Curcumin (CUR) and resveratrol (RES) are classified as natural polyphenolic compounds that have been demonstrated to have anticancer activity against CRC and are associated with minimal side effects. By regulating select target genes involved in several key signaling pathways in CRC, in particular, the Wnt β-catenin signaling cascade, the course of CRC may be positively altered. In the current review, we focused on the therapeutic effects of CUR and RES in CRC as they pertain to modulation of the Wnt β-catenin signaling pathway.Colorectal cancer (CRC) is the third most common cause of cancer-related death worldwide in terms of both its rates of incidence and mortality. Due to serious side effects associated with conventional chemotherapeutic treatments, many natural products with fewer adverse side effects have been considered as potential treatment options. In fact, many natural products have widely been used in various phases of clinical trials for CRC, as well as in  There a studies , 6.Curcuma longa with proven efficacy to treat a number of diseases and exhibiting minimal side effects [β-catenin signaling pathway, which is associated with its anticancer effects in a variety of human CRC cell lines [Curcumin (CUR) is a natural product derived from the rhizome of  effects , 8. This effects –17. In all lines , 18.β-catenin signaling pathway, and inhibit cell proliferation and induce apoptosis in many solid tumors such as CRC [Resveratrol (RES) is also a polyphenolic compound and is extracted from many plants such as grapes, peanuts, and berries . RES hash as CRC , 26.β-catenin signaling pathway in tumorigenesis and the recurrence of CRC.Wnt signaling is the most common pathway involved in the regulation of the cell cycle, stemness, and cancer . Wnt sigβ-catenin signaling and noncanonical Wnt signaling [β-catenin signaling in CRC is discussed. The regulation of cellular processes induced by Wnt β-catenin signaling is associated with the presence, or absence, of Wnt ligands, which are known to be secreted glycoproteins [β-catenin destruction complex is derived from intracellular molecular signals including adenomatous polyposis coli (APC), Axin (a scaffolding protein controlling β-catenin stability), glycogen synthase kinase 3β (GSK3β), and casein kinase 1α (CK1α) [β-catenin destruction complex, phosphorylation and ubiquitination of β-catenin are mediated by GSK3β and β-transducin repeat-containing protein (β-TrCP), with eventual translocation of β-catenin into the cell nucleus and inhibition in the expression of target genes involved in regulating cellular processes [β-catenin pathway, canonical Wnt signaling is characterized by binding of Wnt proteins to Frizzled receptors (Fzd) and low-density lipoprotein receptor-related protein 5/6 (LRP5/6), phosphorylation of Disheveled (Dvl), inhibition of the β-catenin destruction complex, translocation of β-catenin into the nucleus, and β-catenin and T-cell factor/lymphocyte enhancer factor (TCF/LEF) induced overexpression of target genes such as cyclin D1 and c-Myc, respectively [The Wnt signaling pathway can generally be divided into canonical Wnt ignaling . In the proteins . In the α (CK1α) . Followirocesses . In contectively .β-catenin signaling pathway plays an important role in regulating the cell cycle, as well as the growth, proliferation, and differentiation of cells [β-catenin signaling induced by genetic/epigenetic disorders promotes proliferation and tumor growth of cancer cells, in particular, CRC cells [β-catenin signaling can be inhibited by antibodies [In a state of homeostasis, Wnt of cells . In contRC cells . The dystibodies , shRNA mtibodies , Wnt sigtibodies , and an tibodies .β-catenin transduction [β-catenin signaling components, such as APC and β-catenin proteins located at the crypt base of the colonic epithelium [β-catenin signaling is considered the initial pathway involved in the early stages of CRC, it also plays an important role in regulating gene expression and several cellular processes. For example, these cellular processes include invasion, proliferation, migration, differentiation, and apoptosis from the time of initiation to the time of recurrence of CRC [β-catenin mutation-induced overactivation of Wnt β-catenin signaling also occurs in the early stages of CRC [β-catenin, translocation of the β-catenin into the nucleus, and overexpression of genes associated with the progression of CRC [β-catenin cascade involved in CRC, which include c-Myc [β-catenin signaling in colonic epithelial cells and subsequent development and progression of CRC. Many of the biological and pharmacological factors normally thought of as Wnt antagonists, can affect disturbed regulation of Wnt β-catenin signaling, and can be considered as potential therapeutic agents for various types of colon cancer.Epithelial cells of the colon and small intestine normally comprise the functional unit termed the crypt, which includes the crypt base, transit-amplifying cells, and terminally differentiated cells . The crysduction . In contithelium . Althouge of CRC . On the s of CRC . Along tn of CRC . An incrde c-Myc , Cyclin de c-Myc , matrix de c-Myc , and Musde c-Myc , resultsβ-catenin signaling are CUR and RES. It has been shown that these natural compounds such as micronized resveratrol  [β-catenin signaling associated with the progression of CRC. The characteristics of various natural compounds, especially CUR and RES , as they pertain to modulation of the Wnt β-catenin signaling at different stages of CRC, are summarized in Due to the rate of recurrence of cancer in patients with CRC, therapeutic treatment methods such as biotherapy, radiotherapy, surgery, and curative antibodies in combination with conventional chemotherapy are used . Regardi0920803) , resvera0920803) , geniste0920803) , 5-fluor0920803) , and iri0920803) , can effβ-catenin signaling pathway, which is activated by many tumorigenic factors such as azoxymethane (AOM), dextran sodium sulfate (DSS), and dibenzazepine (DBZ) as determined in various in vitro and in vivo studies [Based on the results of previous studies, curcumin inhibits aberrant activation of several signaling pathways including the Wnt  studies , 60. Reg studies , apoptos studies , cell vi studies , cell cy studies , invasiv studies , and met studies  of varioβ (IL-1β), IL-6, cyclooxygenase-2 (COX-2), β-catenin, and Axin-2, which was mediated by the inactivation of the Wnt β-catenin signaling pathway [β-catenin signaling pathway. Specifically, it was suggested that the mechanism of inhibition of cell proliferation was Wnt β-catenin signaling-mediated overexpression of β-catenin, TCF4, microRNA-21 (miR-21), and miR-130a, in conjunction with downregulation of a negative regulator of this pathway ) [β-catenin signaling including paternally expressed gene 10 (PEG10), SIAH1 gene, Wnt1, Wnt3a, and β-catenin [β-catenin signaling, this natural product may also potentially target miR-491/PEG10 and regulate cell proliferation during CRC recurrence. Accordingly, Li et al. reported that CUR reduced cell proliferation and stimulated apoptosis of HCT116 cell lines by upregulating miR-491 and downregulating PEG10 and Wnt β-catenin signaling [Using a mouse model in which CRC was induced via AOM and DSS, together with real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC), Hao et al. have shown that CUR significantly suppresses tumor cell proliferation of colorectal tissue through the downregulation of interleukin-1 pathway , 67. In  (Nkd2)) . One of -catenin , 70. Sinignaling .β-catenin signaling [β-catenin-induced aberrant cell proliferation of CRC cells. This study employed transfection of Wnt3a-conditioned medium (CM) treated HEK293 reporter (SEAP) cell lines into plasmid expressing hFz-1. Based on the activity of firefly luciferase (FL), it was demonstrated that CUR and its analogs inhibit cell proliferation of several CRC cell lines  by decreasing FL and SEAP activity induced by Wnt3a-CM, reducing the expression and degradation of cytoplasmic β-catenin, and downregulating a positive regulator of this signaling pathway called p-300 activator [β-catenin, Wnt1, and GSK3β, which suggests that dietary consumption of THC may potentially decrease the number of aberrant crypt fuci (ACF) [In addition to CUR, its chemical derivatives, which include demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC), and tetrahydrocurcumin (THC), are also able to inhibit cell proliferation of various cancer cell lines via inactivation of Wnt ignaling , 73. In ctivator . In contctivator , 76. Aloci (ACF) .β-catenin signaling-mediated loss in the expression of tumor suppressor genes, including ten-eleven translocation1 (TET1) and NKD2, as well as an upregulation of β-catenin, TCF4, and Axin [In addition to the antitumor effects of CUR in CRC, this natural compound can effectively modulate drug resistance of some chemotherapy drugs ) during the development of the epithelial-mesenchymal transition (EMT) . One of and Axin . Importa50 of 2.9 and 9.8 μM, respectively [β-catenin signaling-mediated colonic cell proliferation by decreasing the expression of colonic cytokines such as IL-6, IL-1β, and tumor necrosis factor-α (TNF-α), which are overexpressed in individuals with a high degree of adiposity secondary to high-fat diets. This suggests that overactivation of the Wnt β-catenin signaling pathway is accompanied by an increased number of inflammatory cytokines during tumorigenesis of colonic epithelial cells [Since CUR can potentially strengthen the inhibitory effects of 5-FU during EMT, it is possible that CUR may also synergistically augment the antitumor effects of the natural compound quercetin during EMT and thereby suppress the proliferation of CRC cells. Specifically, a previous study reported that the combination of CUR and quercetin synergistically inhibited the proliferation of HCT116 cells in a dose-dependent manner with an ICectively . Just asectively , 82. Regal cells .18)-g-polysuccinimide  and PEG/Hyd-CUR/C18-g-PSI (NFA-Cur), were synthesized to improve the antitumor efficacy of CUR via Wnt β-catenin signaling-mediated cytoplasmic expression levels of β-catenin [β-catenin more effectively than NFA-Cur. Accordingly, these authors proposed that the mechanism responsible for the suppression of cell proliferation by both micellar formulations of CUR was due to a reduction in the expression of β-catenin in the cell cytoplasm and nucleus. They concluded that the anticancer effects of FA-Cur micelles were a promising candidate for colon cancer via the inhibition of the Wnt β-catenin signaling pathway [Biopolymers, such as micelles, have long been investigated to transport various drugs for site-specific drug therapy in an effort to improve drug targeting and efficacy, as well as minimize any side effects , 85. In -catenin . The fin pathway .β-catenin nuclear translocation, Wnt3a, c-Myc, survivin, and cyclin D1. Additionally, when tested on SW620 human colonic adenocarcinoma cells, CUR exposure enhanced the expression of a negative regulator ) in the CDX2/Wnt β-catenin signaling pathway [(+/+)), HCT116(p53(−/−)), and HCT116(p21(−/−)) cell lines. This was suggested to occur due to an increase in caspase-3 expression-induced degradation of β-catenin and downregulation of c-Myc [In contrast to the antiproliferative activity of CUR in regulating the proliferation of CRC cells, it has been reported that CUR also increases apoptosis and reduces cell viability. It has been suggested that CUR mediates increased apoptosis and reduced cell viability by decreasing the expression levels of  pathway . Anotherof c-Myc . In addiof c-Myc , 90. Addof c-Myc .ββ-catenin, and cyclin D1, which is mediated by inactivation of the Wnt ββ-catenin cascade. This would suggest that si-MALAT1 can potentially increase the sensitivity of SW480 cells to CUR [In a study by Zhang et al. curcumin was evaluated for its potential to inhibit tumor EMT through the Wnt signaling pathway in colon cancer cells. Naked cuticle homolog 2 (Nkd2) small-interfering RNA (siRNA) and chemokine receptor 4 (CXCR4) expression plasmid were synthesized and transfected into curcumin-treated SW620 colorectal cancer cell lines, and the NKD2 and CXCR4 expression levels were determined. Their findings showed that curcumin significantly inhibited the proliferation of colorectal cancer cells and upregulated the expression of NKD2 in the cells, which resulted in the downregulation of key markers in Wnt signaling. Moreover, the progression of EMT was inhibited due to an overexpression of E-cadherin and downregulation of vimentin, as well as inhibition in tumor metastasis due to a significant downregulation in the expression of CXCR4 . In addis to CUR .ββ-catenin pathway are essential [β-catenin signaling, which include target genes, β-catenin, and tumor suppressors in an effort to suppress EMT-induced metastasis and invasion of CRC cells [β-catenin using immunofluorescence. Incubation of the cells with varying concentrations of CUR significantly downregulated the expression levels of DNA methytransferase1 (DNMT1) and the methylation levels of the CDX2 promoter in a concentration-dependent manner. In addition, the nuclear translocation levels of β-catenin were also reduced in a concentration-dependent manner. These authors concluded that CUR negatively regulated transcription factors promoting EMT in CRC cells by decreasing CDX2 promoter DNA methylation and consequently suppressing the CDX2/Wnt3a/β-catenin signaling pathway [One of the most effective pathological processes for increasing the malignant nature of CRC is EMT, which plays a crucial role in promoting metastasis and invasion of human CRC cells . To enhassential , 96. CurRC cells . In one  pathway .Citrobacter rodentium infection and were shown to promote cell survival and mucosal regeneration by restoring Wnt β-catenin signaling. These investigators used a mouse model wherein a C. rodentium infection induced transmissible murine colonic hyperplasia (TMCH) and various degrees of inflammation. Chronic administration of dibenzazepine (DBZ) for 10 days was utilized to block both Notch and Wnt signaling, disrupt the intestinal barrier, and induce colitis. Dietary curcumin (4%) restored signaling via Notch and Wnt/β-catenin pathways, which promoted crypt regeneration, replenishment of the mucus layer, and thus, an amelioration of the C. rodentium- and DBZ-induced colitis. Therefore, CUR inhibited TMCH and colitis induced with DBZ administration and C. rodentium infection in mice [In contrast with these results, the anti-inflammatory effects of CUR were evaluated by Ahmed et al. in an animal model of colitis induced by  in mice   and one normal mucosa-derived (NCM460) cell line. RES had no effect on cell proliferation at ≤40 μM (HT29 and NCM460) and ˂20 μM (RKO), although, at concentrations as low as 10 μM, RES significantly decreased the amount and proportion of β-catenin in the nucleus of RKO and reduced the expression of lgs and pygol  in all cell lines. These authors concluded that at low concentrations in the absence of effects on cell proliferation, RES significantly inhibited Wnt signaling in colon-derived cancer cells, which appeared to be due in part to intracellular β-catenin localization [β-catenin signaling pathways, to suppress cell proliferation of CRC cells [β-catenin are all decreased by RES in a concentration-dependent manner. Thus, these authors concluded that the antiproliferative effects of RES in human colon cancer cells may be mediated by regulating separately the PTEN/PI3K/Akt and Wnt β-catenin signaling [Resveratrol is known to inhibit cell proliferation and induce apoptosis in cancer cell lines at concentrations above 50 lization . FurtherRC cells . In factignaling .β-catenin/T-cell factor (TCF) complex in the β-catenin signaling pathway. Using co-immunoprecipitation, together with in vitro binding assays, Chen et al. demonstrated that RES was capable of disrupting the binding between β-catenin and TCF4, which contributes to a decrease in Wnt signaling and Wnt β-catenin-mediated aberrant cell proliferation. Based on these findings, it was shown that RES is capable of identifying, and ultimately destroying, the β-catenin-TCF4 complex to inhibit the growth of P19 and COS-7 CRC cell lines mediated by inactivation of Wnt β-catenin. This process was suggested to be due to downregulation in the expression of Wnt target genes such as cyclin D1, Axin2, ET-1, and c-Myc without altering the expression levels of cytoplasmic and nuclear β-catenin [β-catenin pathway-mediated cell proliferation, it was demonstrated using Wnt signaling-specific microarray analysis and RT-qPCR that low doses of RES-containing freeze-dried grape powder (GP) did not significantly inhibit the Wnt pathway in colon cancer but had significant activity in inhibiting Wnt target gene expression in normal colonic mucosa obtained from eight patients. However, the GP, which contains low dosages of RES in combination with other bioactive components, at 80 g/day can inhibit the Wnt pathway in vivo, and this effect appeared to be confined to normal colonic mucosa [Resveratrol has also been evaluated to determine whether it can target the -catenin . In contc mucosa .β-catenin [β-catenin signaling that mediates CRC cell growth [2 (PGE2) is highly expressed in CRC cells during tumorigenesis and is also considered a target for RES to effectuate inhibition of Wnt β-catenin signaling-mediated cell proliferation [β-catenin, including the phosphorylation of GSK3β, the expression levels of cytoplasmic and nuclear β-catenin, and the expression levels of c-Myc and cyclin D1 [Although RES's antiproliferative activity does not influence cell proliferation during CRC progression, it has been proven that RES can extensively target several mediators in CRC cells that are overexpressed ) and thereby suppress cell proliferation mediated by aberrant activation of Wnt -catenin . One of -catenin . In viewl growth . Similarferation . Indeed,yclin D1 .αvβ3-induced activation of extracellular signal-regulated kinase-1 and kinase-2 (ERK1/2) and stimulation of COX-2 accumulation. Accordingly, apoptosis of various human cancer cells is ultimately induced via upregulation of target genes  involved in apoptosis [β-catenin-induced overexpression of β-catenin and high mobility group protein A2 (HMGA2), as well as increasing both the nuclear accumulation of COX-2 and the expression levels of p53 [β-catenin signaling disorder, such as IGF-1. This leads to suppression of cell proliferation and, ultimately, induces apoptosis of CRC cells. Along these lines, findings from a different study showed that following the addition of IGF-1 to HT-29 and SW480 cells, RES (100–150 μM) exhibited antiproliferative properties in the HT-29 cells even after IGF-1 exposure by arresting the G0/G1-S phase of cell cycle progression via p27 stimulation and cyclin D1 suppression [β-catenin signaling , that enhance the inhibitory effects of RES and induce RES-stimulated apoptosis [β-catenin [According to the findings of several recent studies, the antiproliferative and apoptotic effects of RES are mechanistically dependent on RES-bonded integrin poptosis , 107. Ths of p53 . In addipression . These apression . There apoptosis . In a st-catenin   underlying their effects to regulate both downstream and/or upstream mediators in signaling pathways associated with CRC progression and recurrence is (are) more clearly elucidated.In this review, we have summarized recent findings on the antitumor effects of CUR and RES in the pathogenesis of CRC as it pertains to the Wnt  pathway . CUR and


Aging is associated with cardiac myocyte loss, sarcopenia, and cardiac dysfunction. Adult cardiac myocytes are postmitotic cells with an insufficient proliferative capacity to compensate for myocyte loss. The canonical WNT (cWNT) pathway is involved in the regulation of cell cycle reentry in various cell types. The effects of the cWNT pathway on the expression of genes involved in cell cycle reentry in the postmitotic cardiac myocytes are unknown.The aim of the study was to identify genes whose expression is regulated by the β-catenin, the indispensable component to the cWNT signaling, in the postmitotic myocytes.Myh6-Mcm) mice were used to delete the floxed exon 3 or exons 8 to 13 of the Ctnnb1 gene to induce gain-of-function (GoF) or loss-of-function (LoF) the β-catenin, respectively. Deletion of exon 3 leads to the expression of a stable β-catenin. In contrast, deletion of exons 8–13 leads to the expression of transcriptionally inactive truncated β-catenin, which is typically degraded. GoF or LoF of the β-catenin was verified by reverse transcription-polymerase chain reaction (RT-PCR), immunoblotting, and immunofluorescence. Myocyte transcripts were analyzed by RNA-Sequencing (RNA-Seq) at 4 weeks of age. The GoF of β-catenin was associated with differential expression of ~1700 genes, whereas its LoF altered expression of ~400 genes. The differentially expressed genes in the GoF myocytes were enriched in pathways regulating the cell cycle, including karyokinesis and cytokinesis, whereas the LoF was associated with increased expression of genes involved in mitochondrial oxidative phosphorylation. These findings were validated by RT-PCR in independent samples. Short-term GoF nor LoF of β-catenin did not affect the number of cardiac myocytes, cardiac function, myocardial fibrosis, myocardial apoptosis, or adipogenesis at 4 weeks of age.Cardiac myocyte-specific tamoxifen-inducible MerCreMer (Activation of the β-catenin of the cWNT pathway in postmitotic myocytes leads to cell cycle reentry and expression of genes involved in cytokinesis without leading to an increase in the number of myocytes. In contrast, suppression of the β-catenin modestly increases the expression of genes involved in oxidative phosphorylation. The findings provide insights into the role of β-catenin of the cWNT pathway in the regulation of cell cycle reentry and oxidative phosphorylation in the postmitotic cardiac myocytes. Likewise, myocyte loss is the core component of various myocardial pathological conditions, including myocardial infarction and heart failure, which are major causes of mortality and morbidity in the elderly population. To compensate for the loss, the cardiac myocytes need to replicate. However, cardiac myocytes progressively lose their proliferative capacity and become postmitotic with a renewal rate of < 1% per year. Consequently, there is considerable interest in forcing the postmitotic myocytes to re-enter the cell cycle and proliferate.Aging is associated with a drop in the number of cardiac myocytes, sarcopenia, and mitochondrial dysfunction among others, collectively, leading to impaired cardiac function . The WNT molecules are secreted glycoproteins, whose secretion requires palmitoylation by the porcupine enzyme. Inhibition of porcupine effectively suppresses the WNT pathway and has been used as a potential therapy in cardiomyopathies. The binding of the WNT molecule to its co-receptors induces the formation of the WNT signaling complex. The complex protects the β-catenin from degradation by the destruction complex, enabling its nuclear localization. In the nucleus, the β-catenin binds to the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors and serves as the co-transcriptional regulators of the expression of the cWNT target genes. In the absence of the WNT ligands, the destruction complex, comprised of axis inhibition (AXIN), glycogen synthase kinase 3β (GSK3β), casein kinase 1α(CK1α), adenomatous polyposis coli (APC), target the β-catenin for sequential phosphorylation, initially by CK1α at Ser 45 and subsequently by GSK3β at Thr41, Ser37, and Ser 33 at the N terminal domain. The phosphorylated β-catenin is recognized by the β-transducin repeats-containing protein/Skp-Cullin1-F-Box complex and is subjected to degradation by the ubiquitin-proteasome system (UPS).The WNT/β-catenin pathway, known as the canonical WNT (cWNT) pathway, is an ancient evolutionarily conserved pathway involved in numerous biological processes, including embryogenesis, stem cell fate, cell proliferation, differentiation, cancer, and senescence. The pathway also regulates the responses of the heart to injury, including myocardial fibrosis and cardiac remodeling post-myocardial infarction and pressure overload-induced cardiac hypertrophy,7. Furthermore, the cWNT pathway is implicated in the pathogenesis of fibro-adipogenesis in arrhythmogenic right ventricular cardiomyopathy, a hereditary disease caused by mutations in genes encoding desmosome proteins,8–10. Desmosomes are members of the intercalated discs and are responsible for myocardial mechanical integrity, mechano-sensing, and mechano-transduction, including regulation of the Hippo and the cWNT pathways–13. The β-catenin is a major constituent of the intercalated disc proteins and has functions independent of the cWNT pathway.In the heart, the cWNT pathway is best known to regulate cardiac development and proliferation of myocytes during the embryonic period). Despite the intense interest in the role of the cWNT pathway in myocyte proliferation, differentiation, maturation, aging, and various cardiovascular pathology, the transcriptional signature of this major pathway in mature cardiac myocytes has remained unknown. The purpose of the present study was to identify genes whose expressions are regulated by the cWNT/β-catenin pathway in postnatal quiescent cardiac myocytes using the genetic gain-of-function (GoF) and loss-of-function (LoF) approaches followed by sequencing of the cardiac myocyte transcripts.Several studies have targeted the cWNT pathway for beneficial gains in various cardiovascular pathological states, including cardiomyopathies , stabilizes the β-catenin protein, and activates the cWNT/β-catenin pathway (GoF). In contrast, deletion of the transactivation motif (amino acids 361–692), encoded by exons 8 to 13 in the Ctnnb1 gene, eliminates transcriptional activity of the β-catenin, typically resulting in the degradation of the truncated protein and leading to LoF of the cWNT/β-catenin pathway [Ctnnb1F(ex3)] (a gift from Dr. Takedo) and LoF  mouse models has been established–22. To suppress or activate the cWNT/β-catenin in cardiac myocytes, Myh6-Mcm and Ctnnb1F/F(ex8–13) or Myh6-Mcm and Ctnnb1F/F(ex3) mice were crossed, and the offspring carrying the desired genotypes, designated as Myh6-Mcm:Ctnnb1LoF and Myh6-Mcm:Ctnnb1GoF, respectively, were treated with subcutaneous injection of tamoxifen (30 mg/kg/day) for 5 consecutive days starting from postnatal day 14 to induce deletion of the Ctnnb1 gene specifically in cardiac myocytes  pathway . Fidelitmyocytes . ,23. The list of oligonucleotide primers used for the genotyping is provided in Genotyping was performed by PCR using genomic DNA isolated from the mouse tails, as described–25. In brief, mice were anesthetized using pentobarbital, the heart was excised, cannulated retrogradely, and perfused at a flow rate of 4 mL/min with a digestion buffer containing type 2 collagenase at 2.4 mg/mL concentration (Worthington Cat# LS004176). Upon completion of the enzymatic digestion, the ventricles were freed from the large blood vessels and minced in a stop buffer containing 10% calf serum, 12.5 μM CaCl2, and 2 mM ATP. Then the cell suspension was filtered through a 100 μm cell strainer and centrifuged at 20 G to precipitate the cardiac myocytes. This was followed by the re-introduction of calcium by a stepwise increase in the molar concentration of CaCL2 from 100 μM to 400 μM and to 900 μM in the stop buffer. Upon re-introduction of the last CaCl2 concentration, cardiac myocytes were suspended either in a Qiazol reagent  for RNA extraction or in a protein extraction buffer for immunoblotting.Cardiac myocytes were isolated from 4-week-old mice in the experimental groups as published,26. In brief, total RNA was extracted from mouse ventricular cardiac myocytes using the miRNeasy Mini Kit . RNA extracts with an RNA Integrity Number value of > 8, determined using an Agilent Bioanalyzer RNA chip, were used to construct sequencing libraries. Strand-specific ribosomal RNA depleted sequencing libraries were generated using TruSeq stranded total RNA library preparation kit . The libraries were sequenced as 75bp paired-end reads on an Illumina HiSeq 4000 instrument.To identify genes that are differentially expressed upon suppression or activation of the β-catenin, cardiac myocyte transcripts were analyzed by RNA-Seq, as published. The GENCODE gene model was used to annotate the uniquely aligned read pairs (https://www.gencodegenes.org/mouse/). The differentially expressed genes (DEGs) were identified using the DESeq2 statistical package in R. Benjamini-Hochberg FDR-adjusted P-value of < 0.05 were considered significant. The R packages were used for the visualization of heatmaps and volcano plots (www.rstudio.com). The GOCHORD function in R was used to plot the Circos maps.The quality of RNA sequence reads was assessed by FASTQC, and the STAR program was used for alignment of the sequence reads to the mouse reference genome build mm10 and generation of gene read countshttp://software.broadinstitute.org/gsea/) was used to predict dysregulated biological pathways. Normalized Enrichment Score determined from the Molecular signature database (MSigDB) 3.0 was used to curate gene sets for the Hallmark canonical pathways. The DEGs were analyzed using the upstream regulator analysis function of the Ingenuity Pathway Analysis software  to predict their transcriptional regulators (TRs). IPA was used to identify the targets of the specific TRs. TRs showing a P-value of < 0.05 for overlap with the IPA target genes and a predicted Z score of < −2 or > 2 were considered dysregulated.Gene Set Enrichment .Kaplan Meier survival rates were constructed for the wild type (WT), Body and heart weight were measured at 4 weeks of age, and the ratios of the heart weight indexed to bodyweight were compared among the groups.,26. In brief, B-mode guided M mode images of the ventricles were obtained in mice lightly anesthetized with 1% isoflurane. Anterior wall thickness, posterior wall thickness (PWT), left ventricular end-systolic diameter (LVESD), and left ventricular end-diastolic diameter (LVEDD) were measured using the leading-edge method. The measurements were used to calculate the left ventricular fractional shortening and left ventricular mass. The latter was calculated using the Devereux formula, as LVM=0.8 × [1.04 × (ST + LVEDD + PWT)3 − (LVEDD)3] and indexed to body weight.Echocardiography was performed at 4 weeks of age using a Vevo 1100 ultrasound imaging system , as published,25. The rhythm was recorded using a Power Lab 4/30 data acquisition system, and the data were analyzed using Lab Chart7 software .Two-lead surface electrocardiogram was recorded for about an hour while keeping the mice under light anesthesia, as published,24. CVF was calculated using the Image J software in 8 high magnification microscopic fields (×40) per section, in 6 sections per heart, and a minimum of 5 mice per group.To detect and quantify myocardial fibrosis, thin myocardial sections were stained with picrosirius red, and the percent area stained, as a measure of collagen volume fraction (CVF), was calculated as published. Following overnight incubation, sections were treated with the secondary antibody conjugated to Alexa 594, and the nuclei were counterstained with 4’, 6 Diamidino-2-phenylindole dihydrochloride  at 1 μg/mL concentration. The stained sections were mounted using fluorescence mounting media  and imaged using a Zeiss Axioplan fluorescence microscope. The total number of PLIN1-stained adipocytes were counted in each section, in 6 sections per mouse, and at least 5 mice per genotype. The average number of cells stained positive for PLIN1 expression per myocardial section was determined per group and compared among the genotypes.To detect adipocytes in the heart, thin myocardial sections were incubated in 4% formaldehyde solution, subjected to antigen retrieval in a sodium citrate buffer (pH 6.0), and incubated with an antibody against Perilipin 1 (PLIN1), a marker of lipid-containing adipocytes , as published,24,30,31. In brief, thin myocardial sections (5 μm) were incubated with WGA (1 μg/mL) conjugated to Texas red  to mark the interstitium. Sections were co-stained with an anti PCM1 antibody and DAPI. The WGA and PCM1 co-stained images were analyzed by Image J software (https://imagej.net) to measure the interstitial area and to count the number of cardiac myocytes in each field, normalized to the nuclei stained positive for PCM1 expression. An average of 8 fields per section, 6 sections per heart, representing around 20,000 nuclei per heart was analyzed.To quantitate myocyte cross-sectional area (CSA), thin myocardial sections were stained with wheat germ agglutinin (WGA) to mark the interstitium, and an antibody against pericentriolar material protein 1 (PCM1), which marks the myocyte nuclei , as published,25. The nuclei stained positive for the TUNEL assay were identified in 8 high magnification fields (×40) per section, in 6 sections per heart, and 5 mice per group. About 20,000 nuclei per mouse heart were counted to determine the percentage of nuclei stained positive for TUNEL, and the mean values were compared among the groups.To detect apoptosis, the terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay was performed using In-Situ cell death detection Fluorescein kit , as published,26,32. In brief, to extract proteins, ventricular tissues or cardiac myocytes were homogenized and dissolved in a radioimmunoprecipitation assay buffer (Cat# 974821) containing 0.5% SDS along with protease and phosphatase inhibitors . The lysates were sonicated briefly using a Bioruptor Pico (Diagenode) and centrifuged at 13,000 rpm to precipitate the protein. Aliquots of 50 μg total protein were loaded onto a 10% SDS polyacrylamide gel, electrophoresed, and transferred onto a nitrocellulose membrane. The membranes were probed with antibodies against the target proteins. The corresponding secondary antibodies were used, and the signals were detected using the ECL western blotting detection kit  and were imaged using the LiCOR (Odyssey) imaging system. All antibodies used are listed in Immunoblotting was performed on cardiac myocytes and whole heart protein extracts, as publishedGapdh or Vcl transcript levels. Changes in the transcript levels were calculated using the ΔΔCT method and presented as fold changes relative to the values in the WT mice. The reverse transcription-polymerase chain reaction (RT-PCR) reactions were performed in 5 mice per group. The primers used in the study are listed in the To quantitate transcript levels of selected genes, total RNA was extracted from myocytes using a miRNeasy Mini Kit  and treated with DNAse I  as per the manufacturer’s instructions. Reverse transcription was performed with random primers in approximately 1 μg of total RNA using a high-capacity cDNA synthesis kit . Transcript levels of the selected genes were determined using SYBR green or TaqMan assays in duplicate reactions and normalized to the t-test and among multiple groups by one-way ANOVA followed by Bonferroni pairwise comparisons. Data that departed from the Gaussian distribution were analyzed by the Kruskal-Wallis test. The categorical data were analyzed by the Fisher exact or the Chi-Square test. Kaplan-Meier survival plots were constructed and compared using the Log-rank test. Statistical analyses were performed using Graph pad Prism 8 or STAT IC, 15.1.The data were analyzed by the Shapiro-Wilk normality test, and the normally distributed data were compared between two groups using the Myh6-Mcm:Ctnnb1LoF and Myh6-Mcm:Ctnnb1GoF mice from P14 to P19 [Ctnnb1 gene were also reduced in the Myh6-Mcm:Ctnnb1LoF myocytes, likely reflective of the nonsense-mediated decay of the truncated transcripts [Ctnnb1 gene were unchanged in the Myh6-Mcm:Ctnnb1GoF myocytes, indicating that the loss of exon 3 did not affect the Ctnnb1 transcript stability [The β-catenin was genetically activated or suppressed specifically in the postnatal cardiac myocytes upon daily administration of tamoxifen to 4 to P19 . Postnat4 to P19 . In addinscripts . In conttability .Ctnnb1 gene in the genome browser was significantly reduced in the Myh6-Mcm:Ctnnb1GoF group [Ctnnb1 gene, which was floxed, as well as the non-floxed exons were reduced in the Myh6-Mcm:Ctnnb1LoF myocytes, i.e., the read for all Ctnnb1 exons were lower, consistent with the nonsense-mediated decay of the truncated transcripts [In accord with the RT-PCR data, the number of reads in the RNA-Seq data that was mapped to exon 3 of the oF group . Likewisnscripts .Myh6-Mcm:Ctnnb1GoF mouse hearts, consistent with activation of the cWNT pathway [Myh6-MCM:Ctnnb1GoF mouse hearts. The level of the smaller β-catenin protein was elevated as the deletion removes the phosphorylation sites of GSK3β, which targets the protein for degradation by the UPS. A less intense band representing the full-length β-catenin was also detected, which reflects an incomplete Cre-mediated excision in the Myh6-Mcm:Ctnnb1GoF mouse heart [Ctnnb1 gene corresponding to removal of about 270 amino acids, was not detected in the Myh6-Mcm: Ctnnb1LoF mouse hearts, despite probing with two different antibodies [Myh6-Mcm:Ctnnb1LoF mouse hearts, likely because of incomplete Cre-mediated excision of the floxed exons 8 to 13 of the Ctnnb1 gene [Levels of the β-catenin (CTNNB1) protein were increased markedly in the  pathway  and D. Tse heart . In conttibodies  and D. Tnb1 gene  and D.Myh6-Mcm:Ctnnb1LoF and increased density and size of the β-catenin-stained areas in the myocardium in the Myh6-Mcm:Ctnnb1GoF mouse myocardial sections [Myh6-Mcm:Ctnnb1GoF mouse hearts [Immunofluorescence staining of thin myocardial sections showed reduced localization of β-catenin to IDs in the sections . To assee hearts  and G. We hearts  and G.. Therefore, to identify the transcriptomic signature of the cWNT/β-catenin pathway in cardiac myocytes, transcripts that are known to be affected by the tamoxifen-Cre system were removed before the analysis of the RNA-Seq dataset.To identify genes whose expression levels were altered upon postnatal activation or suppression of the cWNT pathway specifically in cardiac myocytes, ribosome-depleted RNA was isolated from cardiac myocytes and sequenced. Administration of tamoxifen and activation of Cre transiently affect transcript levels of ~350 genesMyh6-Mcm:Ctnnb1GoF forming the most distinct cluster [The average number of total reads (54.3 ± 4.9 million reads), uniquely mapped reads (41.6 ± 4.1 million reads), and percent uniquely mapped reads (76.0% ± 2.3%) indicated a high quality of RNA-sequencing and did not differ among the genotypes . Genotyp cluster . Likewis cluster .Ctnnb1 gene was associated with differential expression of 369 genes (238 upregulated and 131 downregulated), whereas GoF of the Ctnnb1 gene led to dysregulation of ~1700 genes (1050 upregulated and 641 downregulated), as compared to the WT myocytes [Ctnnb1 gene, respectively [LoF of the myocytes  and B. Cmyocytes . Heat mamyocytes –H. Compamyocytes . Likewisectively . IPA of ectively . In the ectively . Pertineectively . The DEGectively . Compariectively .Given the prominent role of DEGs and the predicted transcriptional regulators, such as E2F1 and FOXM1, in the regulation of cell cycle progression, the DEGs were annotated to different phases of the cell cycle to gain insights into the effects of activation of the cWNT/β-catenin pathway on cardiac myocyte proliferation. Transcript levels of over 60 genes involved in the regulation of the cell cycle were increased in the GoF as compared to the WT myocytes  and B. TMyh6-Mcm:Ctnnb1GoF mice compared to the WT myocytes, corroborating the RNA-Seq findings [Myh6-Mcm:Ctnnb1GoF mouse hearts as compared to the WT or Myh6-Mcm:Ctnnb1LoF genotypes [Myh6-Mcm, and Myh6-Mcm:Ctnnb1LoF genotypes [Gene set enrichment analysis showed enrichment of the E2F target genes, which agrees with the pathway analysis of the RNA-Seq data . To testfindings . To furtenotypes –H. Thereenotypes –H. To deenotypes  and J.Myh6-Mcm:Ctnnb1LoF myocytes, as compared to the WT or the Myh6-Mcm:Ctnnb1GoF myocytes [Myh6-Mcm:Ctnnb1LoF myocytes and, conversely, a modest decrease in the Myh6-Mcm:Ctnnb1GoF myocytes [Bioinformatics analysis of the RNA-Seq data suggested activation of OXPHOS and TCA cycle in the myocytes . To testmyocytes . The fin.Given the focus of the study on identifying the transcriptomic changes imparted by the cWNT/β-catenin pathway in the heart, the additional phenotypic data were analyzed at 4 weeks of age, coinciding with the timing of RNA-Sequencing. Long-term effects of activation or suppression of the β-catenin have been studied before and, therefore, were not pursuedMyh6-Mcm:Ctnnb1LoF and Myh6-Mcm:Ctnnb1GoF mice, injected with tamoxifen, were similar to the corresponding WT and Myh6-MCM mice; the latter also were similarly injected with tamoxifen up to 6 months of age .LoF and GoF of β-catenin had no significant effect on survival, as the survival rates in the Myh6-Mcm:Ctnnb1LoF and Myh6-Mcm:Ctnnb1GoF mice. Detailed echocardiographic data are presented in Effects of suppression or activation of the β-catenin on cardiac size and function were determined at 4 weeks of age by echocardiography. Activation or suppression of the cWNT in cardiac myocytes had no significant effect on the LVEDD, LVESD, or LVEF . HoweverMyh6-Mcm:Ctnnb1GoF mice, showed a modest increase in the HW/BW ratio compared to the WT or Myh6-Mcm mice, the latter also treated with tamoxifen [,30,31,34. The myocyte cross-sectional area was increased in the Myh6-Mcm:Ctnnb1GoF as compared to the Myh6-Mcm:Ctnnb1LoF mice but was not different from that in the WT or Myh6-Mcm mice [To determine whether activation or suppression of the β-catenin in quiescent cardiac myocytes affected cardiac mass, heart weight to body weight ratio HW/BW) was determined in the experimental groups. Only GoF of the β-catenin, i.e., the amoxifen  and B. TW was detamoxifen [24,30,31Mcm mice  and E.Myh7b, whose transcript levels were markedly increased in myocytes isolated from the Myh6-Mcm:Ctnnb1GoF mice [To further assess the effects of the GoF and LoF of β-catenin on cardiac size and function, transcript levels of selected markers were assessed by RT-PCR. There were modest but significant changes in the transcript levels of several markers except for the GoF mice .Myh6-MCM:Ctnnb1LoF and Myh6-MCM:Ctnnb1GoF mice [All mice exhibited normal sinus rhythm, sinus arrhythmias, sinus bradycardia, or sinus tachycardia during 2-lead surface electrocardiography recording for 1 h . Rare veGoF mice .Col1a1, Tgfb3, Lrp1, and Mmp2 [To determine the extent of myocardial fibrosis upon suppression or activation of the cWNT/β-catenin pathway in cardiac myocytes, myocardial CVF was determined in the experimental groups upon staining thin myocardial sections with picrosirius red . Neitherand Mmp2 .The number of apoptotic cells in the myocardium was determined upon staining of the thin myocardial sections with the TUNEL assay. Neither LoF nor GoF of the β-catenin affected the number of TUNEL stained myocardial cells  and B. L. Overall, at the young age of 4 weeks, the number of adipocytes in the heart were similar among the experimental groups as the LoF or GoF of the β-catenin had no discernible effect on the number of mature adipocytes in the heart at the young age of 4 weeks [To determine the effects of suppression or activation of the β-catenin on myocardial adipogenesis, the number of mature adipocytes in the myocardium was determined upon staining the myocardial sections with an antibody against PLIN1, which tags the triglyceride-rich adipocytes 4 weeks  and B. F 4 weeks .. Despite the interest in the role of the cWNT/β-catenin pathway in cardiovascular pathology, its effects on gene expression in quiescent cardiac myocytes have remained unknown. Through a set of GoF and LoF studies, we show that genetic activation or suppression of the β-catenin led to altered expression of over 1500 genes in cardiac myocytes. GoF of the β-catenin had the most remarkable effect on gene expression and strongly provoked cell cycle reentry of the quiescent cardiac myocytes through activation of the E2F pathway and others, but did not result in proliferation, as the number of myocytes remained unchanged. The LoF of the β-catenin affected a smaller number of genes and modestly activated the expression of genes involved in OXPHOS. The smaller number of the DEGs upon LoF, as opposed to the GoF in cardiac myocytes, is in accord with the relatively dormant activity of the cWNT/β-catenin pathway in quiescent cardiac myocytes. Collectively, the data emphasize the role of the cWNT/β-catenin pathway in cardiac myocyte cell cycle reentry, albeit without myocyte proliferation, and also suggest a potential role for this pathway in mitochondrial OXPHOS.The cWNT/β-catenin pathway is involved in cardiac myocyte proliferation during embryogenesis and in the pathogenesis of cardiovascular diseases, including cardiomyopathiesMyh6-Mcm:Ctnnb1LoF and Myh6-Mcm:Ctnnb1GoF mice and were corroborated using complementary methods.The fidelity of the genetic LoF and GoF mouse models was established across multiple platforms, including immunoblotting, RT-PCR, and RNA-Seq approaches. Genes regulated by the cWNT/β-catenin pathway were identified in the absence of cardiac dysfunction and altered myocardial architecture, and therefore, were likely primary effects of the cWNT/β-catenin and largely free of the confounding effects of cardiac dysfunction or fibrosis. In addition, the RNA-Seq experiments benefited from robust quality control and a high read number, enabling identification of a large number of DEGs upon activation or suppression of the β-catenin in cardiac myocytes. Furthermore, the phenotypic consequences of genetic activation or suppression of the β-catenin were concordant across cardiac phenotypes in the .Activation of the β-catenin upon deletion of the GSK3β phosphorylation sites provoked activation of the E2F1 and 3 transcription factors and reentry of the postnatal cardiac myocytes into the cell cycle. However, it was insufficient to induce the proliferation of quiescent cardiac myocytes. This is in contrast to some other cell types in which activation of the cWNT/β-catenin leads to proliferation. The mechanism(s) necessary to complete the cell cycle, including karyokinesis and cytokinesis of postnatal cardiac myocytes, were not explored in the present study. In contrast, suppression of the β-catenin upon genetic deletion of the transactivation domain of β-catenin led to a modest increase in the transcript levels of genes involved in OXPHOPS in cardiac myocytes, albeit the changes were modest and mostly not significant. It remains to be seen whether in pathological conditions such as heart failure whereby OXPHOS is suppressed, suppression of the cWNT/β-catenin pathway could attenuate or rescue the heart failure phenotype by upregulating mitochondrial OXPHOS. This notion is in agreement with the results of a recent study showing that pharmacological inhibition of the cWNT by targeting porcupine partially rescues cardiac dysfunction and fibrosis in a mouse model of arrhythmogenic cardiomyopathy,7,36. Nevertheless, the long-term effects of the transcriptional changes induced upon activation or suppression of the cWNT/β-catenin pathway merits further investigation. In addition, cell cycle progression in the postnatal cardiac myocytes upon activation of the cWNT pathway was not investigated in great detail, partly because the number of myocytes, the end product of cell cycle reentry, was unchanged. Furthermore, mitochondrial OXPHOS was analyzed only at the transcript levels and was not pursued further, given the modest nature of the changes. Finally, bioinformatics analysis of the transcripts predicted dysregulation of several biological pathways and regulators of gene expression, which should be considered provisional, hypothesis-generating, and exploratory, requiring validation through alternative methods.The study has several limitations. The study was a short-term study designed to identify the targets of the cWNT/β-catenin pathway in postnatal cardiac myocytes. Consequently, it was not designed to study the effects of the activation or suppression of the cWNT/β-catenin pathway on long-term cardiac structure and function. Previous studies have investigated the long-term effects of activation or suppression of the cWNT/β-catenin in the heartIn conclusion, the data indicate that genetic activation or suppression of the cWNT/β-catenin in postmitotic cardiac myocytes in mice leads to altered transcript levels of more than 1500 genes, particularly those involved in cell cycle regulation and mitochondrial OXPHOS. The findings upon activation of the β-catenin set the stage for delineating the mechanisms involved in myocyte proliferation. In contrast, the data suggesting activation of OXPHOS upon suppression of the β-catenin, if validated in pathological states, would suggest potential salutary effects of the suppression of the cWNT pathway in attenuating and rescuing the cardiovascular conditions whereby OXPHOS is suppressed, such as in heart failure.Supplementary Material


The Cyclin E1/Cdk2 pair promoting G1/S progression was also decreased after PRMT5 inhibition, as was the phosphorylation of retinoblastoma. In the SJL mouse relapsing-remitting model of MS, the highest PRMT5 expression in central nervous system-infiltrating cells corresponded to peak and relapse timepoints. PRMT5 expression also positively correlated with increasing CD4 Th cell composition, disease severity and Cyclin E1 expression. These data indicate that PRMT5 promotes G1/S cell cycle progression and suggest that this effect influences disease severity and/or progression in the animal model of MS. Modulating PRMT5 levels may be useful for controlling T cell expansion in T cell-mediated diseases including MS.Multiple Sclerosis (MS) is a debilitating central nervous system disorder associated with inflammatory T cells. Activation and expansion of inflammatory T cells is thought to be behind MS relapses and influence disease severity. Protein arginine N-methyltransferase 5 (PRMT5) is a T cell activation-induced enzyme that symmetrically dimethylates proteins and promotes T cell proliferation. However, the mechanism behind PRMT5-mediated control of T cell proliferation and whether PRMT5 contributes to diseases severity is unclear. Here, we evaluated the role of PRMT5 on cyclin/cdk pairs and cell cycle progression, as well as PRMT5’s link to disease severity in an animal model of relapsing-remitting MS. Treatment of T helper 1 (mTh1) cells with the selective PRMT5 inhibitor, HLCL65, arrested activation-induced T cell proliferation at the G1 stage of the cell cycle, suggesting PRMT5 promotes cell cycle progression in CD4 Multiple Sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) that often starts in young adulthood and currently has no cure. The majority of patients are diagnosed with relapsing-remitting MS (RRMS), characterized by episodes of disease activity and recovery. Heterogeneous symptoms include but are not limited to fatigue, vision problems, muscle weakness/paralysis, emotional changes/depression and speech/swallowing problems . It is uProtein arginine N-methyltransferases (PRMTs) are enzymes that catalyze the addition of methyl groups onto arginines on histones and other proteins. They play a role in many cell functions including cell signaling, proliferation and differentiation . One way+ T lymphocyte cell cycle progression and the cyclin/cdk pairs that control the cell cycle. In addition, we also study how PRMT5 is linked to disease severity in an animal model of relapsing-remitting MS. We find that PRMT5 promotes the CyclinE1/Cdk2 pair and G1/S progression and that PRMT5 and Cyclin E1 expression in CNS infiltrates is significantly linked to clinical EAE disease severity. Overall, these results show that PRMT5 promotes G1/S cell cycle progression and provide a link between PRMT5 and disease severity and/or progression in the animal model of MS.Here, we examine the role of PRMT5 in CD41-11 T cell receptor (TCR)-transgenic mice  and myelin basic protein (MBP) Acribed in ) were br139-151, CS Bio) in complete Freund’s adjuvant supplemented with heat-killed Mycobacterium tuberculosis for a final concentration of 4 mg/ml (both from Difco/Sigma)/PBS per site. From day 7 through the end of the experiment, mice were monitored daily for weight and EAE score. Mice were scored by Hooke Labs scoring system; briefly, 0 - no disease, 1 – limp tail, 2 – hind limb impairment, 3 – hind limb paralysis, 4 – complete hindlimb paralysis and partial front limb paralysis, 5 – death. This is a relapsing-remitting form of EAE. Mice were deemed in remission when their score dropped by at least 1.5 from peak. Mice were considered in relapse when their score rose by at least 1 from lowest score in remission. Mice were sacrificed at various stages of disease, including peak, remission and relapse. Sham mice anesthetized and injected with PBS were used as controls. Mice were euthanized by injection with 25 mg/ml ketamine and 4 mg/ml xylazine (150 ml/20 g mouse) and perfused with PBS. Brains and spinal cords were combined together (termed CNS throughout the paper) from representative mice and processed as described in (9-week SJL/J mice were subcutaneously injected at two sites on their backs with an emulsion of 0.1 mg of proteolipid peptide (PLPribed in  for flowAc1-11 stimulation (CS Bio) and irradiated splenocytes in the presence of rhIL-2 (Miltenyi) every 7-10 days. Flow cytometry and ELISA characterization of Th1 cells cytokine profile showed that they were 98% positive for the Th1 cytokine IFN-γ (Murine T helper 1 (Th1) cells were generated from MBP TCR-transgenic mice  as descrResting or activated T cells were collected at various timepoints and the cell pellets were frozen at -80°C until analysis. At that time, cells were lysed in RIPA buffer  containing protease and phosphatase inhibitors (Thermo Fisher Scientific). A BCA assay (Thermo Fisher Scientific) was run to determine protein concentrations and 5-20 µg of protein was run per sample on a 10% or 14% SDS-PAGE gel and transferred to PVDF or nitrocellulose membrane. Blots were incubated with Odyssey blocking buffer (Li-cor) or 1% non-fat milk in TBS with 0.05% Tween-20 for 30 min and then probed with primary antibodies for 3 h at room temperature or overnight at 4°C in blocking buffer or 1% non-fat milk in TBS with 0.05% Tween-20. Primary antibodies: PRMT5 , H4R3me2s , pRb , Rb , Cyclin E1 , Cdk2 , Cdk4 , Cyclin D1  and β-actin . Blots were then washed and incubated in secondary antibodies  at 1:20,000 each, 1h, RT. An Odyssey-CLx (Li-Cor) was used to image all blots and Image Studio software (Li-Cor) was used to quantify results.CNS infiltrating cell protein from individual mice or pooled from multiple mice (for conditions with low infiltrating CNS cell numbers) were used as samples for protein analyses. A minimum of ~400,000 cells per sample were solubilized for protein analyses. 5-15 ug of protein were loaded for analyses. Equal amounts of protein were loaded and analyzed in individual experiments and normalized to sham. Two to three experiments were combined  for 10 min at 4°C and then primary antibodies for 15 min at 4°C. Primary antibodies included: CD3 , CD4 . Samples were run on a FACSCalibur with DxP multicolor upgrades  and analyzed using FlowJo software .Isolated CNS  infiltrating cells were used directly 3H)-thymidine at 6, 30 or 56h post-plating and harvested at 24, 48 or 72h onto a Unifilter-96 well plate . MicroScint-20 scintillation fluid was added and plates were run on a TopCount (PerkinElmer).mTh1 cells were plated at 100,000-125,000 cells/well in triplicate on anti-CD3/-CD28 coated plates with either DMSO vehicle control, 5µM HLCL65 or 5µM EPZ015666. Cells were pulsed with 1µCi of tritiated (6) were plated on anti-CD3/CD28 coated 48-well plates for 48h in the presence of DMSO or 2.5µM HLCL65. Cells were incubated with 10µM BrdU for the last 4 h at 37°C . For controls, 3µM aphidocolin for G1/S arrest or 0.25µM paclitaxel for G2/M arrest were added 15 min prior to addition of BrdU. Cells were stained as described in manufacturer’s instructions. Briefly, cells were blocked 10 min at 4°C in FC block (Biolegend). Primary antibodies, CD4-e450  and CD44-FITC  were added and incubated 15 min at 4°C. Cells were washed, fixed with BD Fixation Solution for 20 min at 4°C, then washed with BD Perm Wash and stored overnight in FACS. The next day, they were washed in BD Perm Wash, incubated in BD CytoPerm Permeabilization Buffer Plus 10 min at 4°C, refixed with BD Fixation solution, 5 min at 4°C, incubated with DNase solution 1h at 37°C, washed and stained with BrdU 20 min at RT, washed and resuspended with 7-AAD to stain total DNA. Cells were then run on a FACSCanto flow cytometer (BD) and analyzed by FlowJo software.mTh1 cells , biotinylated (13-7021-85), purified (14-7022-85)]. ELISA was performed as previously described  and Spearman correlation was used for non-parametric data (scores vs. expression).via a different binding mechanism, namely substrate binding competition. While EPZ015666 also reduced proliferation at all timepoints examined, it did not modulate IL-2 expression and the anti-proliferative effect was more muted compared to HLCL65 (PRMT5 promotes proliferation in mouse and human T helper (Th) cells , 17, 20,via CD3/CD28, it progresses through several cell cycle phases, including Gap1 (G1), Synthesis (S), Gap2 (G2) and Mitosis (M)  model of MS. In this model, SJL mice immunized with PLP+ Th cells within those infiltrating cells  in Jurkat T cells and we have subsequently shown that PRMT5 inhibition or deletion suppresses IL-2 production in primary T cells. Therefore, PRMT5 may control T cell proliferation through control of IL-2 production, although direct effects on the cell cycle could additionally play a role. The T cell model used in this study were Th1 cells, which produce large amounts of IL-2 upon T cell stimulation. In contrast, IL-2 is generally undetectable in the supernatants of activated Th2 cells, which are still sensitive more resistant to proliferation inhibition with PRMT5 inhibitors. This suggests that control of IL-2 is one of the major mechanisms by which PRMT5 controls T cell proliferation. However, high dose PRMT5 inhibitors may affect proliferation independently of IL-2 as seen in Th2 cells. It would be interesting to evaluate whether similar PRMT5 inhibition cell cycle and cyclin changes as those observed in Th1 cells occur in Th2 cells, which produce much lower amounts of IL-2. Interestingly, effects of PRMT5 inhibition on IL-2 secretion are already observable at 8 hours, prior to substantial induction of PRMT5 in T cells. This suggests that the low basal levels of PRMT5 expression may result in the PRMT5 activity that results in IL-2 production. This raises the question of how T cells suppress these effects prior to T cell activation. We speculate this may occur PRMT5 methylates a number of histone, splicing, transcription factor and other protein targets, any of which could directly or indirectly contribute to Cyclin E1/Cdk2 expression and proliferation. In cancer cells, a number of cell cycle mediators have been shown to interact with, be directly methylated by or be regulated by PRMT5. For instance, PRMT5 overexpression has been shown to increase CDK4 levels  similar + T cells in the infiltrating population suggests that T cell PRMT5 contributes to disease severity during the course of the disease. However, the data here provided are correlative, rather than causal. We speculate that PRMT5+ T cells in the EAE CNS are proliferating, and the linked Cyclin E1 expression supports this idea. In addition, Cyclin E1 expression was the most highly correlated marker we evaluated to disease severity. Since Cyclin E1 mediates proliferation, it is tempting to speculate that T cell proliferation is an important driver of disease. Cyclin E1 has been linked to EAE in another study, which found that disease suppression by 3,39-Diindolylmethane treatment resulted in low expression of the gene encoding Cyclin E1  at The Ohio State University.MG-d-A formulated the hypothesis and designed the experiments. SA and WO performed the experiments and analyzed the data. SA and MG-d-A drafted the manuscript. MG-d-A revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by funds from the National Institutes of Health National Institute of Allergy and Infectious Diseases Grants R01AI121405 and 1R21AI127354 (both to MG-d-A), OSUCCC’s Drug Development Institute funds and The Ohio State University School of Health and Rehabilitation Sciences start-up funds (MG-d-A).MG-d-A has a PRMT5 inhibitor patent pending and is a PRMT5 inhibitor inventor on a licensing deal with Prelude Therapeutics.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rationale: Glucose oxidase (GOx) has gained tremendous research interest recently as a glucose-consuming enzyme for tumor starvation therapy, while its in vivo applications are strictly limited by rapid deactivation, as well as side effects of non-specific catalysis.Methods: To address these issues, here we report a protective nano-shell to encapsule GOx for localized melanoma therapy delivered by dissolving microneedles (MNs). Inspired by cell membrane that separates and protects cell organelles and components from outside environment while selectively ingesting nutrition sources, we designed polydopamine (PDA)-structured nano-shell to allow free transportation of glucose for catalytic reaction, while impede the penetration of GOx, proteinase, and other GOx-deactivating macromolecules across the shell membrane.Results: GOx was well protected in core layer with persistent catalytic activity for at least 6 d under various biological matrixes  and surviving different harsh conditions . Such long-acting nano-catalyst can be easily integrated into MNs as topical delivery carrier for effective glucose consumption in melanoma tissue, achieving significant tumor growth inhibition via starvation therapy with minimized side effects as compared to systemic administration.Conclusion: This work provides an elegant platform for in vivo delivery of GOx, and our cell-mimicking nano-system can also be applied for other enzyme-based therapeutics. For instance, GOx has been combined with other enzymes Metabolic pathways are important therapeutic targets for tumor therapy in vivo delivery, thus requiring frequent administration to achieve the desired efficacy However, GOx-based tumor therapy is still in its infancy, and there exist significant problems for its potential clinical applications. Since the substrate of glucose and oxygen are ubiquitous in body, non-specific GOx catalysis could inevitably cause unwanted side effects, such as glycopenia, tissue hypoxia, and reactive oxygen species (ROS) generation Since systemic administration of GOx-loading vehicles may catalytically cause glycopenia, several studies chose intratumoral injection 2+ ion in vivo. The MNs were applied in B16F10 tumor-bearing mice model, and robust tumor growth inhibition was achieved with excellent biosafety.Here, we designed and fabricated dissolving MNs to achieve highly efficient topical delivery of GOx for melanoma therapy Scheme . Such MN2+  in 600 s  into glucuronic acid (GA) to cut off the supply of energy source, accompanied by the generation of toxic Hin vivo digestion. To explore this protection effect, we challenged the GOx@PDA with various physiological conditions, including PBS buffer, 10% serum, and cell lysate from B16F10 melanoma cells. After different incubation periods, the GOx@PDA was collected and tested for relative activity by examining O2 consumption. Among the tested buffer, the cell lysate had the most significant influence on GOx@PDA activity, while it still remained 70% activity after 24 h incubation  to damage cells 2O2 could also damage cells 2O2 generation was monitored by using DCFH-DA probe that can emit green fluorescence in response of H2O2. The cells with PDA shell treatment did not show any fluorescent signal, while bright fluorescence was observed inside cells after GOx@PDA treatment  and polyvinylpyrrolidone (PVP) as matrixes owing to their excellent biocompatibility, mechanical property, and tailored cross-linking density 2 patch with center-to-center interval of 600 μm . Disodium hydrogen phosphate (Na2HPO4), and sodium dihydrogen phosphate (NaH2PO4) of reagent grade quality were obtained from Hong shun . 8-hydroxy-5-quinolinesulfonic acid (HQS) was from Sigma-Aldrich , and 4-(2-hydroxyethyl)- piperazine-1-ethanesulfonate (HEPES) was from Mandel Scientific . Silica gel, glucose and dimethyl sulfoxide (DMSO) were purchased from Sinopharm Chemical Reagent Co., Ltd . BCA protein quantification kit was obtained from Beijing Dingguo Changsheng Biotech Co., Ltd . Trypan Blue and Tris (hydroxymethyl) aminomethane (Tris) were purchased from Soleibao Technology Co., Ltd . Dulbecco's Modified Eagle Medium (DMEM), Roswell park memorial institute 1640 medium (RPMI-1640), 3--2,5-diphenyltetrazolium bromide (MTT), penicillin-streptomycin solution, and fetal bovine serum (FBS) were purchased from Gibco Life Technologies . Live/dead viability/cytotoxicity assay kit was obtained from Nanjing KeyGEN Biotech. Co., Ltd. . The 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was obtained from Macklin Co., Ltd . ATP assay kit was purchased from Beyotime Institute of Biotechnology . B16F10, 4T1, A549 and MDA-MB-231 cells were obtained from Xiangya cell center .Polydimethylsiloxane (PDMS) were purchased from Dow Corning Corp. . Hyaluronic acid , polyvinyl alcohol , polyvinyl pyrrolidone , folic Acid (FA), glucose Oxidase (GOx), dopamine (DA), protease K, and 3--2,5-diphenyl tetrazolium bromide (MTT), ZnCl2 (8 mM) was added under vigorous stirring, followed by sonication for 5 min. Then, 400 μL Tris  was added to adjust the pH to 7.6, and DA (5 mg/mL) was added for 48 h self-polymerization. NCPs/GOx@PDA was collected via centrifugation, and dialyzed by phosphate-buffered saline  for 1 h to etch the NCPs core. The resulting GOx@PDA was obtained by centrifugation, and dispersed in water for further use. Morphology of the nanoparticles was observed by transmission electron microscopy  at 200 kV. ζ-Potential, size, and size distribution of were measured with a dynamic laser scattering . UV-vis absorption measurement was performed on a UV-vis spectrophotometer . The loading amount of GOx was analyzed by bicinchoninic acid (BCA) protein assay (Beyotime).FA , GOx  and PVA  were dissolved in 0.5 mL ultrapure water, and 0.5 mL ZnCl2+ were mixed with 2 mM HQS , and the fluorescence was measured by plate reader  to build the standard curve for Zn2+ quantification. Likewise, the standard curve for FA was built by dissolving folic acid in HEPES buffer (pH 7.4 10 mM) with a series of concentrations, which were measured by using high performance liquid chromatography (HPLC). To extract Zn2+ and FA, the nanoparticles were treated with 20 mM GSH for 2 h, and the supernatants were collected by centrifugation  for subsequent quantification using the standard curves as described above.Various concentrations of Zn2+ release was assessed by dialysis. NCPs/GOx@PDA were dissolved in 10 mM phosphate buffer solutions (pH 7.4). At predetermined intervals, sample was withdrawn and centrifuged, and Zn2+ concentration in supernatant was detected by HQS.The ZnGOx@PDA (3.5 mg/mL) was mixed with glucose (2.5 mg/mL), and the real-time pH change and oxygen consumption was monitored by a pH meter  and a dissolved oxygen meter , respectively. To explore the protection effect, free GOx or GOx@PDA  was mixed with cell lysate, 10% serum or phosphate-buffered saline (pH 7.4). After different incubation time periods at 25 °C, the catalytic activity was measured by monitoring the oxygen consumption. To investigate the protection ability against proteases, free GOx or GOx@PDA  was treated with proteinase K (5 mg/mL) at 37 °C for 2 h under gentle shaking. Then, the activity was measured by monitoring pH variation and oxygen decrease. Likewise, the enzymatic activity of free GOx or GOx@PDA under harsh conditions was also measured by treating the samples with phosphoric acid solution (pH 4.0), phosphoric acid solution (pH 5.5 and 6.5), or ammonia solution (pH 10).The release of GOx was measured by a membraneless dissolution method. Typically, 200 μL GOx@PDA was mixed with 800 μL release medium under gentle shaken at 37 °C. Samples were taken at predetermined time points, and the concentration of GOx was analyzed to calculate cumulative drug release.2.B16F10 cells, A549 cells and MDA-MB-231 cells were incubated in DMEM medium containing 10% FBS and 1% antibiotics , while 4T1 cells were incubated in RMPI 1640 medium containing 10% FBS and 1% antibiotics . The cells were cultured at 37 °C in a humidified atmosphere containing 5% COThe cells were seeded into 96-well plates at a density of 5000 cells per well for 24 h culturing. Then, gradient concentrations of GOx@PDA were added for 48 h incubation, followed by adding MTT . After another 4 h incubation, the media were replaced with 100 μL of DMSO, and the absorbance was measured using a microplate reader . The cell viabilities (%) were calculated as follows: 100 × OD (samples) / OD (control), where OD (control) and OD (samples) represent the absorbance at 490 nm for the cells with different samples treatments and non-treatment control, respectively.4 cells/cm2. When the cell confluence reached about 70%, the cells were incubated with PBS (control), PDA shell (4 μg/mL), or GOx@PDA (4 μg/mL) for 24 h, respectively. Subsequently, the cells were collected and counted to ensure that each sample contained the same number of cells. The intracellular ATP content was measured by following the user manual of the ATP assay kit.B16F10 cells were seeded into a 6-well plate with an initial cell density of 2 × 104 cells per well. After 48 h, the cells were treated with PDA shell or GOx@PDA for 6 h. Then, 10 μM of DCFH-DA was added for 30 min incubation. The cells were then imaged by fluorescent microscopy.The B16F10 cells were seeded onto 24-well plates at density of 4 × 10B16F10 cells were seeded into the 35 mm × 12 mm CLSM cell culture dish . When the cell confluence reached about 70%, the original culture medium was replaced by a fresh one containing PBS (control), PDA shell (4 μg/mL), or GOx@PDA (4 μg/mL), followed by 48 h incubation. Then, the cells were stained with Calcein AM/Propidium Iodide (PI) for 15 min and analyzed using a confocal microscope .To prepare the blank MNs, PVP K30 (40%) and HA (15%) were poured into the PDMS mold under vacuum, and the sample was dried in a sealed desiccator overnight, followed by peeling off from the molds, and stored in a desiccator. To prepare drug-loaded MNs, GOx@PDA solution was first covered and filled into a microneedle mold under vacuum. Thereafter, redundant solution was removed to make sure each cavity was filled with the same volume of drug solution. Then, the solution was evaporated for a short period to concentrate GOx@PDA on MN tips before filling the PVP K30 and HA matrixes to form the base. Morphologies of MNs were observed by a scanning electron microscope . To determine the drug loading, MNs were dissolved in a certain volume of phosphate buffer, and GOx were quantified by measuring its catalytic activity.The mechanical strength was measured using a tensile load frame. The tensile force was continuously monitored as a stainless-steel plate compressing arrays of microneedles along the y-direction on a stress-strain gauge. The initial gauge was set at 2.00 mm between the MN tips and the stainless-steel plate, the speed of the top stainless steel plate movement towards the MN-array patch was 0.1 mm/s. The failure force of MNs was recorded as the needle began to buckle.To assess the skin insertion capability, MNs were applied onto the dorsal skin of a mouse for 5 min. Then, the mouse skin was retrieved and stained with 0.4% trypan blue solution for 1 h, followed by washing with water. Then the skin was observed under an AxioCam MRc5 microscope , and pictures were taken with a high-definition mobile phone (Huawei P30). The insertion ratio is equal to the number of dots on the skin surface divided by the number of needle tips on each microneedle. The depth of the microneedles inserted into the skin was measured on frozen tissue sections.2) with receiving cell containing 2.1 mL phosphate-buffered saline solution. After 0.5 h incubation , the receiving solution was collected to quantify the GOx@PDA penetration.The skin penetration of the MNs were evaluated by using Franz transdermal diffusion cell. Briefly, the MNs were applied to depilated back skin via thumb insertion for 5 min, and then the skin was transferred to Franz transdermal diffusion cell , the mice were randomized into four groups: (1) control; (2) blank MNs without GOx loading; (3) GOx/PDA MNs loading of physical mixture of GOx and PDA shell; (4) GOx@PDA MNs. Each MN contained 105 μg GOx. All the groups received treatments on day 0, 3, 6. Body weights and tumor volumes (0.5 × length × width2) of the mice were recorded every day. The mice were sacrificed on day 10, and the tumors as well as the main organs were harvested for subsequent analyses, including weighting, hematoxylin & eosin (H&E) staining, and Ki67 immunofluorescent staining.C57 (4 to 5 weeks old) were obtained from Changzhou Cavens Lab animal Co., Ltd. . The animals were maintained in a sterile environment and allowed free access to food and water. All animal experiments were approved by the Experimental Animal Ethics Committee of Central South University and carried out in accordance with the requirements of the National Act on the use of Experimental Animals (people's Republic of China). The mice were seeded with B16F10 cells at a density of 1 × 102 for 1 min).For photothermal imaging, the mice were pictured using an FLIR C2 camera at 0.5 h post-injection of GOx@PDA MNs after laser irradiation  control (no treatment); (2) intravenous injection of GOx@PDA NPs ; (3) topical administration of GOx@PDA MN (700 μg MNs). At pre-determined timepoints, 25 μL blood sample was taken from the tail vein, and the blood glucose concentration was measured by a blood glucose meter.All quantitative data were expressed as mean ± SD from at least triplicate measurements. Differences between two comparative groups were assessed using the Student's t-test, and the significance among multiple groups was examined by the one-way analysis of variance (ANOVA). Significance was measured at the following thresholds: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.2+-coordinated NCPs were employed as templet for GOx encapsulation and self-polymerization of PDA shell layer, and then NCPs nano-core was etched to improve the biocompatibility of the nanosystem. The resulting GOx@PDA can retain its catalytic activity for at least 6 d in presence of biological matrixes including serum and cell lysate, and resist various harsh conditions , all of which were benefited from the protective effect of PDA nano-capsule. In vitro, GOx@PDA showed excellent anti-tumor effect against various types of tumor cells via catalytic consumption of glucose to cut off the energy supply. For in vivo application, GOx@PDA was integrated into dissolving MNs, and the MNs were well-characterized in terms of micro-structure, drug loading, skin penetration, and biocompatibility. Upon transdermal administration, significant tumor growth inhibition was achieved without any noticeable side effects, while accompanied by angiogenesis in tumor tissue. Therefore, it shows the potential of combining GOx@PDA with antiangiogenic drugs for better therapeutic outcomes. This work addresses the key limitations of GOx for in vivo applications, and pointing out the future direction of GOx-based starvation tumor therapy for enhanced efficacy.In summary, we designed and fabricated a PDA-based nano-capsule to load GOx for long-lasting catalytic starvation therapy of melanoma via MNs-based transdermal delivery. FA/ZnSupplementary figures.Click here for additional data file.


Epoxy with low viscosity and good fluidity before curing has been widely applied in the packaging of electronic and electrical devices. Nevertheless, its low flexibility and toughness renders the requirement of property improvement before it can be widely acceptable in dynamic loading applications. This study investigates the possible use of 2-hydroxyethyl methacrylate (HEMA) toughening agent and nano-powders, such as alumina, silicon dioxide, and carbon black, to form epoxy composites for dynamic property improvement. Considering the different combinations of the nano-powders and HEMA toughener, the Taguchi method with an L9 orthogonal array was adopted for composition optimization. The dynamic storage modulus and loss tangent of the prepared specimen were measured by employing a dynamic mechanical analyzer. With polynomial regression, the curve-fitted relationships of the glass transition temperature and storage modulus with respect to the design factors were obtained. It was found that although the raise in the weight fraction of nano-powders was beneficial in increasing the rigidity of the epoxy composite, an optimal amount of HEMA toughener existed for its best damping improvement. Epoxy is one of widely used synthesized resins for general purposes and industrial applications. Due to its low viscosity and good fluidity before curing and excellent strength and stiffness after curing, epoxy resin is commonly thought of as a good candidate for packaging use in electrical and electronic applications. However, with the increase in degree of crosslinking among its molecular chains after the reaction with hardener agent finished, the cured epoxy often becomes too brittle to be applied in some circumstances which require material toughness, such as the environments involving dynamic loading or vibrations.2 nano-powder has 3D network molecular structure and forms a floccular ball-like particle, which is commonly used as a reinforcement in composite [2 nano-powder and glass fiber in a composite laminate has been reported to raise the fatigue life by 3–4 folds compared to its pristine epoxy resin counterpart [2 nano-powders. However, the glass transition temperature was lowered if the content of SiO2 nano-powder was greater than 5 wt.%.By adding some “reinforcements” into the epoxy resin to form composites, the mechanical properties of the resin can be tuned. Liu and coworkers  investig3AlC2 and Ti3C2Tx) to treat the acidified short graphite fibers and reported the 100% and 67% increases in tensile and flexural strengths of the epoxy composite, respectively. The SiO2 nano-powders also had good compatibility with the epoxy resin and its epoxy composite showed the improvements in tensile compressive strengths and fracture toughness [In addition to simply adding inorganic powder for reinforcement, the interface usually needs treatment to improve the compatibility between phases. Liu and coworkers  used MXeoughness .The other approach for toughening the epoxy resin is by modifying its structure with interpenetrating polymer networks (IPNs). This structural modification down to the molecular level with uniform interlacing of toughening phase and the crosslinked epoxy phase could promote the material compatibility. Around the 1980s, Sperling and coworkers  used difThe further modification on the microstructure of IPNs was proposed by Hsieh and Han  with theMimura and coworkers  reported2O3 and SiO2, to tune the thermal conductivity of the epoxy resin. However, considering the Al2O3 filler had higher specific gravity than SiO2, less volumetric fraction of the former could have similar weight fraction with the latter. The partial substitution of Al2O3 for the SiO2 would keep the epoxy resin lower in viscosity or higher in fluidity, which was beneficial in packaging. The other report by Khalil et al. [2O3 nano-powder within 2.0 wt.% in epoxy would improve the wetting behavior and tensile shear strength of the epoxy resin. Thus, the addition of nano-powders within 2.0 wt.% in the epoxy resin was adopted in this study.In the electronic packaging applications, the use of different materials to tune the mechanical property was a common practice. However, the collateral effect arisen from the mismatch in the thermal expansion of its constituents could worsen or even fail the packaging function . The othl et al.  illustra2O3, SiO2, and carbon black were chosen to be the reinforcements of the epoxy resin in addition to a HEMA toughener. The design of experiments by using the Taguchi method was adopted and the analysis of variables (ANOVA) was performed to find the optimal compositions for the dynamic property improvement. The prediction of the dynamic properties of the prepared epoxy composite was established for the design purpose in packaging.As reviewed from the previous studies, the incorporation of inorganic nano-powders in epoxy resin seems beneficial to its packaging applications. However, the underlined mechanisms have not been well investigated. Moreover, the reliability performance of epoxy composites with different compositions are susceptible to the service temperature and loading conditions. Therefore, by using the dynamic mechanical analyzer, the main focus of this study is to investigate the viscoelastic behavior and energy dissipation from the internal friction within its microstructure of the epoxy composite over a specified temperature range. Three nano-powders of AlIn this study, a simultaneous polymerization reaction was adopted to prepare the epoxy with IPNs. Three reactions were proceeded simultaneously in the crosslinking of epoxy, as presented in Secondly, benzoyl peroxide as a thermal initiator disintegrated into two free radicals upon being heated and are described in A dynamic mechanical analyzer (TA DMA 2980) was used in this study to measure the dynamic properties of the prepared specimens in different time, temperature and loading frequency. In the measurement, the specimen was installed in a specified configuration, such as three-point bending and cantilevered fixture, and was subjected to a vibrational stress or strain. The responses of the specimen in deformation or loading were recorded both in magnitude and phase. Accordingly, the storage modulus E′, loss modulus E″, and coefficient of loss tangent tanδ of the specimen can be calculated as follows.Storage modulus E′ is calculated as follows.The loss modulus E″ is calculated as follows.The coefficient of loss tangent, tanδ, is calculated as follows.In the above equations, ε and σ represent the amplitudes of the applied strain and the measured stress, respectively. The larger the peak of tan, the more viscous the material behaves. In other words, the specimen with larger tanδ has more damping in response to dynamic loading. Moreover, the temperature where the tanδ attains its peak magnitude denotes the corresponding glass transition temperature of the specimen.9(34) orthogonal table for this study. There were four design factors, i.e., the weight contents of A: nano-alumina powder; B: nano-silica powder; C: nano-carbon black powder; and D: HEMA toughener, respectively. Three levels for each factor were selected, which were 0, 1, and 2 wt.% and 0, 5, and 10 wt.% for the nano-powders and the toughener, respectively. Accordingly, the dynamic properties of the specimens were measured by using the DMA analyzer and the S/N ratios were calculated from the quality equation.The-higher-the-better (HB) characteristics were employed with decibels (db) as its unit, shown in the following.X and S are the mean and standard deviation for each set of measurements.Three specimens for each measurement were employed in this study in order to evaluate the variation of the results. Therefore, the coefficient of variance (C.V.) was calculated accordingly:i2 and ci1 are the coefficients for the quadratic and linear terms, respectively. The constant term for the relationship is summed up in c0. The object functions that will be evaluated later include the storage moduli.With the storage moduli as a substitute for the machining process factor, the regression fit of the object function could be preceded. For simplicity, the dependence of the object function on the factors was assumed to be quadratic and the coupling among the factors was neglected. These assumptions will be checked with the verification example presented later in this study. Thus, the regression function Y is written as follows:2 was adopted. The larger the R2 is, the more accurate the regression function becomes. The coefficient of determination was defined as the ratio of regression variance to total variance. It can be expressed as follows:T is the total sum of squares and SSR is the regression sum of squares. The magnitude of R2 lies between 0 and 1, i.e., 0 < R2 < 1. The closer the R2 approaches 1, the better the regression function represents the physical relationship.The validity of the regression function needs to be examined. Herein, a coefficient of determination R2/g, an average primary particle diameter of 14 nm, and a tapped density  of 60 g/L. For the other nano-powders, the aluminum oxide  had an average particle diameter of 50 nm and density 2.414 g/cm3. The nano carbon black powder was with a specific surface area (BET) of 90 m2/g, average particle diameter of 28 nm, and density of 1.719 g/cm3. Moreover, the toughening agent was 2-hydroxyethyl methacrylate (HEMA) with a mass density of 1.106 g/mL, which was an acrylate with polar substations that was purchased from TCI Co. Ltd. Benzoyl peroxide (BPO) was used as a thermal initiator. It should be mentioned that all the purchased materials were used as-received without further purification.A diglycidyl ether of bisphenol A type epoxy resin  was purchased from Chang-Chun Plastics Co. Ltd. . The epoxy resin was cured with the incorporation of an amine type epoxy curing agent . When the nano-powder used was a silicon dioxide (silica) powder, the surface of the fumed silica was chemically modified with a poly(dimethyl siloxane) coupling agent. The hydrophobic fumed silica had a specific surface area (BET) of 100 mFirstly, the epoxy resin and HEMA toughener with specified weight fraction were put in a container of a deaerator and thoroughly mixed. Subsequently, the nano-powders with specified weight fractions were added and agitated mechanically. The well mixed solution, which was mixed with an ultrasonic and centrifugal mixer, was obtained as the pre-mixture for next process. In our previous study , the pre9(34) orthogonal array of the Taguchi method. Four control factors were employed in the experiments: nano-alumina powder, nano-silica powder, nano-carbon black powder, and HEMA toughener. Three levels for each factor were selected, which were 0, 1, 2 wt.% and 0, 5, 10 wt.% for the nano-powders and the toughener, respectively.Dynamic mechanical analysis (DMA) was performed according to ASTM D4065-01 to determine storage modulus (E′), loss modulus , and glass transition temperature (Tg) of the epoxy composites. The tests were conducted in the dual cantilever beam mode with a vibration frequency of 1 Hz and a displacement amplitude of 10 μm in a DMA analyzer . The temperature was ramped from 30 to 140 °C at a rate of 2 °C/min. At least three specimens of each type were tested and the data were analyzed. The DMA testing purpose was to simulate the loading conditions of the epoxy composite used in the packaging and subjected to periodic vibration from the surroundings. g. As far as the dynamic property is concerned, the loss tangent denotes the damping coefficient of the material. Larger material damping is always required for controlling the vibration of structure near resonance.g’s at around 67 °C and 83 °C. The appearance of two loss tangent peaks is related to the HEMA toughener added in the epoxy composite, which will be discussed in more detail afterwards.Similar test results are shown in g moving to lower temperatures. Similar measurements for the rest of the six specimens were also performed and the Tg’s are summarized in As observed from g are specimens without adding the HEMA toughener. The specimens with the inclusion of HEMA toughener in the compositions revealed the lower Tg peak. In more detail, Specimens T2, T6, and T7, which all have a 5 wt.% of HEMA, had lower Tg’s at 67.42, 68.68, and 65.92 °C, respectively. Moreover, with further increases in HEMA content to 10 wt.%, Specimens T3, T4, and T8 lowered their corresponding Tg’s to 52.60, 55.93, and 54.05 °C, respectively. It is known that the glass transition temperature reflects the cross-linking density of its molecular chains inside the polymer material. Those specimens without HEMA inclusion in their compositions revealed only one Tg peak. The introduction of a thermoplastic HEMA into the microstructure somewhat hindered the cross-linking of epoxy during curing and the molecular chains of HEMA started to move locally at lower temperature. Minor or shoulder peaks may appear due to the phase separation between the epoxy resin and the toughening agent or inhomogeneous crosslinking density [g peak at higher temperature around 78.77~90.26 °C, which reflected the movement of the molecular chains in the microstructural regions unaffected by the HEMA. With more HEMA contents, the regions affected by the HEMA enlarged and, consequently, further lowered the Tg. Of course, the movement of the molecular chain was still influenced by other nano-powders included in the composite. Therefore, the variation in Tg was measured for specimens with other different compositions but with less significance. The lowering of the resistance for the molecular chains to move at lower temperatures created more flexibility to the material and, therefore, alleviated the brittleness. That is the mechanism for using toughener in epoxy resin to raise its toughness. Since the main purpose of this study is to investigate the effects of the nano-powders on the dynamic property of the epoxy composite, only the second Tg is referred to specifically afterwards. density . However2 had the most significant effect while Al2O3 had the least. It is worth mentioning that the amount of HEMA doping decreased the glass transition temperature drastically, which was mainly caused by the rotation and vibration of the organic molecules of HEMA toughener under the influence of heating. Moreover, the reaction from the added toughener in the epoxy also hindered its crosslinking and reduced the crosslinking density between the molecular chains. Therefore, the molecular chains had more space for moving and the Tg decreased accordingly. These nano-powders usually have higher stiffness than the epoxy matrix. The reinforcement demonstrates its effect with the raise in glass transition temperature. On the other hand, the HEMA toughener showed an opposite trend because of its interference with the cross-linking density, as discussed previously. Among the control factors, HEMA had the highest influence on the glass transition temperature followed by silica, alumina and carbon black, sequentially.With the obtained experimental data presented in In the above equation, 2O3 has the least effect. However, the HEMA toughener played a different role of lowering the storage modulus with increasing doping content. The lowering effect was more pronounced than its nano-powder counterparts in raising the storage modulus of the epoxy composite as the S/N ratio was twice larger in magnitude. As mentioned previously, the incorporation of thermoplastic HEMA molecules into the epoxy polymer chains impedes the cross-linking and reduces the local cross-linking density. Thus, the storage modulus reduced with higher HEMA contents. On the other hand, the nano-powders filled in the epoxy matrix act as the reinforcement phase in the composite. Moreover, the dispersion of hard inorganic nano-powders in the microstructure of epoxy also serves as the pin-point sites to increase the resistance of chain movement. Thus, deformation reduced and the stiffness of the composite increased. It should also be mentioned that the insertion of HEMA molecules in blocking the crosslinking of the epoxy network and the incorporation of nano-powders in the microstructure could interfere with crack propagation in the epoxy composite. Therefore, the toughness of the epoxy composite can be enhanced.The storage modulus of the material used in packaging denotes the material’s measure to withstand deformation due to dynamic loading. The higher the storage modulus, the better the resistance to dynamic deformation. Similar regression fit using SPSS software was conducted relative to the relationship between the storage modulus and the control factors. The storage moduli measured from the nine specimens were employed in the regression fit with the content wt.% of the control factors. The obtained equation is listed as follows.In order to check the accuracy of the above prediction function for the storage modulus, a specimen with the optimal combination of control factors (A3 B3 C3 D1 from g and the storage modulus, while the addition of HEMA toughener lowered both dynamic characteristics. With only the incorporation of each nano-powder of silica, alumina, and carbon black by 2 wt.%, the storage modulus of the epoxy composite at 30 °C showed a 34% increase from its pristine counterpart. On the other hand, the addition of 10 wt.% of HEMA into the epoxy could widen the damping plateau of the loss tangent spectrum from a temperature span around its Tg from 20 °C to 50 °C. This wider plateau denoted that the material damping could be operative in larger temperature span or frequency span. Finally, the prediction equations for the glass transition temperature and storage modulus were obtained from regression fits of the measured data, respectively. These equations could be used for design purpose and showed <1% deviation from the measurement within the range of control factors investigated in this study.When material is used in vibrational environment, the capability to estimate its dynamic mechanical property is critical during the design to prevent the unexpected dynamic response from external excitation. The dynamic property such as the stiffness and damping coefficients are crucial for the packaging materials used in the delicate electronic devices. This study explored the feasibility to predict the storage modulus and glass transition temperature of an epoxy composite that incorporated the nano-powders of silica, alumina, and carbon black in a HEMA toughened epoxy matrix. The use of nano-powders as reinforcements was shown to increase both the T