, and all threshold settings were kept the same between samples when running the Imaris Filament Tracer software. Processes shorter than 5 μm were excluded from the analysis. Sample sizes were between 17 and 46 neurons from 5 biological replicates for each condition.To validate antibodies and shRNA knockdowns, primary rat hippocampal neurons were transfected with shRNA to knockdown each p120-subfamily catenin at 3 DIV, then were fixed at 7 DIV. Samples were incubated with antibodies targeting the respective endogenous catenin, then imaged. Fiji was used to measure the fluorescent intensity of endogenous catenin in cells that were transfected with shRNA and were compared against cells that were transfected with GFP as a control.post-hoc test. Statistical significance for antibody and shRNA knockdown validation was determined using unpaired t-tests with Welch’s correction. Significance was assigned at P < 0.05 for *, P < 0.01 for **, P < 0.001 for ***, and P < 0.0001 for ****.Data were analyzed using GraphPad Prism. Statistical significance for all endogenous localization study and morphological analysis comparisons were determined using a two-way Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 multiple comparisons The original contributions presented in this study are included in the article/The animal study was reviewed and approved by the Institutional Animal Care and Use Committee.MD: concept of study, research design, data collection and analysis, and manuscript preparation. YS: data collection and protein modeling. CD: hippocampal culture preparation and experimentation. AP-H: experimentation, imaging, and analysis. MW: concept of study, research design, and manuscript review and editing. PM: concept of study, research design, manuscript preparation, and review and editing. All authors contributed to the article and approved the submitted version.


Sorafenib improves the overall survival in patients with advanced hepatocellular carcinoma (HCC). Dickkopf-1 (DKK1) is commonly overexpressed in HCC. In this study, we investigated whether the inhibition of DKK1 enhances the anti-tumor efficacy of sorafenib in HCC.HCC cells were treated with sorafenib and WAY-262611, which is an inhibitor of DKK1. Transgenic mouse models were also developed using hydrodynamic tail vein injection. Mice were orally administered with sorafenib (32 mg/kg), WAY-262611 (16 mg/kg), or sorafenib + WAY-262611 for 10 days. Mechanisms of sorafenib and WAY-262611 were explored via western blotting, immunostaining, and RNA sequencing.P < 0.05). Compared with sorafenib alone, sorafenib + WAY-262611 significantly inhibited the cell viability, invasion, migration, and colony formation by promoting apoptosis and altering the cell cycles in HCC cells . Moreover, sorafenib + WAY-262611 decreased the p110α, phospho-Akt , active β-catenin  and phospho-GSK-3β (Ser9) expression levels, while increasing the phospho-GSK-3β (Tyr216) expression levels compared with those in the sorafenib alone in vitro and in vivo. In addition, sorafenib + WAY-262611 inhibited tumor progression by regulating cell proliferation and apoptosis, significantly better than sorafenib alone in mouse models.DKK1 was significantly overexpressed in patients with HCC than in the healthy controls and patients with liver diseases except HCC (all Our results indicate that DKK1 inhibition significantly enhances the anti-tumor efficacy of sorafenib by inhibiting the PI3K/Akt and Wnt/β-catenin pathways via regulation of GSK3β activity, suggesting a novel therapeutic strategy for HCC.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-023-01355-2. Hepatocellular carcinoma (HCC) is the most common type of primary liver malignancy and is the fourth leading cause of cancer deaths worldwide , 2. ChroSOR was the first tyrosine kinase inhibitor (TKI) approved for the treatment of advanced HCC , 13. SORDickkopf-1 (DKK1), a secretory inhibitor of the Wnt/β-catenin pathway, plays a crucial role in the induction of head formation during vertebrate development –19. DKK1Mutations are commonly observed in the Wnt/β-catenin pathway components, which result in constitutively activated β-catenin in HCC . AlthougIdentification of synergistic agents for SOR is important to increase its sensitivity and therapeutic efficacy, however the association between SOR and DKK1 remain ambiguous –32. TherDKK1 mRNA expression was analyzed using publicly available databases TCGA projects-HCC datasets. In total, 373 HCC and 50 non-tumor samples were obtained from TCGA website .All human samples were obtained with the approval of the Institutional Review Board (IRB) of Severance Hospital . This study adhered to the ethical guidelines of the Declaration of Helsinki 1964. Human serum samples were obtained from the Yonsei Liver Blood Bank of the Division of Gastroenterology and Hepatology, Department of Internal Medicine (IRB numbers 4-2018-0537 and 4-2018-1036). Liver tissues were obtained during liver transplantation from the Department of Transplant Surgery, Yonsei University College of Medicine, Severance Hospital, Seoul, Korea (IRB number 4-2016-0323).Serum DKK1 concentrations were measured using ELISA kits , according to the manufacturer’s instructions.IHC and IF staining were performed as previously described . DetaileAll experiments involving animals were performed in accordance with the Guidelines and Regulations for the Care and Use of Laboratory Animals at AAALAC-accredited facilities. This study was approved by the Animal Policy and Welfare Committee of the Yonsei University College of Medicine (Permit number: 2020-0290 and 2020-0299). Four-week-old C57BL/6 and Balb/c nude male mice were purchased from Orient Bio, Inc.  and Central Lab. Animal, Inc. , respectively. The mice were randomly treated with vehicle , SOR (32 mg/kg) , WAY (16 mg/kg) , and SOR + WAY for 10 d. SOR was dissolved in a stock solution containing 75% ethanol and Cremophor EL  (1:1). Subsequently, the stock solution was dissolved in a saline solution (1:9). WAY was dissolved in DMSO (0.02 g/mL) as a stock solution, which was dissolved in polyethylene glycol (Sigma-Aldrich) and saline water.G12V, pT2/short-hairpin RNA down-regulation p53 (shp53), pT2/c-Myc, pT3/EF5a TAZS89A, pT2/EGFP, pT2/Smad7, pT3-EF1a HrasG12V-microRNA down-regulating p53 (miRp53), and pT2/PI3KE545K as previously described , compared to Hep3B wild type (WT) cells [mean 3.00 ± 0.26 μM (24 h) and 3.08 ± 0.14 μM (48 h)]    , but decreased the percentage of G0/G1 (P < 0.05) and S cells, which was significantly better than SOR treatment alone, indicating that SOR + WAY treatment induced G2/M cell arrest in Hep3B cells   and volume (P < 0.05), compared to SOR treatment alone in xenograft mice generated using Hep3B cells  were significantly lower than in SOR-treated mice , and phosphorylation of GSK3β at Ser 9 (p-GSK3β-Ser9), compared to SOR treatment alone in Huh7 cells , compared to SOR treatment alone, however no difference in p-GSK3β-Ser9 levels was observed , whereas significantly increased the expression of phosphorylation of GSK3β at Tyr 216 (p-GSK3β-Tyr216) , compared to SOR treatment alone in Huh7 and Hep3B cells , growth arrest and DNA damage-inducible 45, and superoxide dismutase 2 , whereas it significantly decreased the expression levels of the Wnt/β-catenin pathway target genes, such as c-Myc, Twist, and matrix metalloproteinase 2, compared to SOR treatment alone in Huh7 and Hep3B cells    . SOR + LY294002 treatment not only changed the expression of the PI3K/Akt pathway molecules, such as p110α, p-Akt and p-GSK3β-Ser9, but also the expression of the Wnt/β-catenin pathway molecules, such as active β-catenin and p-GSK3β-Tyr216, significantly better than SOR treatment alone in Huh7 and Hep3B cells , SOR (n = 4), WAY (n = 5), or SOR + WAY (n = 5) for 10 d    , whereas significantly decreased Ki-67 expression (P < 0.05), compared to SOR treatment alone , active β-catenin and p-GSK3β-Ser9 (P < 0.01), whereas increased p-GSK3β-Tyr216 expression, compared to controls in Huh7 cells , compared to controls (P < 0.05)  (n = 5) showed tumor formation significantly more than mice injected with HrasG12V and miRp53 (Hras/miRp53) (n = 5) (P < 0.05) , p-Akt, active β-catenin, cyclin D1 (P < 0.01), and DKK1 (P < 0.05), compared to HrasG12V and miRp53 transfected mouse liver tumors  Fig. D. Simultors Fig. E. In HraG12V, miRp53, and PI3KE545K were orally administered SOR and/or WAY for 10 d   increased active β-catenin expression through increases of p-GSK3β-Ser9 in mouse liver tumors, suggesting that PI3K/Akt pathway regulated Wnt/β-catenin pathway through GSK3β. Therefore, our results support the hypothesis that PI3K/Akt pathway are connected with Wnt/β-catenin pathway.Fifth, we found that the PI3K/Akt pathway are correlated with Wnt/β-catenin pathway through GKS3β in HCC. Kaidanovich-Beilin et al.  found th50 values in the same tumor cell lines and the increased tumor cell density could be associated with increased chemo-resistance [P < 0.05), significantly better than LEN treatment alone, but no difference in the Wnt/β-catenin pathway was observed in Huh7 cells, and LEN + WAY treatment had no effects on PI3K/Akt and Wnt/β-catenin pathways in Hep3B cells. Although WAY significantly enhanced the anti-colony formation ability of LEN (Supplementary Fig. Despite several important results, this study has some limitations. First, our results showed that Hep3B is more sensitive to sorafenib compared to Huh7, whereas some studies showed that sorafenib is more sensitive to Huh7 cells than Hep3B cells . This phsistance . Thus, mIn conclusion, we found that DKK1 inhibition significantly enhanced the anti-tumor efficacy of SOR by inhibiting the PI3K/Akt and Wnt/β-catenin pathways and both pathways were connected via GSK3β in HCC. Therefore, inhibition of DKK1 may be a novel therapeutic strategy for HCC.Additional file 1: Supplementary Figure 1. The effects of LEN, WAY or their combination treatment on cell viability or colony formation in HCC cells. Supplementary Figure 2. Combination effects of SOR + WAY treatment on tumor progression in xenograft mouse model. Supplementary Figure 3. SOR + WAY treatment regulates PI3K/Akt and Wnt/β-catenin pathways in HCC. Supplementary Figure 4. The combination effects of SOR + WAY treatment under condition of PI3K activation. Supplementary Figure 5. Correlation between DKK1 expression and immune cell infiltration. Supplementary Table 1. Primary and secondary anti-bodies used in this study. Supplementary Table 2. Primer sequences used in the qRT-PCR.Additional file 2.Additional file 3.


The experimental results have shown that the average diameter and particle size distribution of silicone particles depend on the chemical structure and architecture of the compatibilizers. One copolymer has been identified as the best compatibilizer, allowing a lower mean diameter and particle size distribution in addition to the best mechanical properties of the final network (less brittle character). This study has consequently evidenced the possibility of creating in situ silicone capsules inside an epoxy network by adding tailored compatibilizers to epoxy/silicone formulations.The objective of this study was to prepare crosslinked epoxy networks containing liquid silicone particles in order to improve their mechanical properties and obtain less brittle materials. Different copolymers were used as compatibilizers. These copolymers vary in their chemical composition and structure. All of the copolymers contain hydrophobic (PDMS sequences) and hydrophilic groups. The effect of their chemical structure and architecture on the morphology of the dispersed phase, and on the final physico-chemical and flexural characteristics of epoxy/silicone blends, was explored. The morphology of crosslinked formulations was studied by scanning electron microscopy (SEM), and the thermal characteristics (glass transition temperature, T Epoxy resins form a class of widely used high-performance thermosetting polymers, due to their excellent mechanical properties, moisture and chemical resistances, heat and dimensional stabilities, very good electrical insulation, and very good adhesion to different substrates ,2,3. Theg). One of the first approaches has been to add reactive liquid elastomers such as carboxyl-terminated butadiene-acrylonitrile copolymer (CTBN rubber) to the uncured epoxy formulations and to cure these reactive systems [Several strategies have been developed to consistently increase the epoxy toughness without a significant lowering of the overall mechanical performance or the glass transition temperature  and a shell able to ensure strong bonding to the resin matrix. Since this strategy generates similar biphasic final morphologies as in the case of reactive liquid rubbers, it was assumed that the mechanisms described for rubber-toughening are also valid here ,17,18,19A very recent strategy to epoxy-toughening suggests using self-organizing thermoplastic copolymers that are miscible with the uncured epoxy matrix, and conduct phase-separation during epoxy curing, generating different nanostructured elastomeric domains inside the 3D epoxy network. The major advantage of this approach consists of the much lower viscosity of the epoxy formulation, making it easier to process ,21,22,232 [Other recent exploratory strategies for epoxy-toughening propose using nanoparticles such as silica ,25,26, g2 ,29, and 2 ,31,32,332 ,34,35,362 ,37,38 of2 ,24,39.In addition to the interest in improving epoxy toughness and impact resistance, some certain new challenges have appeared in recent years, based on the great potential of epoxy resins to produce innovative advanced functional materials exhibiting self-healing, self-cleaning, shape memory, or other functional properties such as transparent-to-opaque transitions ,42,43,44In the present work, we investigated the possibility of preparing 3D epoxy networks containing liquid silicone particles and tailoring their biphasic morphology by choosing an appropriate compatibilizer . Using a−1. Consequently, the exact EEW and 1H NMR data [The equivalent epoxy weight (EEW) of a DGEBA resin represents very important data for developing formulations with a stoichiometric epoxy/hardener ratio. For the present work, the DGEBA supplier’s data only indicated a batch with NMR data ,48, know1H NMR spectrum, as follows [pR is the ratio between the proton signals associated with the aromatic rings of DGEBA repetitive units, (a+bI) and the proton signals corresponding to the terminal epoxy units (g+h+eI), and tR corresponds to the same ratio in the case of the DGEBA oligomer (when n = 0).On the other hand,  follows ,48:DP¯n=1H NMR spectrum of DGEBA used in this study.−1, well below the 700 g·mol−1 indicated by the supplier. Consequently, for all formulations, the experimental EEW value was taken for the DGEBA/IPD ratio.The results show that the DGEBA resin used in this study has a The results of the miscibility tests are presented in The last column from Preliminary DSC measurements have shown that the neat DGEBA/IPD epoxy system reached full crosslinking after one hour of curing at 150 °C see . Based oThe main chemical reactions for DGEBA + IPD resins are presented in −1) offered additional heat for epoxy and diamine to regain thermodynamic mobility and continue the crosslinking reactions. The post-curing stage for one hour at 150 °C was intended to fully crosslink the epoxy network.During the curing of all of the epoxy systems, the formation of the first crosslinks induced an increase in the matrix viscosity and glass temperature transition. The curing phase at a slow temperature ramp (2.5 °C·min−1. The onset of cure (Tonset), the exothermic peak temperature (Tpeak), and the heat of cure ΔHcure of the epoxy formulations were determined from the DSC curves.The curing behavior of epoxy reactive systems (with and without silicone) was observed by non-isothermal DSC, from room temperature to 210 °C, at 10 °C·minonset or the temperature of the maximum curing rate, Tpeak. For instance, The presence of liquid inclusions of 5 to 10 wt% of chemically inert silicone did not change the large exothermic profile due to the curing of the epoxy matrix see . When cocure with the addition of silicone that corresponds to the decrease in epoxy matrix concentration. Similar results have been obtained for the formulations containing the other compatibilizers.The only difference concerns the decrease in ΔHThe morphological analysis of cured systems was performed by SEM. Cured neat epoxy resins presented a homogeneous transparent aspect. At the end of the curing procedure, all epoxy/silicone formulations containing PDMS and the compatibilizers presented biphasic morphologies with well-dispersed spherical particles inside a continuous rigid epoxy network. The SEM images show that all of the compatibilizers assured a good epoxy/silicone compatibilization, especially for 5 wt% silicone see . All invAt equal concentrations , the most efficient compatibilizers for epoxy/silicone resins based on DGEBA are coPo1 and 3, which gave fine morphologies with narrow particle size distributions. Both copolymers present linear hydrophilic polyether chains which are OH terminated.g values obtained by DSC and DMA, which are not unusual since these two methods are not describing the same physical phenomena from the sample, no clear dependence could be detected depending on the particle sizes of the silicone inclusions or the effectiveness of the different compatibilizers.crosslink) of epoxy/silicone blends. According to the theory of rubber elasticity, the storage modulus G’ from the rubbery plateau  is directly proportional to the crosslink density of cured resins but can also be influenced by the presence of PDMS inclusions [However, DMA data allowed further evaluation of the effect of silicone and compatibilizers on the thermomechanical characteristics and crosslink density , this study suggests that the most appropriate compatibilizers for reaching this goal are coPo1 and coPo3.Since coPo1 and coPo3 exhibit the best compatibilizing capacities, their mechanical properties will be compared. An increase in the flexural strength (at break) is observed for the blend containing coPo1, for both PDMS contents, contrary to the blend containing coPo3 for which a decrease in the flexural strength can be noticed. For the other formulations, no significant trend is observed, proving that the mechanical properties depend not only on the dispersion quality but also on other parameters such as the chemical architecture  or the chemical composition . However, some of them prove to be more effective. In particular, coPo1 and coPo3 induce smaller particle sizes and a narrower distribution, for both of the PDMS concentrations studied. These two linear copolymers with polyether/silicone molar ratios close to one and OH terminal groups are consequently the most efficient as compatibilizers. The ether and hydroxyl groups allow a good compatibility with DGEBA chains, which also possess OH and ether groups, while their linear PDMS sequences allow a good compatibility with the PDMS dispersed phase.The experimental results show that all copolymers allow a stable dispersion of liquid silicone inclusions inside the epoxy network , which proves that both PDMS and copolymers macromolecules are well included in the network, avoiding any migration at the surface. This property is important for coating or bonding epoxy surfaces.The study of the flexural properties, in particular the deformation at break, indicates an improvement (less brittle character) for all of the copolymers, which was the objective of this study. Nevertheless, coPo1 makes it possible to obtain the most interesting properties, with a higher deformation at break.w = 28,000 g·mol−1) was purchased from Alfa Aesar, Illkirch, France. The different linear di-functional or multi-pendant silicone polyethers used as compatibilizers  was purchased from Huntsman Advanced Materials, Europe, Basel, Switzerland. The curing agent, isophorone diamine (IPD), was purchased from Sigma-Aldrich, Saint-Quentin-Falavier, France. Trimethylsiloxy-terminated polydimethylsiloxane  and mixed for another 10 min at 33 °C. Thermosetting formulations were poured into different silicone molds to obtain rectangular specimens for physico-chemical and mechanical characterizations. The formulations were pre-cured in an oven at 33 °C for 60 min, then heated with a temperature ramp up to 150 °C at 2.5 °C·min1H NMR analyses were performed with a Bruker 300 UltraShield spectrometer, Bruker, Marne La Vallée, France, at 300 MHz, driven by the XWIN-NMR V3.5 software. DGEBA epoxy resin was dissolved in deuterated chloroform at a concentration of 10 wt%.Miscibility tests of the different compatibilizers with either DGEBA or silicone were performed at room temperature and 33 °C (in a heating bath), to determine the best pre-mixing procedure for optimally incorporating all the components of the epoxy formulations. Equal quantities of silicone–polyether copolymers and either DGEBA or PDMS were vigorously mixed during 10 min, and the visual aspect of the mixture was recorded for five hours.Morphological analysis of epoxy formulations was performed by scanning electron microscopy (SEM). The morphology of fractured specimens of cured resins was also investigated by SEM, by means of a JEOL JCM 6000, Jeol, Croissy-sur-Seine, France. The fractured specimens of cured resins were previously immersed in butyl acetate (a good solvent for PDMS) for 24 h, at room temperature, to remove the silicone contamination from the surface and potentially from a few microns in depth. After removal from butyl acetate, the samples were dried and analyzed by SEM. The particle size distribution and average diameter value of inclusions were determined by image analysis, after verifying the homogeneity of the morphological patterns over 3 samples for each specimen.g, and to study the influence of silicone inclusions on the curing kinetics of epoxy materials. The DSC analyses were carried out with a TA Instruments’ DSC-Q200, TA Instruments, Guyancourt, France under a nitrogen atmosphere, using hermetically sealed aluminum pans.DSC was used to investigate the curing of epoxy formulations, to determine the T−1. In the case of cured samples, the first heating scan was used to verify whether the curing was complete or not, and the second to determine the Tg of the cured samples. The first heating scan applied on uncured epoxy formulations indicated the onset temperature of curing, and the exothermic peak temperature. The area under the exothermic peak due to the crosslinking reaction gave, by integration, the heat of cure, ΔHcure (J/g). The second heating scan was used as previously, to determine the Tg of in situ cured formulations.A first heating scan was applied on uncured and cured samples, from 25 to 200 °C, followed by cooling down to 25 °C, and a second heating scan from 25 to 200 °C; all scans were performed at constant rate of 10 °C·min−1. The viscoelastic moduli, storage (G′) and loss modulus (G″), and mechanical loss tangent (tan δ) were obtained for a frequency of 1 Hz. The glass-transition temperature values were assumed as the temperatures at the maximum tan δ peak.Dynamic–mechanical analyses were performed on a modular Anton Parr MCR 302 rheometer, Anton Paar, Les Ulis, France. Cured rectangular specimens (40 mm × 12 mm × 2.3 mm) were mounted in single cantilever bending clamps and submitted to a temperature scan from 25 to 180 °C, with a heating rate of 2 °C·minThe flexural properties of cured epoxy formulations were measured using a Testometric M500-30AT universal testing machine, Testometric, Rochdale, UK. Three-point bending tests were performed on rectangular specimens (142 mm × 12 mm × 2.3 mm). The measurements were performed with a crosshead speed of 0.5 mm/min, at room temperature. Seven specimens were tested for each epoxy formulation.Contact angle measurements were performed to evaluate the water wettability of cured epoxy resins with and without silicone in the formulation. The experiments were carried out with a Krüss DSA100 Drop Shape Analyzer, Krüss, Villebon sur Yvette, France using the sessile drop method. Water droplets of 0.2 µL were deposited on epoxy surfaces (both upper and lower surfaces of rectangular specimens), with a 0.53 mm diameter syringe, at a 500 µL/min flow rate. At least 10 measurements were conducted for each surface.This study has evidenced the possibility of creating in situ liquid silicone capsules inside an epoxy network and to control their size and distribution by adding compatibilizers to epoxy/silicone formulations.The experimental results have shown that the average diameter and particle size distribution of silicone capsules depend on the chemical structure and architecture of the silicone-compatibilizers. The copolymers coPo1 and coPo3 were the most efficient as compatibilizers. CoPo1 appears to be the best choice to decrease the brittle character of epoxy networks while improving the hydrophilicity of the cured sample. The next step will be to study aging by following the evolution of properties over time and under aggressive conditions .


Genome instability in cancer cells causes not only point mutations but also structural variations of the genome, including copy number variations (CNVs). It has recently been proposed that CNVs arise in cancer to adapt to a given microenvironment to survive. However, how CNV influences cellular resistance against ionizing radiation remains unknown. PRMT5 (protein arginine methyltransferase 5) and APE1 (apurinic/apyrimidinic endonuclease 1), which enhance repair of DNA double-strand breaks and oxidative DNA damage, are closely localized in the chromosome 14 of the human genome. In this study, the genomics data for the PRMT5 and APE1 genes, including their expression, CNVs, and clinical outcomes, were analyzed using TCGA’s data set for oral squamous cell carcinoma patients. The two genes were found to share almost identical CNV values among cancer tissues from oral squamous cell carcinoma (OSCC) patients. Levels of expression of PRMT5 and APE1 in OSCC tissues are highly correlated in cancer but not in normal tissues, suggesting that regulation of PRMT5 and APE1 were overridden by the extent of CNV in the PRMT5-APE1 genome region. High expression levels of PRMT5 and APE1 were both associated with poor survival outcomes after radiation therapy. Simultaneous down-regulation of PRMT5 and APE1 synergistically hampered DNA double-strand break repair and sensitized OSCC cell lines to X-ray irradiation in vitro and in vivo. These results suggest that the extent of CNV in a particular genome region significantly influence the radiation resistance of cancer cells. Profiling CNV in the PRMT5-APE1 genome region may help us to understand the mechanism of the acquired radioresistance of tumor cells, and raises the possibility that simultaneous inhibition of PRMT5 and APE1 may increase the efficacy of radiation therapy. Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. A recent estimation of the annual incidence of cancer of the oral cavity and pharynx  in the US is about 54,000, and the estimated mortality is 11,580  . More thCells possess two main DSB repair (DSBR) pathways, i.e., DSBR via homologous recombination (HR) and non-homologous end joining (NHEJ). Enhancement of either DSBR pathway may confer resistance to radiation. Owens et al. recently reported that protein arginine methyltransferase 5 (PRMT5) is a crucial activator of both HR and NHEJ DSBR . AccordiOn the other hand, oxidative DNA damage is repaired by the DNA base excision repair (BER) pathway, in which apurinic/apyrimidinic (AP) endonuclease 1 (APE1) converts AP sites and 3′-blocking SSBs to 3′-OH termini, which are absolutely required for DNA synthesis . APE1 wahttps://www.genecards.org, accessed on 2 September 2023). Simultaneous down-regulation of PRMT5 and APE1 synergistically sensitized OSCC cell lines in vitro and in vivo. Shih et al. recently identified that cancer cells take advantage of particular copy number variations (CNVs) in their growth environments [It was separately shown that down-regulation of either PRMT5 or APE1 sensitized cells to IR ,19,22. Ironments . We detehttps://portal.gdc.cancer.gov/legacy-archive/search/f, accessed on 2 September 2023). They include RNAseq, SNP array, and clinical records from 522 patients. De-identified clinical records in json format were used to extract cross-referenced numbers (patient_id) that were linked to the genomics data, pathological cancer stages, information regarding radiation treatment (labeled “radiation_treatment” and “radiation_dose”). Dates of death and last dates for follow-ups were combined to build survival records.RNAseq and clinical records of OSCC patients were obtained from TCGA  were consolidated to a table containing all RNA expression intensities and the patient_id numbers. There are genes of which expression was almost undetectable, resulting in 0 RNAseq intensities for most of the specimens. Such genes with low RNAseq intensities (0 intensities in 10% or more of the specimens) were ignored and not analyzed in this study. Through this process, data of 15,858 genes were used for further analyses. Patients whose tumors were pathologically diagnosed as T4 stage and who underwent radiation therapy were selected and analyzed for this study. Relevant transcription table and clinical records are provided as p-values were calculated with a log-rank test using the survdiff function in R’s survival package [RNAseq data were linked to the survival data of the corresponding individuals. For each gene, two RNAseq  datasets, QTR1 and QTR4, were grouped based on their expression levels. QTR1 (the lower quartile) consists of those of the minimum to the first quartile, and QTR4 (the upper quartile) contains data from the third quartile to the maximum in terms of gene expression intensities. Then, difference in the survival between the QTR1 and QTR4 groups was examined using a Kaplan–Meier estimator.  package .https://docs.gdc.cancer.gov/Data/Bioinformatics_Pipelines/CNV_Pipeline/, accessed on 5 October 2023), for 522 OSCC patients versus their normal controls. Based on a manifest file that included a file name and patient id as well as specimen tags, the copy number data files were separated to tumor- and normal- (obtained from blood) segment copy files. Each file, consisting of lines starting and ending with GRCh38 genomic coordinates, number of probes, and the segment mean values, were consolidated into location-specific mean CNV values at the transcription starting positions of the analyzed genes, including PRMT5 and APE1.The transcription start sites of genes were obtained from the Entrez database based on GRCh38, and were used as genetic coordinates. Masked copy number segment values were determined using data from Affymetrix SNP array 6.0  supplemented with 10% fetal bovine serum , 20 mM L-Glutamine (Gibco/Thermofisher), and penicillin/streptomycin mix .An hypopharynx squamous cell carcinoma cell line FaDu  was purc7 cells/mL, 100–200 µL cell suspension was mixed with siRNA oligonucleotides at the final concentration of 200 nM. The siRNA were purchased from Ambion/Thermofisher   and IDT . The mixtures were then transferred into 0.4 cm EBC electroporation cuvettes, and electroporated using ECM830 (BTX) at 900 V, 120 µF, with 2 pulses of 0.1 s interval, which induced no detectable cytotoxicity to FaDu cells. Cells were then left at room temperature for 15 min, and then plated in dishes containing the complete medium. Cells were then incubated for 48 h, and then replated for a survival assay or protein analysis next day .Transient down-regulation of APE1 and PRMT5 expression were achieved with electroporation of specific siRNA oligonucleotides. Plates containing 80–90% confluent FaDu cells were washed with phosphate-buffered saline (PBS) twice and trypsinized, and suspended in the complete medium. Adjusted to 1 × 10AgeI-EcoRI sites or NheI-EcoRI sites, respectively, in the vectors above. HEK293Ta cells were transfected with the shRNA vectors with helper DNA (pMD2.G and psPAX2), using ViaFect transfection reagent . Media from these transfection dishes were then used to infect the cells. Selections of stable derivatives were made with appropriate antibiotics: 1 µg/mL puromycin  and 5 µg/mL blasticidine (Invivogen/Thermofisher).Lentiviral vectors from Addgene were used to generate FaDu derivatives stably expressing shRNA specific to PRMT5 and APE1 transcripts. The vectors were pLKO.1-puro , pLKO.1-blast (26655). Oligonucleotides for shRNA were inserted into Cells were washed in PBS twice, and then lysed in a lysis buffer  with sonication for 10 s. Protein concentrations were determined using Bradford reagent , and adjusted to an equal concentration among the sample groups. The cell lysates were run in 4–20% gradient SDS/PAGE (Bio-Rad). The proteins were then electroblotted on PVDF membranes (Bio-Rad). Immunoblot assays were then carried out to detect APE1 and PRMT5 using the method previously described . The pri4-acetate/ethanol solution for 30 min at room temperature, as described in the vendor’s manual, followed by incubation in 70% ethanol for 30 min. The gels in the slides were then dried, and stained in a SYBRgold solution  in TE buffer. After rinsing in H2O briefly, the slides were completely dried and were covered with an anti-fade solution . The comet signals were photographed using a Nikon TE microscope  in the tag image file format (tiff) at 1024 × 1024 resolution, and then analyzed using Comet Assay software (R&D Systems). The parameter of tail moment (the product of distance and intensity integrated over the tail length to assess the severity of damage) is defined using this software.The comet assay  reagents2 incubator for 9–10 days. After colony formation, cells were fixed and stained in a crystal violet solution [A colony formation assay was performed to determine survival fractions for FaDu cells. One day before X-ray exposure, cells were spread on 6-well culture dishes at 1000 cells per well for FaDu. Cells were then incubated for 24 h. X-ray exposure was carried out using a 225 kV orthovoltage X-ray irradiator, as described above. Cells were then incubated in a COsolution . Having AluI restriction endonuclease, which was provided to produce DNA fragments of 200–300 bp size on average, according to the manufacturer’s protocol. The digested DNA was then analyzed in nCounter Sprint using the Oncogenomics core facility of the Markey Cancer Center. The copy number variations in OSCC tissues were determined with a Nanostring CNV cassette  customized to detect the APE1, PRMT5, and other genes inside of the APE1-PRMT5 genome region, using a method described recently . Briefly6) were injected subcutaneously into the mouse flank on a warming platform and under anesthesia with oxygenated isoflurane. When the tumors reached a size of approximately 200 mm3, the mice were anesthetized with ketamine (100 mg/Kg)/xylazine (10 mg/Kg) and were irradiated with a total of 12 Gy (4 Gy × 3 day) at the tumor sites in the X-ray Service Center at the Department of Toxicology and Cancer Biology of the University of Kentucky, using PXi 225XL-RAD [The animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Kentucky. Tumor growth using athymic mice was carried out using FaDu and its derivatives. FaDu derivatives expressing single shRNA  and both shRNA (shPRMT5 and shAPE1) were established to down-regulate these factors stably, using the lentiviral vectors described as above. The cells •(shorter length)/2. Tumor growths among groups were compared for their longitudinal growth difference based on a linear mixed model with Bonferroni adjustment using the R-package TumGrowth [p-values were adjusted with Holm’s method for multiple comparisons, as shown in All in vitro experiments were repeated at least three times. Each survival treatment was triplicated, and the reproducibility of the results was assessed by repeating each experiment at least three times. To statistically evaluate the comet assay results, tail moment values obtained using Comet Assay software (R&D Systems) were compared among the cell lines with Welch’s umGrowth . For CNVp-value = 0.57). Similarly, when patients with all stages were considered, there was no advantage of the higher-dose treatment over the lower-dose treatment or vice versa. These observations indicated that the variance in radiation dosage did not result in a significant difference in the survival outcome for this group of patients. Although cancers of other organs are also treated with IR frequently, OSCC cases provide a large volume of data for patients who have undergone radiation therapy, making them a valuable source of information for studying the resistance of tumors to radiation treatments.There were 522 OSCC cases deposited in TCGA, with gene expression profiles and clinical records. Based on information regarding radiation dosage in the clinical record “rad:radiations”, there were 308 cases with radiation treatment. There were 97 Stage 4 cases  that were treated with radiation. However, two individuals in this group underwent radiation in unusually low doses (0.5 and 0.6 Gy). These two cases were considered outliers and were excluded from this study. The mean and median doses of X-ray in the remaining 95 cases were 60 and 58.36 Gy, respectively. The mean dosage was almost identical to those of all stages, but with a lower s.d. than those of the T4 stage (standard deviation: 11.3 for T4 versus 15.2 for other stages), indicating that the radiation exposure method is more consistent among T4-stage patients than those of all stages. Additionally, the “radiation_therapy” tag in the clinical sheet listed 53 cases with “YES”, which were included in the 95 cases described above. The mean dosage of the 53 cases was 60.23  Gy. Because both groups were treated with similar doses of radiation, the following analyses were carried out with the groups of both 53 and 95 patients unless otherwise noted. To understand whether the survival of patients with lower doses radiation differed from that of those with higher doses, patients who received doses of the minimum to 25th percentile (QTR1) and those with 75th to the maximum dosage (QTR4) were compared using a Kaplan–Meier survival estimator. There was statistically no significant difference between the two groups  were tested for their sensitivity to X-ray . FaDu deTo elucidate the effect of PRMT5 and APE1 kd on the efficiency of DSBR after X-ray radiation, single-cell gel electrophoresis (Comet assay) was carried out . The extTo test the effect of the PRMT5-APE1 down-regulation in vivo, a stable down-regulation of the PRMT5 and APE1 was necessary for tumor growth. Therefore, stable derivatives of FaDu cells harboring the control, shRNA for PRMT5 down-regulation, and shRNA for APE1 down-regulation  were generated. The FaDu derivatives were transplanted into athymic mice, and the effectiveness of fractionated X-ray irradiation  in controlling tumor growth was examined  via a loUsing a NanoString CNV panel with a code set specific to the APE1-PRMT5 gene region, we determined CNV values using genomic DNA extracted from formalin-fixed, paraffin-embedded OSCC tissues . The NanIR generates clusters of DNA lesions composed of DSB and oxidative damage ,36. WhilGenes highly expressed in cancer cells are often associated with aggressive cell growth and survival. PRMT5 and APE1 are among these genes ,24, at lBased on TCGA RNAseq data, a high expressional correlation between PRMT5 and APE1 was found in OSCC tumor tissues. Such high expressional correlation was not observed with the matched normal tissues. Seeking a molecular mechanism that explains the tumor-specific high correlation between PRMT5 and APE1 expression, we consider it significant that the genomic locations of PRMT5 and APE1 are very close to each other at chr 14q11.2 .CNV frequently occurs in the cancer genome, and the chance of sharing an equal CNV value in a cell is higher between two genes that are closely localized in a chromosome. The extent of CNV was almost identical between the PRMT5 and APE1 genes. Importantly, there were high correlations between RNAseq and CNV values for both APE1 and PRMT5. These observations suggest that the extent of CNV in the APE1-PRMT5 genome region is a critical factor in the co-regulation of APE1-PRMT5. DNA replication stress due to uncontrolled cell growth and/or DNA damage via therapeutic procedures are expected to increase genome instability, which may be the cause of increased CNVs in cancer cells. Our assessment of TCGA data and our CNV analysis of FFPE tissues indicate that sub-populations of cells in tumor tissues contain copy number increases in the APE1-PRMT5 region. We speculate that these cells gain growth and survival advantage against IR treatment to affect the outcome of the radiotherapy significantly, as high expression levels of PRMT5 and APE1 have been associated with poor outcomes of radiation therapy in oral cancer patients. Previous studies are consistent with this prediction that high expression of APE1 in OSCC and glioblastoma were linked to tumor resistance to alkylating reagents, reactive oxygen species ,38, and 6A) [The chromosomal locus contains other genes that have been associated with DNA repair . Among t6A) . TherefoFinally, it should be noted that CNV only partially explains the mechanism of the co-regulation of the PRMT5 and APE1 genes, because it does not explain expression patterns significantly different from those of the normal (diploid) cells. We speculate that the loss of normal gene regulation, presumably due to epigenetic alterations during tumorigenesis, results in increased reliance on gene dosage (CNV) alone. Establishing this mechanism may open up opportunities to simultaneously regulate these DNA repair factors in the genome region, which should improve therapeutic strategies for patients who may otherwise have a poor prognosis of radiation treatment due to an increase in copy number in the PRMT5-APE1 locus.