Protein arginine methyltransferase 5 (PRMT5) catalyzes mono-methylation and symmetric di-methylation on arginine residues and has emerged as a potential antitumor target with inhibitors being tested in clinical trials. However, it remains unknown how the efficacy of PRMT5 inhibitors is regulated. Here we report that autophagy blockage enhances cellular sensitivity to PRMT5 inhibitor in triple negative breast cancer cells. Genetic ablation or pharmacological inhibition of PRMT5 triggers cytoprotective autophagy. Mechanistically, PRMT5 catalyzes monomethylation of ULK1 at R532 to suppress ULK1 activation, leading to attenuation of autophagy. As a result, ULK1 inhibition blocks PRMT5 deficiency-induced autophagy and sensitizes cells to PRMT5 inhibitor. Our study not only identifies autophagy as an inducible factor that dictates cellular sensitivity to PRMT5 inhibitor, but also unearths a critical molecular mechanism by which PRMT5 regulates autophagy through methylating ULK1, providing a rationale for the combination of PRMT5 and autophagy inhibitors in cancer therapy. Protein arginine methyltransferases (PRMTs) serve as writers to catalyze the transfer of methyl groups to arginine residues, thereby generating three types of methylarginines: monomethylarginines (MMA), asymmetric dimethylarginines (aDMA), and symmetric dimethylarginines (sDMA). In mammals, nine PRMTs are grouped into three categories based on their products: type I PRMTs  catalyze the formation of MMA and aDMA, while type II PRMTs (PRMT5 and PRMT9) register MMA and sDMA. PRMT7 is the sole member of type III enzyme that generates only MMA,6. Dysregulation of PRMTs has been associated with many human diseases and has become attractive therapeutic targets–10.Arginine methylation has emerged as one of the common posttranslational modifications (PTMs) and plays crucial roles in controlling protein stability, localization, protein–protein interaction, and enzymatic activity. PRMT5 regulates transcription by depositing sDMA on histone –14 and transcription factors–17, while it controls DNA damage response in both transcriptional-dependent and -independent mechanisms–20. PRMT5 is also a critical regulator of RNA splicing,22 and cell signaling transduction–25. Clinically, PRMT5 overexpression has been observed in a variety of cancers,27. Moreover, elevated PRMT5 expression is associated with poor prognosis and chemotherapeutic resistance in breast cancer patients,29. Multiple PRMT5 inhibitors have been developed and are currently being evaluated in clinical trials. Interestingly, preclinical studies showed that breast cancer cells display diverse sensitivity to PRMT5 inhibitors with triple negative breast cancer (TNBC) cells generally being relatively resistant,32. However, the underlying causes for this variation in sensitivity to PRMT5 inhibitors remain elusive.PRMT5 is the main type II enzyme with numerous substrates that are involved in fundamental cellular processes,34. The autophagy process includes five sequential steps: initiation, nucleation, elongation/expansion, autophagosome fusion, and degradation in autolysosome. Each step is executed by distinct complexes that are formed by evolutionarily conserved autophagy-related (ATG) proteins–37. For example, the ULK kinase complex, which is composed of ULK1 or ULK2, ATG13, FIP200, and ATG101, is required for the initiation of autophagy, while the class III PI3K complex I consisting of VPS34, VPS15, and Beclin 1 is essential for nucleation. During autophagy, the ATG4-processed form of LC3 (LC3-I) is further converted to the PE-conjugated form (LC3-II) by ATG7-ATG3,41, while the adaptor proteins p62/SQSTM1 is degraded,43. LC3-II accumulation and p62 degradation are widely accepted as autophagy markers.Autophagy is a self-degradative process that delivers cytoplasmic materials to lysosomes for degradation and serves as a key recycling factory to maintain cellular homeostasis. Autophagy has both tumor suppressive and tumor promoting roles depending on cancer types and stages. Moreover, autophagy can be induced by therapeutic agents and plays a prosurvival role to confer drug resistance,48. Therefore, targeting autophagy is a promising strategy to enhance therapeutic efficacy. Indeed, combination of autophagy inhibitors with different cancer treatments is currently undergoing evaluation in numerous clinical trials.Dysregulation of autophagy has been linked to various human diseases, particularly neurodegenerative diseases and cancersIn this study we demonstrate that PRMT5 inhibition induces cytoprotective autophagy and thereby decreases sensitivity to PRMT5 inhibitor in TNBC cells, and that PRMT5 negatively regulates autophagy in part by methylating ULK1. Thus, our study provides a molecular basis and rational for targeting both PRMT5 and autophagy as a potential option for TNBC treatment.. We observed that ER+PR+ and HER2+ breast cancer cells, and one TNBC cell line (MDA-MB-468) were sensitive to GSK3326595, whereas the other four TNBC cell lines and a widely used non-malignant breast epithelial cell line (MCF10A) were relatively resistant to GSK3326595, which was defined by IC50 < 4 μM and IC50 > 4 μM as previously described 6. We obs,48. We reasoned that cytoprotective autophagy contributes to the observed resistance to PRMT5 inhibitor in TNBC cells. To test this idea, we treated TNBC cells with GSK3326595 and chloroquine (CQ), the only FDA-approved autophagy inhibitor that functions by preventing lysosomal degradation. By treating cells with different doses of CQ, we determined cell line-specific doses that were used in the combination treatment  and red (RFP+) puncta indicates enhanced autophagosome formation, while only accumulation of yellow puncta suggests impairment in autophagosome-lysosome fusion and degradation. Notably, a significant accumulation of both yellow and red puncta of LC3 was observed in PRMT5-depleted cells . Consistent with the finding in cells . In vitro kinase assay showed that ULK1-R532K displayed higher kinase activity than ULK1-WT towards phosphorylating Beclin 1 Next, we sought to investigate how ULK1-R532K enhances its kinase activity. Both ULK1-WT and ULK1-R532K bound to FIP200 and ATG13 at a comparable level  cells, Huh7 hepatocellular carcinoma (HCC) cells, and human oral keratinocytes (HOKs) in hypoxic environment, but not in normoxic conditionAll cells were obtained from American Type Culture Collection (ATCC). HEK293T, MDA-MB-231, MCF7, Hs 578T and their derived cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) . T-47D, MDA-MB-453, MDA-MB-468, BT-549, HCC70 and their derived cell lines were maintained in RPMI 1640 medium . 10%fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin were supplemented in the medium. GSK3326595 (HY-101563), MRT68921 dihydrochloride (HY-100006A), and Chloroquine (HY-17589A) purchased from MedChemExpress.For protein expression, transfection was performed using Lipofectamine 3000  according to the manufacturer’s instructions. For lentivirus production, target constructs containing sgRNA or cDNA were co-transfected with packaging plasmids (pMD2G and pSPAX2) into HEK293T cells with Polyethylenimine . Twenty-four hours post transfection, fresh medium was replaced. Virus containing supernatants were harvested at 48 h post transfection and filtered with 0.45 μm PES filter. Targeted cells were infected with virus and selected with hygromycin (200 μg/ml), puromycin (1–2 μg/ml) or blasticidin (10 μg/ml) for 4 days to eliminate the non-infected cells.https://www.synthego.com and were cloned into lentiCRISPR v2 vector . Sequence of sgRNAs is listed Supplementary Table Flag-PRMT5, Flag-MEP50 were generated by cloning the corresponding cDNA into the pRK5-Flag vector while HA-PRMT5 and HA-ULK1 cDNA were cloned into the pRK5-HA vector. Myc-PRMT5, Myc-ULK1, Myc-Beclin 1, and Myc-Ambra1 were generated by cloning the corresponding cDNA into the pRK5-Myc vector. GST-Beclin 1 (1–86 aa) and GST-ULK1 (1–649 aa) were generated by inserting the cDNA into pGEX-6P-1 bacteria expression vector. Myc-ATG13 (#31965), Flag-FIP200 (#24300), GFP-LC3-RFP (#84573) were purchased from Addgene. Lentiviral HA-ULK1 and HA-PRMT5 were generated by cloning the corresponding cDNA into pLenti-HA-hygro vector or pLJM1-HA-puro vector. PRMT5-E444Q, ULK1-R532K and various ULK1 mutants were generated using the QuikChange XL site-directed mutagenesis kit. Various single guide RNAs (sgRNA) were designed at All primary antibodies were diluted with 5% non-fat milk in TBST buffer for Western blot. Anti-ULK1 (8045), anti-Myc (2278), anti-cleaved Caspase 3 (9661), anti-AMPKα (5831), anti-Raptor (2280), anti-pS757-ULK1 (14202), anti-pS15-Beclin 1 (84966), anti-LC3B (3868), anti-ATG13 (13468), anti-PRMT1 (2449), anti-PRMT5 (79998), anti-S6K1 (9202), anti-HA (3724), and anti-pT389-S6K (9234) were purchased from Cell Signaling Technology. Anti-Tubulin (66240-1-lg) and anti-Beclin 1 (11306-1-AP) were purchased from Proteintech. Rabbit anti-FLAG (F7425), mouse anti-FLAG (F3165), peroxidase-conjugated anti-mouse secondary antibody (A4416), and anti-rabbit secondary antibody (A4914) were purchased from Sigma. Monoclonal anti-HA (901503) was purchased from BioLegend. Anti-PRMT7 (A12159) and anti-p62 (A11483) were purchased from ABclonal. Anti-pS318-ATG13 (600-401-C49) was purchased from ROCKLAND. Anti-MMA was a gift from Dr. Mark Bedford at MD Anderson Cancer Center.2, 1 mM EDTA and 1% Triton X-100) supplemented with protease inhibitor  and phosphatase inhibitors . The cell lysates were centrifuged at 13,200 r.p.m. at 4 °C for 10 min. The protein concentration of lysates was determined using Nanodrop by Bio-Rad protein assay reagent. Equal amounts of whole cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For IP, 2000–5000 μg lysates were incubated with agarose conjugated antibodies for 3–5 h at 4 °C. Immunoprecipitants were washed three times with NETN buffer  or Triton buffer before being resolved by SDS-PAGE. Anti-HA agarose beads (A2095) and anti-FLAG agarose beads (A2220) were purchased from Sigma-Aldrich. Anti-Myc agarose beads (658502) were purchased from BioLegend. Some blots were cut prior to hybridization with primary antibodies, but one full-length original, unprocessed blot for each antibody was provided in the Supplementary Materials.Cells were rinsed with ice-cold phosphate-buffered saline (PBS) and lysed in EBC buffer  or Triton buffer  Escherichia coli transformed with corresponding constructs. Single colony was grown in 7 mL Luria–Bertani (LB) medium overnight at 37 °C. The culture was then inoculated into 400 mL LB medium until an optical density of 0.5–0.6. The protein expression was induced by 0.1 mM IPTG  at 25 °C for 16 h. The bacteria cells were collected and re-suspended in GST buffer  and sonicated. After centrifugation, the supernatant was incubated with glutathione sepharose beads for 3 h at 4 °C. The protein-bound glutathione beads were washed three times with GST buffer and recombinant GST proteins were eluted with elution buffer .2, 1 mM β-mercaptoethanol, 100 mM sucrose) with 1 μL of adenosyl-L-methionine, S-[methyl-3H]  at 30 °C for 1 h. The reactions were stopped by 3 × SDS loading buffer. The samples were resolved by SDS-PAGE and transferred to PVDF membrane, which was then sprayed with EN3HANCE (Perkin Elmer) and exposed to X-ray film.3 μg recombinant GST-ULK1 truncated proteins were incubated with HA-PRMT5/MEP50 in the methylation buffer  for 30 min at 30 °C. The reaction was stopped by adding 2 × SDS loading buffer. Samples were incubated at 100 °C for 5 min and resolved by SDS-PAGE. Phosphorylation of GST-Beclin 1 was detected by anti-pS15-Becllin 1 antibody.3 μg of bacterially purified GST-Beclin 1 recombinant proteins were incubated with HA-ULK1 purified from HEK293T cells in the kinase reaction buffer . Images were taken by Leica SP8 Confocal microscope and puncta were counted manually.m/z window with a maximum injection time of 50 ms. Tandem mass spectra were acquired using higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) for each peptide precursor in an alternating fashion. The HCD collision energy was 35% and ETD was performed using the calibrated charge dependent ETD parameters. The fragment ions were detected in the Orbitrap at 15,000 resolution. Spectra were searched against a custom database containing human ULK1 and a database of common contaminants using MaxQuant and Proteome Discoverer. The false discovery rate, determined using a reversed database strategy, was set at 1% at the peptide and modification site levels. Fully tryptic peptides with a minimum of seven residues were required including cleavage between lysine and proline. Two missed cleavages were permitted. Sites of modification were manually verified.HEK293T cells were transfected with HA-ULK1. Forty-eight hours post transfection, the cells were lysed in Triton buffer, followed by immunoprecipitation. The immunoprecipitates were resolved by SDS-PAGE and visualized using GelCode blue staining reagent . The protein band containing HA-ULK1 was excised and digested with trypsin. Peptides were analyzed on an EASY nLC 1200 in-line with the Orbitrap Fusion Lumos Tribrid mass spectrometer (ThermoScientific). Peptides were pressure loaded at 800 bar and separated on a C18 reversed phase column ) (Thermo Fisher) using a gradient of 2–35% B in 180 min  at a flow rate of 300 nL/min at 45 °C. Mass spectra were acquired in datadependent mode with a high resolution  Fourier Transform mass spectrometry (FTMS) survey scan followed by MS/MS of the most intense precursors with a cycle time of 3 s. The automatic gain control target value was 4.0e5 for the survey MS1 scan. Precursors were isolated with a 1.6 Cells were seeded in 96-well plate at 500–1000 cells per well for 24 h and then treated with indicated doses of inhibitors for 4 days. Cell viability was determined using the Cell Titer-Glo cell viability assay kit according to the manufacturer’s instructions .Cells were seeded in 6-well plates at 300–500 cells per well for 24 h and then treated with indicated inhibitors for 8–10 days until visible colonies formation. Fresh medium with inhibitors was replaced every 3 days. Colonies were fixed with 10% ethanol and 10% acetic acid for 30 min and then stained with 0.4% crystal violent in 20% ethanol for 30 min, followed by wash with dH2O and manually counted.t test or two-way ANOVA. P value < 0.05 was considered significant.As indicated in the figure legends, all quantitative data are presented as the mean ± SD of three biologically independent experiments or samples. Statistical analyses were performed using GraphPad Prism 9 and Excel. Statistical significance was determined by two-tailed Student’s Supplementary Information.


Gene Set Enrichment and Ingenuity Pathway Analyses of male placentas showed that EX led to inhibition of signaling pathways, biological functions, and down-regulation of transcripts related to lipid and steroid metabolism, while EX in female placentas led to activation of pathways, biological functions, and gene expression related to muscle growth, brain, vascular development, and growth factors. Overall, our results suggest that the effects of maternal EX on the placenta and presumably on the offspring are sexually dimorphic.While exercise (EX) during pregnancy is beneficial for both mother and child, little is known about the mechanisms by which maternal exercise mediates changes in utero. Six-week-old female C57BL/6 mice were divided into two groups: with  or without  access to voluntary running wheels. EX was provided via 24 h access to wheels for 10 weeks prior to conception until late pregnancy (18.5 days post coitum). Sex-stratified placentas and fetal livers were collected. Microarray analysis of SED and EX placentas revealed that EX affected gene transcript expression of 283 and 661 transcripts in male and female placentas, respectively (±1.4-fold,  The Developmental Origins of Health and Disease (DOHaD) hypothesis posits that the first 1000 days of life (conception through 2 years of age) represents a critical developmental window during which nutritional and environmental influences can potentially impact disease risk throughout life . GreaterClinical studies have reported that maternal exercise during gestation improves pregnancy and birth outcomes ,11,12 anSod3 and vitamin D receptor (Vdr) expression independent of maternal high-fat diet and may be linked to metabolic changes in the offspring’s liver [Maternal health and environmental insults have been shown to contribute to the persistent programming of offspring metabolic phenotypes. The placenta is thought to be a central mediator of developmental programming, as it is the sole site of nutrient exchanges which is necessary in orchestrating growth and development in utero, as well as a conduit to relay metabolic information about the maternal habitus to the developing offspring ,31,32. I’s liver . HoweverThe present study examined the role of maternal voluntary wheel running commencing 10 weeks prior to gestation, and throughout pregnancy, on placental and fetal liver mRNA expression in late gestation. The purpose of this study was to assess early responsiveness to maternal exercise and examine sex differences in the placenta and offspring. The placenta serves as the principal interface between maternal and fetal circulation and serves as the hub for nutrient sensing and transport, gas exchange, and excretion of waste ,37. Thusp = 0.05), week 7 (p = 0.08), and week 10 (p = 0.05) in EX dams as compared SED dams, shown in p = 0.07) and a higher percentage of lean mass (p = 0.09) after 10 weeks of wheel running as compared to SED dam counterparts  with reduced fetal weights in females (p = 0.05) and a trend towards reduced weight in males (p = 0.08) at dpc 18.5. There was a main effect of maternal exercise (p = 0.004) for decreased placental efficiency that was driven by the reductions in fetal weights and a significant reduction in males (p = 0.01) and a similar decrease in females (p = 0.06) via pairwise comparisons.Access to running wheels 10 weeks prior to conception and throughout pregnancy decreased male and female fetal weights. p < 0.05, p < 0.05), suggesting a larger impact of maternal exercise on global gene expression in placentas (Lpl), acyl-CoA oxidase (Acox), and cytochrome P450 (Cyp)11a1. In female placenta transcripts involved in muscle differentiation that included Notch1 and Hdac 4 and 9, several myosin heavy- and light-chain isoforms were up-regulated in EX as compared to SED  within each treatment group, 56 transcripts were differentially expressed in the EX group, and 42 differentially expressed genes (DEGs) were altered in the SED group  of 1.3 or higher), indicating the over-representation of molecules in the DEG lists. If a given entry had sufficient information, then IPA reports directionally (activation/increase or inhibition/decrease), expressed as a z-score (|z| > 1.96 is considered significant). The reported results for UR analysis excluded exogenous chemicals, drugs, and toxins, focusing on biologically expressed molecules. The reported results from DF analysis excluded diseases, thereby focusing on “biological functions.” IPA also supported GSEA results, as male placentas from EX dams showed changes to URs and BFs related to lipid metabolism and steroid metabolism/transport  of 1.3 or greater). For example, the CP analysis, LXR/RXR activation, production of nitric oxide and reactive oxygen species in macrophages, superpathway of cholesterol biosynthesis, estrogen biosynthesis and xenobiotic metabolism displayed significant z-scores indicating inhibition , using canonical pathway (CP), disease and functions (DF), and upstream regulator (UR) analysis tools . Signifiransport A. Overalhibition A. For URhibition A. The mohibition A.In female placentas, IPA revealed 73 CPs, 639 URs, and 391 biological functions that were significantly affected in the females from EX compared to SED dam, including activation of wound healing, regulation of epithelial mesenchymal transition by growth factors, ILK signaling, activin inhibin signaling, hepatic fibrosis, actin cytoskeleton signaling, and ERK/MARPK signaling for CPs B. Some oFinally, we utilized IPA comparison analyses to directly compare the altered pathways, regulators, and functions in males and females when comparing the effect of maternal EX . From thApoC1, Cyp11a1, Cyp24a1, and Acox as shown in Cyp11a1, Lrp1, Acox, and Scd2 were all increased in female placentas in EX and SED supporting the microarray transcript changes. In addition, we also revealed significant main effects for sex for Apoe, Cyp11a1, Lipin1, Acox, and Scd2 transcripts. Lrp1 showed both sex and exercise effects on mRNA expression while Apoc1 showed a significant interaction effect (Npp (p = 0.03) and Tph1 (p = 0.04). Moreover, Bmp4 mRNA expression showed a significant sex effect (p = 0.005) with increased expression levels in females compared to male placentas as shown in Hsd3β due to maternal exercise only in male placentas (p = 0.11)  B.Apoe (p = 0.01 SED and p = 0.01 EX), Lrp1 (p = 0.0002 SED and p < 0.0001 EX), and Scd2  were increased in the fetal liver of females from both EX and SED as compared to male fetal livers (Apoc1 (Ex effect p = 0.028) and Lipin1 (Ex effect p = 0.013) occurred in placentas from both males and females  and down-regulation of transcripts related to lipid and steroid metabolism following maternal EX. Second, there was an activation of signaling pathways and functions related to muscle growth, vascular development, and growth factors and up-regulation of transcriptions involved in muscle differentiation in female placentas only with maternal EX. Third, our studies suggest an inhibition of upstream X-linked regulator, KDM6A, in male placentas, and epigenetic regulators DNMTs (DNMT1 and DNMT3B) and HDAC1 as significant upstream regulators in female placentas and not male placentas.Hsd3β, an enzyme that has an important role in steroid production, in male placentas with maternal EX. These results are in line with other clinical studies and animal models that have shown improved circulating lipids and lipid metabolism within adipose and liver tissue of offspring with maternal EX [Apoc1 and Apoe in the placenta of males compared to females with maternal EX. Together, these results provide evidence that lipid and steroid metabolism regulation with maternal exercise in the placenta differs with sex and is independent of dietary intake.A notable finding from these studies was the inhibition of lipid- and steroid-metabolism-related gene expression, pathways, and biological functions of the male placenta exposed to MAT EX and not the female placentas. More specifically, at the gene level, we found a significant reduction in the expression of ernal EX ,42,43,44ernal EX . In addiernal EX ,47. Pregernal EX ,49,50. Oernal EX ,52. Furternal EX ,54. Addiernal EX  and may ernal EX ,57. ThisSex steroids and changes in lipoproteins have also been previously associated with changes in placental efficiency and fetal growth ,59. ThesCollectively, these changes in lipid metabolism and steroid metabolism of male placentas with maternal exercise could point to a potential mediating role that maternal exercise has on the placenta. The placenta is a key regulator of fetal growth and health, which occurs in large part through nutrient transport through the placenta. Among the key substrates that are transported through the placenta are lipids and fatty acids. In the context of maternal obesity, exercise during pregnancy can decrease excess lipids in the placenta . In our Bmp4, which is also associated with obesity and energy metabolism, to reveal a significant sex effect, where levels were increased in females compared to males. In relation to muscle growth and vascular development, both factors have been previously shown to be altered with exercise and have shown sex-specific differences, similar to our results [In female placentas, maternal EX was associated with activation of signaling pathways and functions related to muscle growth, vascular development, and growth factors, and up-regulation of transcripts involved in muscle differentiation but not seen in male placentas. The transforming growth factor-beta family is known for its role in placentation and establishing the fetal–maternal interface ,65. In o results ,70,71,72Another interesting observation from this study was the potential epigenetic regulators identified by IPA upstream regulator analysis. In male placentas from EX dams, we found inhibition of X-linked KDM6A as an upstream regulator in male placentas, but it was absent in females. In females, we identified significant upstream regulators; DMNT1, DNMT3B, and HDAC1 were regulated in female placentas with maternal EX but were not present in male placentas. KDM6A is a demethylase encoded by a gene with female-biased expression due to its escaping X-inactivation and function as an eraser of the repressive H3K27me3 mark . In a clIt is important to note the limitations of the current study. First, the use of microarray analyses instead of RNA-sequencing was used on placenta tissue. We acknowledge that RNA-seq is a much more robust and sensitive method compared to microarray; nevertheless, this method was used to conduct these analyses at the time. However, the data still provide several novel findings to be followed up using RNA-seq and other methods. To improve the rigor of our studies, we employed three different analyses of DEGs and were able to find similar pathways and biological functions across all three platforms. A second limitation of this study is that we are not able to isolate the effects of preconception versus gestation exercise effects, as our maternal exercise intervention commenced prior to conception including both exercise during the pre-conception and gestation time. A third limitation is that no follow-up in offspring at later time points was conducted except for fetal livers at dpc 18.5. However, the primary objective of these experiments was to study the effects on the placenta, and therefore, offspring were not studied. Nonetheless, follow-up studies examining the long-term effects of maternal exercise on offspring health and development would provide beneficial information on exercise’s effects on developmental programming.In conclusion, our results describe molecular changes in the placenta associated with maternal EX, which show pronounced sexual dimorphism. Our study identified novel pathways, functions and upstream regulators that are differentially regulated in the placenta of males compared to females. These results provide potential mechanisms for future studies to elucidate sex-specific developmental programming associated with prenatal exercise.Virgin female C57BL/6 mice were obtained from the Jackson Laboratory . Animals were housed in an AAALAC-approved animal facility at the Arkansas Children’s Research Institute in a temperature- and light-controlled room (12 h light/12 h dark cycle). All experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences. Unless specified, all chemicals were obtained from Sigma-Aldrich Chemical Co., .g @ 4 °C for 10 min) to obtain serum and was stored at −20 °C. Serum insulin was measured using an ELISA kit following the manufacturer’s protocol . Triglycerides , Total Cholesterol , Glucose , and non-esterified fatty acids  were assessed in the serum by colorimetric assays following standard procedures.Six-week-old female C57BL/6 mice were divided into 2 groups: with  or without  access to voluntary running wheels. EX was provided via 24 h access to wheels for 10 weeks prior to conception until late pregnancy (18.5 days post coitum). Each female mouse was individually housed and allowed ad libitum access to a purified AIN-93G  diet ,82. Runnp-value < 0.05 (Student’s t-test) between EX and SED dam offspring. Enrichment of gene ontology (GO) terms for differentially regulated genes was performed using GoRilla [Total RNA from individual whole placentas was isolated using TRI Reagent and purified using RNeasy columns  including on-column deoxyribonuclease digestion. Six microarrays  were used for each group, representing biologically distinct pools of RNA from placentas from different dams. Briefly, 5 μg of purified RNA was used to synthesize first- and second-strand cDNA. Labeled aRNA (amplified RNA) was synthesized from double-stranded cDNA using the GeneChip IVT labeling kit  according to the manufacturer’s protocol. The probe array was scanned with a GeneChip Scanner 3000 (Affymetrix) and .CEL files were generated using the GeneChip Command Console (Affymetrix). Data analysis was performed using functions in the affy, limma and sva packages in R ,84. CEL  GoRilla . Gene Sehttps://www.qiagenbioinformatics.com/products, accessed on 18 July 2023). QIAGEN  is a unique program that provides a breadth of analysis options compared with other programs [In addition, we performed Ingenuity Pathway Analysis (IPA) analyses between the male and female placenta differentially expressed gene (DEG) lists  using RNeasy mini columns  including on-column DNase digestion. One microgram of total RNA was reverse transcribed using iScript cDNA synthesis kit . Real-time PCR analysis was performed as described previously using an Applied Biosystems Prism 7500 Fast instrument . Gene-specific primers were designed using Primer Express Software v3.0 .For all placenta and fetal weight calculations, we took the average of all placentas and/or fetus from each individual dam. Then, the average for each dam was used for statistical analyses. Data are expressed as means ± SEM, significance was set at


SCN5A cause Brugada syndrome (BrS), an arrhythmic disorder that is a leading cause of sudden death and lacks effective treatment. An association between SCN5A and Wnt/β-catenin signaling has been recently established. However, the role of Wnt/β-catenin signaling in BrS and underlying mechanisms remains unknown.Mutations in the cardiac sodium channel gene SCN5A gene were recruited in this study. Control and BrS patient-specific induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts using nonintegrated Sendai virus. All iPSCs were differentiated into cardiomyocytes using monolayer-based differentiation protocol. Action potentials and sodium currents were recorded from control and BrS iPSC-derived cardiomyocytes (iPSC-CMs) by single-cell patch clamp.Three healthy control subjects and one BrS patient carrying a novel frameshift mutation (T1788fs) in the v1.5) loss-of-function as compared to control iPSC-CMs. Interestingly, the electrophysiological abnormalities and Nav1.5 loss-of-function observed in BrS iPSC-CMs were accompanied by aberrant activation of Wnt/β-catenin signaling. Notably, inhibition of Wnt/β-catenin significantly rescued Nav1.5 defects and arrhythmic phenotype in BrS iPSC-CMs. Mechanistically, SCN5A-encoded Nav1.5 interacts with β-catenin, and reduced expression of Nav1.5 leads to re-localization of β-catenin in BrS iPSC-CMs, which aberrantly activates Wnt/β-catenin signaling to suppress SCN5A transcription.BrS iPSC-CMs exhibited increased burden of arrhythmias and abnormal action potential profile featured by slower depolarization, decreased action potential amplitude, and increased beating interval variation. Moreover, BrS iPSC-CMs showed cardiac sodium channel (NaSCN5A-related BrS and point to Wnt/β-catenin as a potential therapeutic target.Our findings suggest that aberrant activation of Wnt/β-catenin signaling contributes to the pathogenesis of The online version contains supplementary material available at 10.1186/s13287-023-03477-3. Brugada syndrome (BrS) was first reported in 1992 and is a primary electrical disorder characterized by ST-segment elevation in the right precordial electrocardiogram (ECG) leads . It is wSCN5A, which encodes the α-subunit of the voltage-gated cardiac sodium channel (Nav1.5) and has been identified in 20–25% of BrS patients }, in which V1/2 is half-maximum (in)activation potential and k is slope factor. For analyzing time constants of inactivation, sodium currents were evoked by depolarizing cardiomyocytes from a holding potential of − 120 mV to test potentials from − 110 to − 20 mV with 10 mV increments. Recovery from inactivation of sodium currents was measured by a two-pulse protocol containing the prepulse at − 20 mV for 20 ms, then followed by various intervals before stepping to the test pulse at − 20 mV for 20 ms. Time constants (τfast and τslow) of inactivation or recovery from inactivation were analyzed by curve fitting to double-exponential equation: I/Imax = Afast[1 − exp(− t/τfast)] + Aslow[1 − exp(− t/τslow)], in which Afast and Aslow are the fractions of the fast and slow components of inactivation or recovery from inactivation, and τfast and τslow are the time constants of the fast and slow components of inactivation or recovery from inactivation, respectively.Sodium current was recorded from single iPSC-CMs using the ruptured patch clamp technique with conventional voltage clamp protocols. Bath solution contained: 50 mM NaCl, 110 mM CsCl, 1.8 mM CaClAll the chemicals used in the electrophysiological studies were purchased from Sigma-Aldrich. CHIR99021 was purchased from Axon Medchem, and stock solutions were prepared in 10 mM in dimethyl sulfoxide (DMSO) . IWR-1 was purchased from Calbiochem, and stock solutions were prepared in 10 mM in DMSO.t-test to compare two groups and by One-way ANOVA to compare multiple groups. A p value of < 0.05 was considered statistically significant. Data were shown as mean ± sem and analyzed by GraphPad Prism (GraphPad Software).Statistical significance was determined by unpaired two-tailed Student’s SCN5A gene , which results in a frameshift starting at amino acid 1788 , and this mutation was not found in controls in Exome Sequencing Project, 1000 Genomes Project, or Exome Aggregation Consortium, the pathogenic level of this novel SCN5A mutation was therefore classified as “likely pathogenic” according to the American College of Medical Genetics and Genomics (ACMG) guidelines (PS2 + PM2)  [One BrS patient and three healthy control subjects were recruited in this study Fig. B, and ecoderate) .Fig. 1ClSCN5A T1788fs mutation existed in patient iPSCs but not in control iPSCs  in BrS iPSC-CMs, whereas beating rate and action potential duration (APD) were comparable between control and BrS iPSC-CMs   Fig. D–I. Impov1.5 affected the expression of β-catenin [v1.5 and β-catenin. To test this hypothesis, co-immunoprecipitation (co-IP) studies were conducted to test whether there was an interaction between Nav1.5 and β-catenin in iPSC-CMs. Notably, reciprocal co-precipitation of Nav1.5 and β-catenin was observed using respective antibodies  in the  studies , 26, anaSCN5A-related BrS, featured by the loss-of-function of Nav1.5, is still unknown. In our work, a novel and important finding is the aberrant activation of Wnt/β-catenin signaling in BrS iPSC-CMs, which has not been previously reported.Wnt/β-catenin signaling is involved in various physiological and pathological activities . When thSCN5A, resulting in the reduction in sodium current [SCN5A transcription in both direct and indirect ways, which has been elucidated using neonatal rat ventricular myocytes [SCN5A and subsequently inhibit the transcription. This complex can also increase the expression of Tbx3, a known suppressor of SCN5A. Although the regulation role of Wnt/β-catenin signaling on the expression of SCN5A is known, we found an unexpected phenomenon that the level of Nav1.5 can in turn affect the activity of Wnt/β-catenin signaling. To our knowledge, we demonstrated the interaction between Nav1.5 and β-catenin for the first time, and we suggest this novel link contributes to the redistribution of β-catenin in BrS iPSC-CMs with reduced expression of Nav1.5.Previous studies have shown that activation of Wnt/β-catenin signaling inhibits the transcription of  current , 13. Wntmyocytes . BrieflySCN5A in the iPSC-CM model.It has been known that both the morbidity and mortality of BrS are high . NeverthThere are several limitations to our work that warrant caution and future work. First, Wnt signaling pathway can be classified as β-catenin-dependent pathway  and β-catenin-independent pathway  . In thisSCN5A-related BrS and point to Wnt/β-catenin as a potential therapeutic target.In conclusion, our findings suggest that aberrant activation of Wnt signaling contributes to the pathogenesis of Additional file 1. Supplemental Material.


The treatment of bone defects is a difficult problem in orthopedics. At present, the treatment mainly relies on autologous or allogeneic bone transplantation, which may lead to some complications such as foreign body rejection, local infection, pain, or numbness at the bone donor site. Local injection of conservative therapy to treat bone defects is one of the research hotspots at present. Bone marrow mesenchymal stem cells (BMSCs) can self-renew, significantly proliferate, and differentiate into various types of cells. Although it has been reported that CK1ε could mediate the Wnt/β-catenin pathway, leading to the development of the diseases, whether CK1ε plays a role in bone regeneration through the Wnt/β-catenin pathway has rarely been reported. The purpose of this study was to investigate whether CK1ε was involved in the osteogenic differentiation (OD) of BMSCs through the Wnt/β-catenin pathway and explore the mechanism. We used quantitative reverse transcription-polymerase chain reaction (qRT-qPCR), Western blots, immunofluorescence, alkaline phosphatase, and alizarin red staining to detect the effect of CK1ε on the OD of BMSCs and the Wnt/β-catenin signaling pathway. CK1ε was highly expressed in BMSCs with OD, and our study further demonstrated that CK1ε might promote the OD of BMSCs by activating DLV2 phosphorylation, initiating Wnt signaling downstream, and activating β-catenin nuclear transfer. In addition, by locally injecting a CK1ε-carrying adeno-associated virus (AAV5- CK1ε) into a femoral condyle defect rat model, the overexpression of CK1ε significantly promoted bone repair. Our data show that CK1ε was involved in the regulation of OD by mediating Wnt/β-catenin. This may provide a new strategy for the treatment of bone defects. Bone defects are one of the most complicated problems in clinical orthopedics. It can be caused by many factors such as trauma, infection, and tumor resection . The treBMSC is a subgroup of non-hematopoietic stromal cells residing in bone marrow, capable of self-renewal and differentiation into multiple cell types, as well as the ability to self-renew and significantly proliferate . BMSCs hin vitro and in vivo. Qiong et al. [Wnt signaling is the main evolutionarily-conserved signaling pathway that controls embryonic development and adult tissue homeostasis. The Wnt/β-catenin pathway, the classical Wnt signaling pathway, plays an important role in regulating OD, and the formation of bone matrix and mineralization , 19. Dysg et al.  reported2. Cells collected from a rat were cultured in a culture bottle. The culture medium was replaced every other day until reaching 80–90% cell confluence. These subcultured cells were designated as the first passage (P1), and P3 was used for subsequent experiments.This experiment selected four 4-week-old SPF grade SD male rats, weighing approximately 300 g, purchased from Changzhou Cavens Experimental Animal Co., Ltd. . The animal program has been approved by the Ethics Committee of the First Affiliated Hospital of Soochow University, with an animal ethics approval number of 2022-552. All animal surgical procedures were performed according to the protocols approved by the Ethics Committee of the First Affiliated Hospital of Soochow University. Sprague-Dawley (SD) rats were euthanized by cervical dislocation and soaked in 75% alcohol for 15 min. Bilateral femurs were removed under sterile conditions, and the bone marrow cavity was repeatedly washed by aspirating with Dulbecco’s modified Eagle Medium  using a 1-mL sterile syringe, and collected into a centrifuge tube. The bone marrow cell suspensions were centrifuged. Bone marrow mononuclear cells were removed, washed, and enriched for BMSCs by culture in a culture bottle containing DMEM/F12 and 10% fetal bovine serum  at 37°C and 5% CO2 until reaching 70–80% cell confluence. Then, the initial culture medium was removed, and an osteogenic induction culture medium , 20 mL; OriCell®Supplement for rat bone marrow mesenchymal stem cells OD, 3 mL; alizarin red stain, 10 mL; Gelatin, 10 mL) was added. Si-NC (20 nM), CK1ε siRNAs (20 nM), pcDNA3.1 (2 μg/mL), and pcDNA3.1-CK1ε (2 μg/mL) were purchased from Tsingke Biotechnology Co., Ltd. . Due to the time of osteogenic induction and the aging of transfection, OD induction was initiated 24 hours after transfection, and transfection was performed again on the 5th and 10th day, respectively, to maintain the efficiency of transfection. The proliferation efficiency and morphology of BMSCs did not change significantly after the first transfection. 24 h after transfection, OD culture began. At this time, the proliferation efficiency of BMSCs began to decline, and the morphology of some cells gradually changed from spindle to polygon. After the second and third transfection and OD induction culture, the cell proliferation efficiency was further reduced, and the cell morphology was completely polygon. 7 days later, nodular structures gradually began to appear in the cultured cells. In addition, DKK-1  at a concentration of 0.5 μg/mL and pcDNA3.1-CK1ε were co-transfected to verify the interaction between CK1ε and Wnt/β-catenin. Transfections were carried out using lipofectamine 2000  [Routine OD induction culture procedure: BMSCs were grown in DMEM/F12 containing 10% FBS, 50 ug/mL of streptomycin, and 100 U/mL of penicillin at 37°C and 5% COMA, USA) . IWP-O1 After exposure to OIM for two weeks, the medium was removed. The BMSCs were washed with phosphate-buffered saline (PBS) twice and then fixed in 4% paraformaldehyde for 30 min. BMSCs were washed with double-distilled water and stained with 0.2% alizarin red stain  for 30 minutes at 37°C. The staining results were observed under a microscope . For quaCalcium concentrations were measured using a calcium colorimetric assay kit , according to the manufacturer’s instructions. Briefly, the cells were washed with PBS and suspended in buffer. Working solution (150 μL) was added to each well and the cells were incubated at 37°C for 5 min. Absorbance at 575 nm was measured using a SpectraMax M2 spectrometer  , 31.−ΔΔCt method [After the osteogenic induction culture of BMSCs for 14 days, total RNA was extracted using TRIzol . Osteoblast-related genes RUNX2, OCN, SP7, COL1A1, CK1ε, β-catenin, and GAPDH were assayed using the mRNA PCR Detection Kit (Takara). The 2t method  was usedBMSCs cultured in different groups for 14 days were collected and lysed. After centrifugation, ALP activity was assayed in the BMSC lysates using a commercial ALP assay kit, , and the results were read on a microplate reader  . BMSCs wThe total proteins in the BMSCs of the different groups were lysed using RIPA lysis buffer and extracted. The bicinchoninic acid (BCA) protein assay kit  was used for measuring protein concentrations. Each protein sample underwent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and washed with TBST. The membranes were incubated with primary antibodies overnight at 4°C and then with horseradish peroxidase-conjugated secondary antibodies for two hours at 37°C . FinallySince mature BMSCs do not express CD11b but express CD44, both CD44 and CD11b were analyzed by flow cytometry using CD44-fluorescein isothiocyanate (FITC) and CD11b-FITC antibodies  according to the manufacturer’s protocol .After two weeks of incubation, BMSCs in the different groups were fixed in 4% paraformaldehyde for 15 min at room temperature, then permeabilized in 0.1% Triton X for 20 min. To block nonspecific antibody binding sites, the BMSCs were incubated with 1% bovine serum albumin (BSA) for 20 min, then with primary anti-β-catenin antibodies (1: 200) overnight at 4°C for eight hours. The cells were washed three times with PBS and incubated with cy3-labeled rabbit secondary antibody  diluted 1: 500 for one hour. To observe β-catenin nuclear translocation by fluorescence microscopy, the cells were stained with 4′-6-diamidino-2-phenylindole . Finally, images were obtained on image management software .SD rats were purchased from Changzhou Cavens Experimental Animal Co., Ltd. All rats were males, 8–10 weeks old, and weighed about 300 g to 350 g. All surgical procedures and perioperative management complied with Chinese laws and the regulations of the local ethics committee.n = 10), control group , AAV5 group  lentivirus vector treatment, n = 10), and AAV5-CK1ε group . AAV5 lentivirus vector was purchased from OBiO Technology  [in vivo. First, a 2% sodium pentobarbital solution was injected intraperitoneally at a dose of 2.5 mL/kg. The femoral condyle was exposed by carefully and passively separating the muscle space to avoid damaging the surrounding blood vessels and soft tissue. Bicortical perforation was performed with a standard 3-mm drill at low speed, and normal saline was injected at the same time to avoid bone necrosis caused by high temperatures. Finally, to verify the potential target for minimally invasive injection therapy, the muscles, fascia, and skin of the rats were sutured layer by layer, and the AAV5 lentivirus vector was injected through the skin. After surgery, penicillin was routinely injected intramuscularly to prevent infection. The animals were kept at 25°C and 55% humidity, with 12-hour alternating light and dark cycles. The rats were provided with standard laboratory feed and tap water [The rats were randomly divided into four groups: normal group  . The ratap water –40.SD rats were euthanized four weeks after surgery, and femoral specimens were taken and fixed in a 10% formalin solution. First, the specimen was imaged and evaluated. Micro-computed tomography  was used to analyze the repair of the femoral defects. The micro-CT parameters were 18 μm, 65 kV, and 385 mA. Scanning parameters were 1 mm thick, rotation step 0.7 deg, average 1 frame, sync with event 5 ms and list Mode 2 frame, using SkyScan software. Appropriately 3-mm cylindrical regions were designated as regions of interest using Dataview and CT analyzer software and analyzed for bone mineral density (BMD), bone volume fraction (BV/TV), trabecular separation (TB.sp) and trabecular thickness of bone (TB.th).Decalcified specimens were scanned by micro-CT after incubating in xylene solution at room temperature, gradient dehydration in ethanol, and embedding in paraffin. The embedded femurs were sectioned sagittal to a thickness of 5 μm using a microtome. Tissue sections were stained with hematoxylin and eosin (H&E) and Masson’s stain to analyze tissue regeneration in the defect areas. CD31 immunofluorescence staining , CK1ε, osteocalcin  and osteopontin  immunohistochemical staining were performed on the tissue sections to evaluate angiogenesis and bone repair.t-test was used to compare the differences between two groups. One-way analysis of variance (ANOVA) was used to compare differences between multiple groups using SPSS 19.0  or GraphPad Prism 9.0. A p-value of < 0.05 indicated statistical significance.All cell experiments were performed in triplicate. The data were measured three times and expressed as means ± standard deviations (SD). The quantitative analysis of immunohistochemical and immunofluorescence staining was performed on five representative images per group using ImageJ software. The Student’s All remaining data are available within the article, or available from the corresponding authors upon request.P < 0.01). ARS was used to observe the calcification of the BMSCs after osteogenic induction culture. The results in P < 0.001), which indicated that the osteogenic induced group showed a higher degree of calcification after the osteogenic induction culture, which also suggested the success of OD. In order to further verify the success of the OD of the BMSCs, relative mRNA and protein expressions of OD related factors, including RUNX2 [P < 0.01). The Western blot gel images and semi-quantitative analysis results further confirmed the higher relative protein expression levels of these related proteins in the osteogenic induced group, with all of the P values < 0.05 . Subsequently, si-CK1ε and pcDNA3.1-CK1ε were constructed and transferred into the BMSCs to down-regulate and up-regulate the expression of CK1ε, respectively. The outcome, as presented in P < 0.05). ALP activity was detected to further verify the OD state and the ability to mineralize. The results further verified that the pcDNA3.1-CK1ε group had higher osteogenic activity, when compared with the other two groups (P < 0.001). Based on the above outcomes, we proposed the hypothesis that the overexpression of CK1ε may promote the OD of BMSCs.The role of CK1ε in the OD of BMSCs is the next focus of our study. After two weeks of induction, qRT-PCR and Western blot results showed that CK1ε and β-catenin relative mRNA and protein expressions were up-regulated in the osteogenic induced group when compared with uninduced group (P < 0.01). Next, to further study the exact relationship between CK1ε and Wnt/β-catenin, DKK-1  was used to interfere with the BMSCs during the whole process of induction. After seven days of incubation, ARS showed reduced calcification levels in the DKK-1+pcDNA3.1-CK1ε group compared with the pcDNA3.1-CK1ε group, which means that blocking the Wnt/β-catenin pathway significantly reduced the level of calcification (P < 0.01). Moreover, the results were more obvious on the 14th day (P < 0.01). Then, we estimated the ALP activity in all the groups. Combined with ALP staining and the activity results, we found that overexpression of CK1ε significantly increased the activity of ALP, while low expression of CK1ε decreased the activity of ALP. Meanwhile, DKK-1 could also reverse the increased ALP activity caused by CK1ε overexpression (P < 0.01).Then, we examined the mineralization of different groups. The cells were allowed to grow for two weeks and related events were observed every seven days. Consistent with our hypothesis, the up-regulation of CK1ε significantly increased the calcification state of cells compared to groups with lower or normal expression of CK1ε. The pcDNA3.1-CK1ε group demonstrated the most obvious staining changes on both day 7 and 14, when compared with other groups , 3D. TheWe further verified the results of osteoblast-related protein expression between the different groups. At day 7, pcDNA3.1-CK1ε significantly increased the relative protein expression levels of the OD markers  compared with the control group and the si-CK1ε group, while this trend was reversed by DKK-1 . HoweverIn order to further explore the relationship between CK1ε and Wnt signaling pathway, we conducted follow-up experiments. The stimulation of the Wnt/β-catenin pathway by CK1ε was further observed by immunofluorescence staining on the seventh day after induction, and the results showed that CK1ε promoted the expression of β-catenin. P < 0.05). These results further demonstrate that CK1ε can activate the Wnt/β-catenin pathway when overexpressed, and its target location is upstream of β-catenin. By competitively binding to the LRP5/6 receptor, DKK-1 blocks the down transmission of the Wnt signal and reduces the Wnt signal entering the cytoplasm normally, which may be the reason why the expression of intracellular CK1ε itself is not inhibited. To explore the relationship between CK1ε and specific targets in the upstream region of the Wnt/β-catenin signaling pathway, we introduced the DVL protein inhibitor IWP-O1 . IWP-O1 can specifically inhibit Dvl2 phosphorylation in cells [P < 0.05). The previous experimental results combined with the competitive binding effect of DDK-1 on low-density lipoprotein receptor-related protein (LRP) and the direct effect of DVL blocker, suggested that CK1ε may activate the Wnt/β-catenin pathway through direct interaction with DVL2 and activation of DVL2 phosphorylation.We further verified the relative protein expression of CK1ε and β-catenin on day 7 and 14 after BMSC osteogenic induction by Western blots. At the same time, the relative protein expression changes of C-JUN, C-MYC, LEF1, and TCF7 in the downstream of Wnt/β-catenin pathway at day 14 were also verified. in cells . Our resWe established a rat model of a femoral condylar defect with a local injection to verify the effect of CK1ε intervention on bone defect healing in rats. The rats survived well and were killed 4 weeks after the operation. Specimens were collected for further experiments. First, Micro-CT was conducted, and histological images and data were collected for analysis. Bone defect reconstructions were the worst in the AAV5 and control groups . The defWe obtained sagittal sections from the specimens collected at 4 weeks and performed H&E and Masson staining to analyze the effect of femoral condyle repair. in vivo at 4 weeks. The expression of osteoblast-related markers OPN and osteocalcin were verified (P < 0.01). The signal of OPN and Osteocalcin in the AAV5-CK1ε group was significantly stronger than that in the control group and AAV5 group, and this result was verified by semi-quantitative analysis (P < 0.01). Western blot analysis was used to detect CK1ε protein in samples from different groups, and the outcomes indicated that CK1ε relative protein expression in the AAV5-CK1ε group was significantly higher than that in the control and AAV5 groups (P < 0.001). These results suggest that CK1ε can promote the healing process of femoral condylar defect in rats by accelerating the differentiation of osteoblasts.Secondly, the specimens were stained by immunohistochemistry. Immunohistochemical staining of CK1ε showed that compared with the AAV5 and control groups, the AAV5-CK1ε group was still significantly expressed P < 0.01). These results confirmed the mechanism of AAV5-CK1ε in promoting angiogenesis at the molecular level. In vivo, CK1ε has been shown to promote local angiogenesis, possibly through the Wnt/β-catenin signaling pathway. These results provide a good basis for understanding long-term bone tissue repair.The early angiogenesis and maturation of vascular sprouting are important driving factors for tissue repair. CD31, also known as a platelet-endothelial cell adhesion molecule, is an important indicator of angiogenesis. In this study, four weeks after injury, the CD31 signal in the AAV5-CK1ε group and the normal group was significantly stronger than that in the control group and the AAV5 group . The quain vitro study demonstrated that CK1ε intervention may promote BMSC OD through a variety of experimental methods, and this result may be achieved by activating DVL2 phosphorylation and thereby activating Wnt/β-catenin signaling. Further, in vivo studies have shown that CK1ε intervention has better efficiency of new bone formation in a femur condylar defect model, as well as enhanced local osteoblast action and angiogenesis  and single-pass low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) combine with Wnt ligands to form a Wnt–FZD–LRP5/6 trimeric complex , which iHowever, our research had some limitations. As mentioned above, the Wnt/β-catenin signaling pathway is an important pathway in the OD of BMSCs, and after inhibiting this pathway, the role of CK1ε in promoting OD was significantly attenuated. However, we did not determine whether there are other signaling pathways or factors, as well as other cells in the local microenvironment, involved in the regulation of the OD of BMSCs, which requires further study. Secondly, a knockout animal model would bring a higher level of evidence to our animal model results. Once again, we did not conduct further research on animal locomotor ability and behavior.In summary, our results suggest that CK1ε can promote the OD of BMSCs by activating DVL2 phosphorylation and promoting β-catenin nuclear transfer, thus activating the β-catenin/Wnt signaling pathway. CK1ε may be a potential agent of choice for promoting bone defect healing. Overall, the importance of these findings is that they provide a potential strategy for treating bone defects and promoting bone regeneration.Supplementary Figures


In vitro, we treated rat proximal renal tubular epithelial cell line (NRK-52E) with cisplatin to mimic in vivo models of AKI. The NRK-52E cells were treated with or without 160 μg/mL hucMSC-Ex, and 1 µg/mL cisplatin was added after 9 h. Cells were harvested after 24 h. In the IRI group, the levels of serum creatinine (Scr) and blood urea nitrogen (BUN) were increased; renal tubules were dilated, epithelial cells were vacuolated, and collagen fibers were deposited in the renal interstitium. After treatment with cisplatin, the NRK-52E cells displayed pyroptotic morphology characterized by pyroptotic bodies. The protein expression levels of fibronectin, α-smooth muscle actin (α-SMA), vimentin, gasdermin D (GSDMD), caspase-1, interleukin-1 (IL-1β) and NLRP3 in IRI tissues and in cisplatin treatment NRK-52E cells were significantly upregulated. However, after the hucMSC-Ex intervention, kidney injury was effectively improved in vivo and in vitro. The current study shows that pyroptosis is involved in AKI and that hucMSC-Ex improves AKI by inhibiting pyroptosis.Human umbilical cord mesenchymal stem cells (hucMSCs) have been shown to improve kidney injury. Exosomes have been indicated to be important mediators of renal protection in MSC therapy. In spite of this, the mechanism remains unclear. Our study investigated how exosomes derived from hucMSCs (hucMSC-Ex) improve acute kidney injury (AKI). Exosomes were extracted by using an ultracentrifugation technique and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. Twenty-four male SD rats were randomly divided into four groups: sham group, sham + hucMSC-Ex group, ischemia–reperfusion injury (IRI) group, and IRI + hucMSC-Ex group.  In alloStem cells are pluripotent cells that have remarkable abilities to self-renew, regenerate, multiply, and differentiate ,6. AdditLiving cells secrete exosomes, which are small vesicles with a diameter of about 30–150 nm. They have a lipid bilayer structure and are important information carriers that regulate cell function and gene expression. Substances such as microRNAs and noncoding RNAs are transferred into recipient cells . ComparePyroptosis is a form of proinflammatory programmed cell death characterized by nuclear shrinkage, the formation and rupture of plasma membrane pores, and inflammatory response ,16. It i2.2.1.2, saturated humidity, 37 °C incubator. After 3 h, the culture was continued by adding 15 mL of F12 medium (BI) containing 10% fetal bovine serum . The culture medium was replaced on day 6 of culture. Passage was performed when several blocks of tissue were found to be surrounded by large cell colonies. The above steps were completed by Chongqing Stem Cell Therapy Engineering Technology Research Center . P2–P6 hucMSCs were incubated at 37 °C, 5% CO2. HucMSCs were routinely cultured in F12 medium  containing 10% fetal bovine serum . HucMSC-Ex was extracted from the supernatant of hucMSCs. When 5th generation hucMSCs reached 80% confluence, the culture was changed into the exosome-free culture medium with 10% serum for 48 h. The medium was collected and centrifuged sequentially at 1000 × g for 10 min, at 2000 × g for 20 min and at 10,000 × g for 30 min. After centrifugation, the upper 80% of the supernatant was transferred to Beckman Ultra-Clear Tubes to remove cell debris and residual cells. The hucMSC-Ex were pelleted by ultracentrifugation at 100,000 × g for 70 min. For further purification, the supernatant was aspirated layer by layer. The remaining small amount of supernatant was thoroughly mixed with hucMSC-Ex, transferred to Beckman Ultra-Clear Tubes, and ultracentrifuged at 100,000 × g for 70 min. The supernatant was decanted, and hucMSC-Ex was suspended in 200 µL of PBS. Large extracellular vesicles in the hucMSC-Ex were removed by filtration through a 0.22 μm pore filter. Determination of HucMSC-Ex protein concentration was carried out using the BCA method according to the directions . The morphology of the collected hucMSC-Ex was observed by TEM, and the size distribution of hucMSC-Ex was quantified by NTA. The expression of Alix , CD63 , and TSG101  was detected by western blotting. HucMSC-Ex was stored at −80 °C for subsequent experiments.The collected umbilical cord was sterilized with 75% alcohol  for 10 s and then rinsed three times with saline. We cut the umbilical cord into small pieces. The chopped umbilical cord tissue was inoculated in 75T (NUNC) culture flasks. They were placed in a 5% CO2.2.g for 70 min. HucMSC-Ex was suspended in 400 µL of PBS and stored at −80 °C for later use. NRK-52E cells were cultivated at 37 °C with 5% CO2 in DMEM  containing 10% fetal bovine serum . NRK-52E cells were cultured in a 24-well plate, and after the cells adhered, PKH26-labeled exosomes were added, and the cells were fixed after 3, 6, 9, 12 and 24 h. The incubation period at 37 °C was 10 min with the addition of Hoechst. Then, 20 μL of anti-fluorescence quencher were added to the glass slide to mount the slide. HucMSC-Ex uptakes by cells were examined under a confocal microscope.First, 400 µg of hucMSC-Ex was resuspended in 400 µL of PBS, and 500 µL of diluent C was added and mixed with hucMSC-Ex. Then, we added 3 µL of PKH26  dye to hucMSC-Ex, mixed well and incubated for 15 min at 37 °C, to label exosomes. The reaction was stopped by adding complete medium. The mixture was placed in a 90Ti tube and ultracentrifuged at 100,000 × 2.3.n = 6) and treated as follows: (1) in the sham group, sodium pentobarbital  was used to anesthetize the rats, and only the bilateral renal pedicles were freed; (2) in the sham + hucMSC-Ex group, the bilateral renal pedicles were dissociated, and 250 µg of hucMSC-Ex was injected into the tail vein immediately afterward; (3) in the IRI group, the renal pedicle was clamped with nondestructive arterial clips for 40 min before reperfusion, and 250 µL of PBS was injected into the tail vein immediately after reperfusion; and (4) in the IRI + hucMSC-Ex group, the renal pedicle was clamped with nondestructive arterial clips for 40 min and then perfused, and 250 µg of hucMSC-Ex was injected into the tail vein immediately after reperfusion. After 24 h, the rats were sacrificed, and kidney tissue and blood were collected for relevant analyses. Bioethical guidelines were followed in all animal procedures, and the Ethics Committee of the Children’s Hospital Affiliated to Chongqing Medical University approved the study (CHCMU-IACUC20220429002).Male SD rats (190–220 g) were purchased from Chongqing Medical University . The rats were fed regular food and water and housed in a standard cage at constant temperature and humidity, with a 12-h cycle of light and dark. The rats were randomly divided into four groups . NRK-52E cells were cultured at a density of 5000 cells per well in 96-well plates. The cells were treated with different concentrations of cisplatin for 24 h. We added 10 μL of CCK-8 reagent to each well. The cells were incubated for 1 h at 37 °C in an incubator. Microplate readers were used to measure optical density (OD) at 450 nm.2.5.g for 10 min at 4 °C. Automatic biochemical analyzers were used to determine the level of serum creatinine (Scr) and blood urea nitrogen (BUN).Serum was obtained by centrifugation of rat blood at 7500 × 2.6.Specimens from each group were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. After sectioning, HE staining, PAS staining, and Masson trichrome staining  were performed and the stained sections were observed and photographed.2.7.After the paraffin tissue sections were deparaffinized, they were placed in citrate buffer solution, heated 20 min and then cooled to room temperature. After being incubated with endogenous peroxidase blocking solution for 10 min, the sections were washed three times with PBS for 10 min each time, 5% BSA was added for 1 h at room temperature, and the following primary antibodies were added and incubated overnight at 4 °C: smooth muscle actin , vimentin , NLRP3 , caspase 1/P20/P10, , and IL-1 beta . The sections were washed three times with PBS for 10 min each time, and the reaction-enhancing solution was added. An appropriate amount of peroxidase-labeled goat anti-rabbit IgG was added and incubated at room temperature for 20 min. After being washed three times with PBS for 10 min each time, DAB was added to the sample for several seconds. After observing the color change under the light microscope, the reaction was terminated by placing the sample in deionized water. The nuclei were stained with hematoxylin, and the slides were examined under the light microscope.2.8.g for 5 min. The supernatant was discarded and fixed by adding 2.5% glutaraldehyde. The supernatant of treated or NRK-52E cells was discarded and fixed with 2.5% glutaraldehyde. The fixed samples were produced and photographed by Lilai Biotech .Kidney tissues were fixed with 2.5% glutaraldehyde. NRK-52E cells digested with trypsin were centrifuged at 1000 × 2.9.g for 20 min at 4 °C. Equal amounts of protein  were separated by SDS-PAGE, the gel was transferred to a PVDF membrane, and blocked with rapid blocking solution (Xin Saimei) for 10 min. The membranes were incubated at 4 °C with primary antibodies against NLRP3 , caspase-1 , GSDMD , IL-1β , α-smooth muscle actin (α-SMA) , vimentin , and fibronectin  overnight. The membranes were washed three times with TBST for 10 min, diluted secondary antibody  was added, and the membranes were incubated at room temperature for 1 h. ECL developer  was evenly dropped on PVDF membranes, developed and saved. The gray level of the spots was analyzed with Image Lab software. β-actin was used as an internal reference.Kidney tissue and NRK-52E cells were lysed in RIPA lysis buffer  containing PMSF and centrifuged at 12,000 × 2.10.p < .05 was set for all statistical tests performed on GraphPad 9.0 .All data from different experiments are expressed as mean ± standard deviation (SD). Data were compared among multiple groups using one-way analysis of variance (ANOVA). In all comparisons, 3.3.1.We extracted exosomes from hucMSC supernatants by ultracentrifugation and found that the exosomes were round or oval vesicle-like structures with diameters of approximately 30–150 nm by TEM . NTA ind3.2.Rats were anesthetized with sodium pentobarbital, and 250 μg of exosomes were injected via the tail vein immediately after IRI. Subsequently, we examined renal function in the four groups. Compared with the sham group, the levels of Scr and BUN were obviously increased in the IRI group and decreased after exosome administration, respectively, indicating that hucMSC-Ex could prevent renal injury in rats . HE and 3.3.To investigate the reason for hucMSC-Ex improving AKI and considering the important role of pyroptosis in AKI, we examined pyroptosis levels in the four groups. First, we used SEM, and observed that the sham group had no significant damage, while the IRI group had obvious damage, disordered cell arrangement, and visible pyroptotic bodies . Subsequ3.4.Cellular uptake is a prerequisite for drug delivery. Since tubular epithelial cells are the main cell type affected during AKI, we used NRK-52E cells to perform subsequent experiments. Therefore, we examined the uptake of hucMSC-Ex by NRK-52E cells. Confocal analysis showed that hucMSC-Ex was easily taken up by NRK-52E cells. The fluorescence intensity of hucMSC-Ex in cells increased with time. At 9 h, most cells had successfully taken up exosomes, and at 6 h, some cells had taken up exosomes . Therefo3.5.in vivo models of AKI and further explore the molecular mechanism of cisplatin toxicity. The viability of NRK-52E cells treated with 1 µg/mL cisplatin decreased to 58% when compared to that of the control group (According to studies, cisplatin can induce NRK cell pro-fibrotic changes. Therefore, we treated NRK-52E cells with cisplatin to mimic ol group . Therefool group , indicat3.6.Moreover, we also discovered that hucMSC-Ex could attenuate the toxicity of cisplatin and increase the survival rate of cells by CCK-8 . After c4.in vitro uptake experiments of PKH26-labeled exosomes demonstrated that hucMSC-Ex could be effectively taken up by cells.Organ transplantation is one of the most dramatic and important achievements in medicine in the twentieth century and the most effective treatment for end-stage diseases . HoweverIn vivo, we used IRI to construct an animal model of AKI. Compared with the traditional UUO model, this model can better simulate AKI caused by IRI after kidney transplantation and is similar to the clinical case study. In vitro, we treated NRK-52E cells with cisplatin to mimic an in vivo model of AKI. Compared with other fibrosis models, this experimental model is more stable. Comparing Scr and BUN at 24 h after surgery, we found that the level of Scr and BUN in the IRI group was increased and decreased after exosome intervention. Moreover, vascular collapse, renal tubular dilation or atrophy, inflammatory cell infiltration, extracellular matrix deposition, and collagen fiber deposition were observed in the interstitial region in rats in the IRI group. HucMSC-Ex can alleviate the renal tubular injury, indicating that hucMSC-Ex has a protective effect against renal injury.Scarring is characterized by fibrosis and results from an increase in activated myofibroblasts that deposit excess extracellular matrix . Activatin vitro experiment, we observed the same outcomes. Therefore, we believe that hucMSC-Ex has a protective effect on AKI by improving pyroptosis.Pyroptosis is characterized by the rupture of the plasma membrane and the release of intracellular proinflammatory substances . NLRP3, 5.The current study shows that pyroptosis is involved in AKI and that hucMSC-Ex improves AKI by inhibiting pyroptosis. Continued attempts to elucidate the target molecules are activated by pyroptosis. It helps to clarify further the cellular and molecular mechanisms of the protective effect of hucMSC-Ex on acute kidney disease.


The thermal stability (5%T > 334 °C and IPDT reached 1482 °C) of APPEN/GF composites was also investigated to further reveal the copolymerization between epoxy resin and aminophthalonitrile, which may be beneficial to the application of epoxy-matrix-based composites in the field of high-performance polymer composites.Self-polymerization epoxy/phthalonitrile (APPEN) pre-polymers were studied systematically, and then, gelation time and differential scanning calorimetry (DSC) were employed to investigate their curing behaviors. Taking advantage of orthogonal test analysis, the key factors that affected the co-polymerization of APPEN were defined and the appropriate pre-polymerization conditions were analyzed. A possible curing mechanism of APPEN was proposed. Then, the thermomechanical and mechanical properties of glass-fiber-reinforced APPEN laminates (APPEN/GF) prepared at 180 °C were analyzed to understand the cross-linked and aggregation structures. Fracture surface of the composite laminates was also investigated to reveal the copolymerization degree and the interfacial binding. The results indicated that APPEN/GF composites exhibit outstanding mechanical and thermomechanical properties (flexural strength, 712 MPa, flexural modulus, 38 GPa, and  As an important thermosetting matrix, epoxy resins possess excellent mechanical properties, good adhesion, low cost, and chemical stability, which means they are applied in various fields, including aerospace, mechanics, electronics, ships, and so on ,2,3. WitTo improve the thermal stability of epoxy resins, researchers have carried out many studies on designing curing agents and fillers. Through the optimization of catalysts and fillers, including carbon fiber , glass fgT), good dimensional stability, superior flame-resistant properties, and so on [In our previous work, various phthalonitriles were designed and prepared. Attributable to their high glass transition temperatures  of the composite material would decrease with an increase in the content of CTBN. Moreover, controlling the content of CTBN could achieve the best toughening effect when the mass fraction of CTBN was 15%. However, the toughening modification will lead to a further reduction in heat resistance due to the poor heat resistance of toughening modifiers themselves. Herein, a novel toughening effect of rigid group-containing thermoplastic resin Poly (arylene ether nitrile) (PP-PEN) was employed and investigated.Considering the general brittleness of thermosetting resins, to improve the toughness of epoxy resins, various thermoplastic polymers ,21, nanoHerein, to expand the various applications of epoxy resin , AG80/APN prepolymer toughening with PP-PEN, labeled as APPEN, was studied in detail. The curing behaviors and possible copolymerization mechanism of the as-prepared prepolymers were investigated. Then, glass-fiber-reinforced matrix laminates (APPEN/GF) were fabricated, and the mechanical properties, as well as thermal stability, were systematically studied. The results indicated that the self-promoted copolymerization between epoxy and amino phthalonitrile resins can be adjusted by controlling the content of phthalonitrile, and their composite laminates possess excellent thermal and mechanical properties.APN was prepared in our lab. Epoxy resin  was kindly supplied by Shanghai Huayi resin Co., Ltd. . Poly (arylene ether nitrile) (PP-PEN) was synthesized according to the reference reported previously . Glass fAccording to the orthogonal test design method, APPEN prepolymers were fabricated. Weight of AG80, weight of APN, and prepolymerization time, with three levels for each factor, are shown in First, APPEN gel solution was fabricated for cloth dipping. Six layers of glass fiber were tailored and impregnated into the gel solution. When the fabrics were evenly impregnated, then the prepregs were placed in a blast oven and treated at 80 °C for 30 min to remove the solvent primarily. Prepregs were pretreated at 160 °C for 10 min to further homogenize the prepolymerization of APPEN. Finally, prepregs were added into the stainless steel mold with the pressure of 20 MPa at 180 °C for 1 h to obtain APPEN/GF composite laminates. The corresponding preparation process is presented in The curing behaviors of the prepolymers were investigated using differential scanning calorimetry (DSC) with a modulated DSC-Q100 . Heating rate was 10 °C/min and a constant nitrogen flow rate of 50 mL/min was set. The samples were tested from 50 °C to 300 °C. Gelation time  was tested by a gel time tester  from Shanhai Yijia Electric Co., Ltd. Structures of APPEN prepolymers, prepregs, and composites were characterized using Fourier transform infrared spectroscopy . A SANS series microcomputer-controlled electronic universal testing machine  was used to detected the mechanical properties of APPEN/GF composite laminates, and the mode used was three-point bending. Fracture surface morphology of APPEN/GF composites was detected with a scanning electron microscope  operated at 20 kV. Dynamic mechanical analysis (DMA) was performed using DMA-Q800  dynamic mechanical analyzer. The composite laminate samples were tailored to the dimensions of 40 mm * 10 mm * 1.5 mm and were heated from 50 °C to 300 °C at a heating rate of 5 °C/min. Thermogravimetric analysis (TGA) of the composite laminates was tested using TGA-Q50 . The samples were heated from 50 °C to 600 °C at a heating rate of 20 °C/min in a nitrogen atmosphere.In this work, a novel epoxy resin and an amino phthalonitrile co-polymerized for toughness with Poly (arylene ether nitrile) (PP-PEN) shown in  were fab1, K2, and K3 (Kb = ∑Xb), the average values (kb = Kb/3) and the range (R = kmax − kmin) of k values at each level were gained.The orthogonal calculation method was applied to reflect the gelation processes of APPEN prepolymers, shown in A2 > kA3 > kA1 is presented, indicating that the level 1 of factor A was most conducive to the polymerization of the prepolymers. That is, by decreasing the content of epoxy resin, the polymerization rate can be elevated. Similarly, it can be determined that level 1 of factor B is the outstanding level, as well as level 3 of factor C. So, considering the prepolymerization conditions, the optimal combination of prepolymerization time and proportion of each component that affected the gelation time of APPEN prepolymer was identified as A5P4T20. The theoretical analysis revealed the primary and secondary order of each factor. As is known, with an increase in the range, the influence degree of the corresponding factors increases. The order of the range was RB > RC > RA, so the content of APN was determined as the most key factor that affects the gelation time of APPEN prepolymer, followed by the prepolymerization time.In our experiment, the content of epoxy resin, amino phthalonitrile, and prepolymerization time were concretely analyzed. For the factor A (content of epoxy resin), kB3 > kB2 > kB1 and kC1 > kC2 > kC3, showing that increasing the content of APN and prepolymerization time were both conducive to promoting the reaction of APPEN prepolymers. However, considering the co-polymerization between epoxy rings and phthalonitrile without catalysis, the content of amino phthalonitrile was selected as a fourth factor. Then, to intuitively analyze the effects of phthalonitrile on the polymerization of epoxy resin, various prepolymers  were designed and the curing processes were discussed.To sum up, the content of APN and prepolymerization time demonstrated remarkable effects on the prepolymerization degree, while the effect of epoxy resin content was not obvious. At the same time, the effects of APN content (factor B) and prepolymerization time (factor C) on the gelation time of the APPEN prepolymers were kTo further evaluate the polymerization processes and processing conditions, the gelation time of the prepolymers treated at 160 °C, 180 °C, and 200 °C was compared and analyzed, as shown in In this work, DSC was applied to analyze the polymerization processes of APPEN prepolymers because DSC curve shapes and enthalpy changes can reveal possible curing behaviors and polymerization mechanisms. 2 (3342 cm−1) and -CN (2231 cm−1) appeared in all of the spectra but decreased in turn, indicating that along with the polymerization, corresponding groups were transformed. It was also obvious that characteristic peaks of triazine rings and phthalocyanine rings appeared at about 1488 cm−1 and 1005 cm−1, indicating the ring-forming polymerization of nitrile groups. Moreover, the peak of C-O-C appeared at 1085 cm−1, and for A5P7T20 composite, the peak became faint. This may be ascribed to the copolymerization between the epoxy ring and amino phthalonitrile, which would restrict the activity of the molecular and nitrile groups.Gelation time analysis and reaction process monitoring indicated that the amino and nitrile groups copolymerized with epoxy rings, and the ring-opening polymerization of epoxy rings was triggered by active amino structures. Therefore, the consumption of amino groups resulted in the sluggish ring-forming polymerization of nitrile groups. In our previous work, the copolymerization between epoxy and phthalonitrile containing benzoxazine was studied, and the results indicated that the introduction of epoxy significantly suppressed the ring opening of benzoxazine rings and ring-forming polymerization of phthalonitrile. Moreover, the active phenolic hydroxyl groups generated from the ring-opening reaction of oxazine rings promoted the polymerization of epoxy rings and formed copolymerized chain segment structures, which possessed outstanding thermal stability. To intuitively illustrate the characters of molecular structures, the FTIR spectra of the A5P4T20 prepolymer, A5P4T20 prepreg, and A5P4T20 composite are presented in In sum, considering the DSC curves and gelation analysis, the possible polymerization processes of APPEN prepolymers can be summarized in Mechanical properties of matrix-based composite laminates dominated the applications of the composites and were usually used to evaluate the molding processes and polymerization effects. Among them, flexural strength and modulus were the fundamental characteristics tested to evaluate the effects of components and prepolymerization time on the mechanical properties. As shown in To intuitively evaluate the interfacial binding between the matrix and fibers, the fracture morphology for the laminates was measured using SEM as shown in To further evaluate the influence of copolymerization between epoxy and amino phthalonitrile resins on the thermomechanical properties of their laminates, the DMA test was employed. Generally speaking, the relationship between the storage modulus and the tangent delta (Tan δ) as a function of temperature would directly indicate the properties of the composites. In δ plots was defined as gT of the composites. In gsT showed the highest values and the second Tan δ transition peaks were most obvious. The first gT1 increased from 175.85 °C (A7P4T60/GF) to 188.90 °C (A5P4T20/GF), and the gT2 possessed almost the same values (214.75 °C). This can be explained as follows: First, the ring-opening polymerization of epoxy rings in the system was promoted by amino groups and then the long backbone chains were formed. Due to the steric hinderance effects, the subsequent ring-forming polymerization of nitrile groups was restricted, showing a relatively low gT. Additionally, by increasing the content of amino phthalonitrile, the degree of copolymerization crosslinking was improved, and then higher gTs was gained. Additionally, it can be observed that by increasing the content of amino phthalonitrile, the peak intensity and peak width of tan δ plots decreased and became narrow. This may be ascribed to the fact that the motion of molecular chains was restricted due to the growth of molecular chains and corresponding steric hindrance, which would directly restrain the increase in polymerization degree. In summary, APPEN/GF composite laminates exhibited outstanding storage modulus and g,T which can facilitate the laminates playing an important role in high-performance mechanical material fields.Generally speaking, the peak temperature of tan T5%) and 10% (T10%), the residual carbon rate at 600 °C (cY), and the integral program decomposition temperature (IPDT)) are shown in IPDT was composed of Formula (1) and is shown in The thermal stability of APPEN/GF composites was evaluated using TGA analysis, as shown in A and K are the area ratios defined by the TGA analysis curve shown in fT is the final experimental temperature (590 °C), and iT is the initial experimental temperature (50 °C). The A and K were calculated by Formulas (2) and (3) [ and (3) ,39,40,415%T of almost all APPEN/GF composites was higher than 334 °C, T10% for all was higher than 360 °C, all cY were up to 50% at 600 °C, and IPDT was higher than 1482 °C, indicating that the cured products possessed outstanding thermal stability. By adjusting the content of epoxy resin and prolonging the prepolymerization time, T5% of A5P4T20/GF composites showed the highest value (348.62 °C), while cY of A7P4T60/GF composites presented the maximum (58.28%). This can be ascribed to the step polymerization between epoxy resin and amino phthalonitrile. As described above, the ring-opening polymerization of epoxy rings was preferentially catalyzed by amino groups and elementary copolymers were formed. Then, long chain stacking and the three-dimensional network structures of the composites were fabricated. During the polymerization processes, the relative content of epoxy resin and amino phthalonitrile would dominate the main molecular chain of the copolymers, which affected the initial decomposition temperature (T5%), and the prepolymerization time directly influenced the aggregation configuration of the main chains and aromatic heterocyclic structures, which dominated the char yield of the composites (cY). Moreover, all of the composite possessed high IPDT values, which also indicated that the epoxy resin and amino phthalonitrile matrix were well combined, and there was no volatilization or degradation at high temperatures, which would result in reduced thermal stability. In sum, the high IPDT and outstanding thermal stability once again proved the good copolymerization between epoxy resin and amino phthalonitrile, which generated a stable aromatic heterocyclic crosslinking network [From  network .gT > 185 °C, T5% > 334.89 °C, Yc, 600 °C > 50%, and IPDT > 1482 °C). In summary, convenient and effective modification methods for the improvement of epoxy matrix high-performance composites and their laminates were provided.In this work, copolymerization between epoxy resin and amino phthalonitrile matrix was designed, and the effect of relative contents and molding processes on the pre-polymerization and polymerization reaction behaviors were systematically analyzed. The results indicated that prepolymerization time and component content played different roles during the polymerization processes, and the component content of amino phthalonitrile exhibited the most significant influence on the polymerization of APPEN composites. The structures of cured APPEN composites mainly consisted of a phenolic epoxy backbone chain, isoindole, triazine ring, and phthalocyanine ring. Ascribed to the improved crosslinking density and polymerization degree of APPEN prepolymers, their glass-fiber-reinforced laminates presented enhanced flexural strength (712 MPa) and modulus (38 GPa). The investigation of fracture morphology indicated that APPEN/GF composites were toughened efficiently, and the interfacial binding between matrix and fiber was intensified by controlling the matrix constituent and introducing the thermoplastic PP-PEN. Additionally, A5P6T20/GF composites possessed outstanding thermomechanical properties and excellent thermal stability (


A wide array of host factors, in addition to the non-structural proteins, leads to effective CHIKV infection , 2. HoweAfter 24 hpt, cells were harvested and subjected to Western blot analysis. The results showed a decrease of 45% and 64.6% in the levels of Catenin-α-1 using 30 and 60 pm concentrations of siRNA, respectively, compared to the scramble siRNA control . Subsequin situ (A previous report suggested that mutation in Catenin-α-1 leads to familial exudative vitreoretinopathy, a severe retinal vascular disease . Anotherin situ . HoweverCell adhesion and the reorganization of the actin cytoskeleton were mediated by Catenin-α-1 in the case of cancer , and CHI