The Wnt/β-catenin signaling pathway plays an important role in embryogenesis and tumorigenesis. In human cancer, abnormal activity of Wnt/β-catenin signaling pathway induces overexpressed of downstream genes, and initiate oncogene. There are several target genes known to be key players in tumorigenesis, such as c-myc, cyclin D1, MMPs or survivin. Therefore, identifying the target genes of Wnt/β-catenin signaling pathway is important to understanding Wnt/β-catenin-mediated carcinogenesis. In this study, we developed a combined bioinformatics and experimental approach to find potential target genes.Luciferase reporter assay was used to analyze the promoter activity of RMI2. WST1 cell proliferation assays and transwell assays were performed to determine the proliferation and migration capacities of RMI2 overexpressing or knockdown stable hepatic cells. Finally, xenograft experiments were performed to measure the tumor formation capacity in vivo.RMI2 mRNA was upregulated after LiCl treatment and Wnt3a-conditioned medium in a culture of SK-hep-1 cell lines. A chromatin immunoprecipitation (ChIP) assay showed that the β-catenin/T cell-specific factor (TCF) complex binds to the putative TCF binding site of the RMI2 promoter. We then found a TCF binding site at − 333/− 326 of the RMI2 promoter, which is crucial for β-catenin responsiveness in liver cell lines. RMI2 was overexpressed in hepatoma tissue and cell lines, and it promoted the migration and invasion of HCC cells. Moreover, RMI2 upregulated the expression of epithelial-mesenchymal transition (EMT) markers and the Wnt3a/β-catenin-related genes, but silencing RMI2 had the opposite effects. Notably, the expression of RMI2 was positively correlated with the clinical data of HCC patients who had significantly shorter overall survival (OS) and disease-free survival (DFS) (Both: P < 0.05). In addition, a total of 373 HCC patients’ data from the Caner Genome Atlas project (TCGA) were used to validate our findings.The results showed that RMI2 was a new target gene of the Wnt/β-catenin signaling pathway. We also found that RMI2 promotes EMT markers, HCC cell invasion, and metastasis, which indicated that RMI2 is a potential target for preventing or at least mitigating the progression of HCC.Taking all these findings together, we determined that The online version contains supplementary material available at 10.1186/s12885-023-10655-2. Deletion or loss of function of its negative regulators, such as APC, glycogen synthase kinase-3β (GSK3β), or axis inhibition proteins (AXINs), results in abnormal stabilization of β-catenin, which enters the nucleus and forms a complex with T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factor. This complex then activates the expression of target genes, the dysregulation of which significantly affects tumorigenesis.The Wnt/β-catenin signaling pathway is important in embryogenesis and in tumorigenesis in a variety of tissue types, including colon and liver –3. In coof cases , 5. In hof cases , 9 that http://www.stanford.edu/~rnusse/pathways/targets.html). Most of these genes have in their promoter one or more TCF/LEF-binding elements composed of a highly conserved consensus sequence: 5′-(A/T)(A/T)CAA(A/T)G-3′ [COX-2 and VEGF have been developed. Inhibiting COX-2 can block cell proliferation, cell migration, metastasis, and angiogenesis, and it can lead to apoptosis [COX-2 inhibitors, such as celecoxib and rofecoxib, were approved by the Food and Drug Administration (FDA) to shrink and abate colorectal polyps in familial adenomatous polyposis (FAP) patients [Survivin and matrix metallopeptidase (MMP)-7 in particular have also shown consistent promise as prognostic markers. For example, survivin expression is absent in most nonmalignant tissue, but it is upregulated in many types of cancer. High survivin expression is associated with a poor prognosis in medulloblastomas [Many β-catenin/TCF target genes have been reported to be critical for carcinogenesis (A/T)G-3′ . During poptosis . COX-2 ipatients . Survivilastomas , HCC [14lastomas , lung calastomas , and brelastomas .Previously we performed a combined bioinformatics and experimental approach to identify novel oncofetal genes using data from expressed sequence tags (EST). Six thousand one hundred eighteen EST cDNA libraries were chosen and classified into “immature”, “mature”, and “tumorous” groups. The calculated frequencies of the AFP gene in each group  were used as references to set the thresholds of bioinformatics analyses to select genes expressed in the “immature”, downregulated in the “mature”, and re-expressed in the “tumorous” groups. The analyses resulted in 29 genes with known functions and 44 genes with unknown functions at that time. Five of the 29 known genes were indeed oncofetal genes, including AFP, IMP-1, FGF18, CGB, and SOX1 . We furthttp://cgap.nci.nih.gov/) [n = 483) from fetal tissue, infant tissue, placenta and cord blood；tumorous group (n = 3911) from benign and malignant tumors；and mature group (n = 1724) including those without information about age or disease status. Gene expression information was obtained from the file Hs_ExprData.dat, containing UniGene cluster ID, library ID and frequency. For example, the calculated frequencies of the AFP gene in each group of the libraries  were used to determine a set of thresholds for the bioinformatics analyses of novel oncofetal genes in our previous study [https://web.stanford.edu/~rnusse/pathways/targets.html>), 65 were present in our expressed sequence tag (EST) database from the 6118 cDNA libraries  . To clasus study . In thisup n = 391 from behttps://asia.ensembl.org/index.html), we obtained sequences 2000 bp upstream of the transcription start sites of the RMI2 gene. The putative binding sites were predicted using ALGGEN Promo .From the Ensembl Genome Browser Database (6 cells in a 96-mm dish) and cultured them for 4 days. We then harvested the CM, centrifuged it at 1000×g for 10 min, and filtered the supernatant through a nitrocellulose membrane. Control CM was prepared from L cells that had been transfected with phosphoglycerine kinase (PGK)-neo only (designated “L-CM).To collect the conditioned medium (CM) from cultures of L cells that produce Wnt-3a (designated “Wnt3a-CM”), we seeded L cells  in accordance with the manufacturer’s protocols. Two micrograms of RNA was transcribed using reverse transcriptase (SuperScript II). The semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) primer sequences are shown in Supplementary Table g, 4 °C for 20 min. Proteins were separated using 12% SDS-PAGE and then transferred to a PVDF membrane. Primary antibodies included mouse anti-β-catenin (BD Transduction Laboratories), rabbit anti-RMI2 (Abnova), rabbit anti-E-cadherin , rabbit anti-vimentin , rabbit anti-TWIST (Genetex), mouse anti-cyclin-D1 (Arigo), rabbit anti-c-Myc (Genetex), and mouse anti-β-actin (Millipore). To hybridize each marker separately, blots were cut based on the molecular weight of the proteins prior to hybridization with primary antibodies. Original blots with membrane edges are included in Supplementary Fig. Collected cells were dissolved using a lysis buffer on ice and then centrifuged at 10,000×2 incubator at 37 °C. Retroviral supernatant was added to 8 ng/mL of polybrene and used to infect HCC cells. Pooled HCC cells expressing either pMSCVpuro or pMSCV-RMI2 were selected using 2 μg/mL of puromycin.pMSCVpuro, pMSCV-RMI2, and pSUPERretro vectors were co-transfected into GP2–293 T packaging cells with VSV-G plasmids using the calcium phosphate method for 48 h. HCC cells were seeded in a 6-cm dish and cultured overnight in a 5% COβ-catenin and RMI2 expression, the shRNAs of β-catenin:and of RMI2:were used. A plasmid pLKO_TRC005 was the negative control.pLKO.1 lentiviral plasmids that expressed small hairpin RNA (shRNA) were purchased from the Taiwan National RNAi Core Facility . Lentivirus particles were produced by the RNAi Core Facility in the Research Center of Clinical Medicine, National Cheng Kung University Hospital (NCKUH). To knock down 6 SK-hep-1 cells were stimulated for 3 h with 20 μM of LiCl, cross-linked with 1% formaldehyde for 10 min at room temperature, lysed and sonicated to average lengths of 200- to 1000-bps. After centrifugation, the samples were diluted 10x to assess input DNA. Anti-TCF-4 (Millipore), normal mouse immunoglobulin G (IgG) (Millipore), and 5 μg of monoclonal anti-β-catenin antibody (BD Transduction Laboratories) were added and incubated with gentle agitation overnight at 4 °C. The DNA-protein complexes were eluted and incubated with proteinase K at 62 °C for 2 h to reverse the formaldehyde cross-link. The primers used in the ChIP assays are listed in Supplementary Table Chromatin immunoprecipitation (ChIP) assays were performed according to manufacturer’s instructions . Briefly, 3 × 10Transfection was done in triplicate using a reagent (Lipofectamin 2000). pRL-CMV plasmid was included as the internal control in every transfection mixture. The luciferase activity was normalized by renilla luciferase to adjust for variations in transfection efficiency. Cells were harvested 24 h after they had been transfected, and the luciferase activity was measured using a kit .RMI2 promoter fragments were PCR-generated from human genome DNA using specific primers with restriction sites and then inserted into the luciferase reporter vector . The mutations in the TBE sites of the RMI2 promoter were generated using a kit . Primer sequences are shown in Supplementary Table RMI2 and β-catenin. The samples were incubated with primary antibodies against RMI2 (1:200) (Abnova) and β-catenin  overnight at 4 °C and washed in PBS. The slides were then treated with a streptavidin biotin immunoperoxidase complex  for 1 h. The slides were lightly counterstained with hematoxylin to visualize immunoreactivity. Finally, the specimens were dehydrated in an ascending alcohol gradient.Five-micrometer-thick paraffined samples with malignant and non-malignant liver parenchyma tissue from each patient were histopathologically and immunohistochemically analyzed. Standard immunostaining procedures were used to determine Cells were fixed in 4% paraformaldehyde for 20 min. Cells were permeabilized with 0.1% Triton X-100 at 4 °C for 10 min. Primary antibody was diluted with phosphate buffered saline (PBS)/4% BSA for 4 °C overnight. After washing by PBS, the Alexa Fluor 555-conjugated secondary antibody was applied for 1 hour at room temperature. After counterstaining by DAPI, images were recorded on a fluorescence microscope.The lysate-antibody mixture was incubated overnight at 4 °C, then 30 μl of beads were added and the mixture was incubated for 3 hours at 4 °C in a tube rotator. The immunocomplexes were subsequently washed three times with 0.5 ml ice-cold PBS buffer and subjected to SDS-PAGE. The antibodies used for coimmunoprecipitation were: RMI2 (Abnova), Dvl-2 (Genetex), pGSK3β  and β-catenin (BD Transduction Laboratories).4 cells/well) for each time point .Huh-7, PLC/PRF/5 stable pools , and HepG2 and Hep3B stable pools  were seeded in 24-well plates for 96 h. Triplicate wells were plated  or without (for migration) a cell culture matrix . The lower chamber was filled with DMEM with 10% FBS. After 24 h culture, noninvasive cells on the upper side of the membrane were removed with a cotton swab. Each transwell was fixed by 4% paraformaldehyde, stained by crystal violet, and counted under a bright-field microscope for 5 randomly selected fields.Transwell migration and invasion assays were done in 24-well 8-μm-pore transwell plates in accordance with the manufacturer’s instructions . 1 × 10https://www.cbioportal.org>) was used to access TCGA mRNA expression data. The provisional RNA-Seq V2 RSEM z-score for each gene was used. Three hundred seventy-three HCC cases had mRNA expression and OS data. The cBioPortal was used to assess the association of OS with the RMI2 z-scores in TCGA data  using the R package on the cBioPortal for Cancer Genomics. Data were accessed from the cBioPortal between May 15 and July 15, 2017.The cBioPortal for Cancer Genomics (<2)/2. After 5 to 6 weeks, the mice were cervical dislocated under anesthesia and the tumors were isolated and weighted. Tumor tissues were confirmed by histopathological analysis. All mouse experiments were done in accordance with NCKU Laboratory Animal Care guidelines and were approved by the Institutional Animal Care and Use Committee in this study (IACUC #: 106129). The study was carried out in compliance with the ARRIVE guidelines, and all procedures for animal experiments followed the ethical standards.Nod/SCID mice  were purchased from National Laboratory Animal Center (NLAC) Taiwan in a standard pathogen-free environment laboratory. Mice were randomly divided into two groups: PLC/PRF/5-RMI2 and PLC-PMSCV-control. Cells were diluted and mixed with Matrigel  at a 1:1 ratio. The mixtures were subcutaneously injected into the left and right flanks of 6-week-old mice. The width and length of the tumors in each group were measured with vernier caliper twice a week. Tumor volume = (length × width2 tests were used to compute the correlation coefficients of the immunostaining scores between two proteins. Significance was set at P < 0.05.χRecQ-mediated genome instability protein 2 (RMI2).The bioinformatics analyses yielded a list of 161 genes , the Wnt pathway’s key kinase: it phosphorylates β-catenin and initiates its degradation. We treated SK-hep-1 (with a wild-type Wnt pathway) with LiCl for various time periods. β-catenin protein levels reached a maximum in 3 h, but the mRNA level did not change. This reflects the accumulation of β-catenin protein because of GSK3β inhibition and degradation. We also used Sk-hep-1 cells exposed for different time periods to 50% CM from L cells that expressed Wnt3a or from parental L cells. The protein level of β-catenin stabilized as early as 3 h after it had been stimulated with Wnt3a-CM, and it remained stabilized for up to 24 h -fold domain. RMI2 has an apparent molecular mass of 18 to 20 kD (SDS-PAGE) [RMI2 promoter, we analyzed the promoter and found at least six consensus TCF-binding elements (TBEs)  within 2.0 kb upstream of the transcriptional start site [RMI2 promoter (pRMI2–2.0) and all 6 TBEs into the pGL3-basic vector fused to the luciferase gene. The Huh-7 cell line had a wild-type Wnt pathway with no accumulated endogenous β-catenin in the nuclei [DS-PAGE) , 23. To art site . The sixe nuclei . When RMe nuclei . We thusP = 0.024) and β-catenin expression (P = 0.043), but not with age, gender, tumor size, or differentiation. HBV infection (P = 0.066) and vascular invasion (P = 0.063) showed a tendency to be correlated with RMI2 expression. However, the mRNA expression level of β-catenin was not significantly correlated with that of RMI2 in TCGA HCC dataset. This finding suggested that the regulation of RMI2 may be based on subcellular localization of β-catenin protein in cytoplasm and nuclei rather than mRNA level  cases, and RMI2 was positively expressed in 23 (38.3%) cases (Table P < 0.05) negatively associated with OS  and the AJCC stage (p = 0.0015), and no association was found between RMI2 expression and vascular invasion . By immunofluorescence staining, the increased nuclear staining of β-catenin upon RMI2 overexpression was observed, while RMI2 silencing had the opposite effect Fig. D, E. TheGiven that high RMI2 expression was significantly correlated with a poor prognosis, we then investigated the biological functions of RMI2 in HCC cells. Cell cycle phase distribution was determined by flow cytometry analysis. Figure P < 0.05).Aberrant activation of Wnt/β-catenin signaling reportedly contributes to the invasion, metastasis and progression of HCC . To furtP < 0.01), and had higher mass  than the control tumors. Next, we examined RMI2 expression in the tumor samples with western blotting. As shown in Fig. In order to test the tumor forming abilities of the stable PLC/PRF/5 cell lines in vivo, we performed Xenograft assays in SCID mice. Six to seven weeks after PLC/PRF/5 cells that overexpressed RMI2 had been injected into the flanks of the mice, the tumors formed by RMI2 overexpressing cells was significantly larger Fig. A and B , Sry-related HMG box 9 (SOX9) and vasodilator-stimulated phosphoprotein (VASP), play in the positive feedback loop to support Wnt activity in various types of human malignancies –30. In tRMI2 was found to be essential for the BLM-Topo IIIa complex [RMI2 with the BLM-Topo IIIa complex is through RMI1 [BLM, a RecQ-like helicase, have been linked to the autosomal recessive cancer-prone disorder Bloom’s syndrome. RMI2 was widely considered to play a crucial role in DNA repair surveillance, and prevents genomic instability [RMI2 was identified as significant cervical squamous cell carcinoma after expression validation and survival analysis [RMI2 was also reported to be associated with the worse prognosis in pancreatic cancer [RMI2 expression to worse prognosis of lung cancer [RMI2 was associated with worse OS in HCC patients. It remains unclear whether RMI2 contributes to tumorigenesis through a BLM-related mechanism, or through dysregulation of the Wnt/β-catenin pathway which may not involve the BLM-Topo IIIa complex. complex , 23 and ugh RMI1 . Mutatiotability . RMI2 waanalysis . RMI2 wac cancer . Recentlg cancer , 35. OurIn Table β-catenin is an important gene in HCC tumorigenesis and progression , 37–41, . RMI2 is a worse prognostic biomarker for HCC. The molecular mechanisms of RMI2 in HCC cell aggressiveness might be associated with the dysregulation of the Wnt pathway.We established a bioinformatics analytical model and found RMI2 as a new direct target gene in the Wnt pathwayAdditional file 1: Supplementary Fig. 1. Mutation study of RMI2 promoter. (A)Diagram of the mutation constructs, mutated sites are as indicated. The core sequence (CAAAG) located between positions-1369 and-1375 was mutated as (GCTAG). β-catenin (S37/A) can activate the mutation of deletion. Deletion and mutation of deletion were cotransfected with increasing amount of β-catenin (S37/A) expression construct . (B) Diagram of the mutation constructs, mutated sites are as indicated. The core sequence (ACTTTG) located between positions − 918 and − 924 was mutated as (GAATTC). β-catenin (S37/A) can activate the mutation of deletion. Transfections were carried out as described in A.Additional file 2: Supplementary Fig. 2. Clinical significance of RMI2.  RMI2 mRNA expression levels in HCC of different pathological stages and lymph node metastasis (p < 0.05).Additional file 3: Supplementary Fig. 3. Correlation of RMI2 and beta-catenin (CTNNB1) expression in the TCGA dataset.Additional file 4: Supplementary Fig. 4. The original images for western blot in article.Additional file 5: Supplementary Fig. 5. The original images for agarose gel in article.Additional file 6. Steps to download X-crunch dataset.Additional file 7: Supplementary Table 1. Target genes of Wnt/beta-catenin signaling from http://www.stanford.edu/group/nusselab/cgi-bin/wnt/.Additional file 8: Supplementary Table 2. (A) Primer sequence used for PCR assays. (B) Primer sequence used for ChIP assays. (C) Primer sequence used for site-directed mutagenesis assays.


The Wnt signaling pathway plays a critical role in regulating normal cellular processes, including proliferation, differentiation, and apoptosis. Dysregulation of Wnt signaling has been implicated in various human diseases, including cancer. β-catenin and LEF1 are key mediators of Wnt signaling, and their dysregulation is a hallmark of many cancer types. In this study, we aimed to identify the deubiquitinases (DUBs) that regulate the Wnt signaling pathway through the essential component LEF1. Screening candidate DUBs from the human DUB library, we discovered that OTUD7B interacts with LEF1 and activates Wnt signaling. OTUD7B and LEF1 interact with each other through the UBA and HMG domains, respectively. Furthermore, OTUD7B promotes the nuclear localization of LEF1, leading to an increased interaction with β-catenin in the nucleus while not noticeably affecting ubiquitination on LEF1. Using qPCR array analysis, we found that OTUD7B overexpression leads to an upregulation of 75% of the tested Wnt target genes compared to the control. These findings suggest that OTUD7B may serve as a potential therapeutic target in human diseases, including cancers where Wnt signaling is frequently dysregulated. LEF1 (Lymphoid enhancer-binding factor 1) is a transcription factor that plays a crucial role in the development of multiple tissues, including the immune system, skin, and hair follicles ,3. It beThe Wnt signaling pathway is a complex network of signaling events that governs cellular processes such as cell proliferation, differentiation, and survival . AbnormaLEF1 activity is modulated by various post-translational modifications, including phosphorylation and ubiquitination ,18,19,20In this study, we aimed to identify the DUBs that regulate Wnt signaling by screening a human DUB library and found OTUD7B as a LEF1-binding DUB. Surprisingly, OTUD7B did not exhibit deubiquitinating activity on LEF1 despite its interaction with LEF1. Instead, OTUD7B activated the Wnt signaling pathway, at least in part, by promoting the nuclear translocation of LEF1 and its interaction with β-catenin. Our data demonstrate the catalytic-independent function of a DUB in potentiating the Wnt signaling pathway. Further investigation is warranted to explore the potential of OTUD7B as a therapeutic target in human cancers by disrupting its interaction with LEF1 and inhibiting Wnt signaling pathway activation.2.The HEK293T cell line was obtained from the American Type Culture Collection and cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were maintained in a 37 °C humidified incubator with 5% COThe full-length LEF1 open reading frame (ORF) amplified from a human cDNA library was subcloned into the MYC-tagged expression vector using the gateway system (MYC-LEF1) . SFB-DUB vectors were constructed as previously described . HA-ubiqCycloheximide and MG132 were purchased from Cayman Chemical . Lithium chloride was obtained from Thermo Fisher Scientific . S-protein agarose beads were obtained from Merck . MYC antibody-conjugated agarose beads were from Thermo Fisher Scientific . The following antibodies were used: anti-FLAG , anti-MYC , anti-OTUD7B , anti-cyclophilin B , anti-HSP90 , anti-HA , anti-V5 , anti-α-tubulin , and anti-lamin B1 .siRNAs targeting human OTUD7B were purchased from GenePharma , and their sense sequences are provided as follows: siOTUD7B #1, 5′-UUCUUGAACGAUGUCAUCCTT-3′; siOTUD7B #2, 5′-CUUCUGUGUAUACCAGCCCTT-3′; siOTUD7B #3, 5′-AGGUCUCUCUCUAUGAAGCTT-3′.MYC-LEF1 and SFB-OTUD7B truncation mutants were generated using the EZchange site-directed mutagenesis kit  following the manufacturer’s protocol.For pulldown of SFB-DUBs, S-protein beads were utilized, and subsequent procedures including cell lysis, pulldown with S-protein beads, and immunoblotting were performed as described . MYC-beaHEK293T cells were co-transfected with MYC-LEF1, SFB-OTUD7B/SFB-GFP, and HA-ubiquitin constructs. Subsequent procedures were performed following the described protocol .HEK293T cells were co-transfected with MYC-LEF1, MYC-GFP, and SFB-OTUD7B/SFB-GFP constructs. After 2 days, the cells were treated with cycloheximide (CHX) for the indicated durations. The cells were harvested, lysed with RIPA buffer using sonication, and subjected to immunoblotting.Western blotting assays following pulldown assays and immunoprecipitation were carried out as previously described .HEK293T cells were plated in quadruplicates in a 24-well plate. The cells were co-transfected with 150 ng of SFB-DUB/SFB-GFP, 100 ng of the firefly luciferase vector (TOPflash or FOPflash), and 1 ng of the Renilla luciferase vector. To co-transfect siRNAs and plasmid DNAs, the cells in the 24-cell plate were initially transfected with 0.17 µg of siOTUD7B #1, #2, #3  using X-tremeGENE siRNA transfection reagent . After 30 min, the cells were further transfected with 100 ng of firefly luciferase vector (TOPflash or FOPflash), and 1 ng of Renilla luciferase vector.After 2 days, the cells were lysed, and luciferase activities were measured using the Dual-Luciferase Reporter Assay System  with the EnSpire Multimode Plate Reader , following the manufacturer’s protocol. The firefly luciferase activity was normalized using the Renilla luciferase activity.HEK293T cells were co-transfected with MYC-LEF1 and SFB-OTUD7B/SFB-GFP constructs. After 2 days, the cells were harvested, and cytoplasmic and nuclear proteins were extracted using the Nuclear Extraction Kit . The proteins were quantified, and the protein samples were subjected to immunoblotting.HEK293T cells were co-transfected with MYC-LEF1, SFB-β-catenin, and OTUD7B-V5/GFP-V5 (in pLX304 vectors) constructs. After 2 days, the cells were harvested. Prior to harvest, the cells were treated with 20 mM lithium chloride for 24 h. Cytoplasmic and nuclear lysates were extracted using the Nuclear Extraction Kit . Each lysate was pulled down with S-protein beads and immunoblotted with the indicated antibodies.https://imagej.nih.gov/ij/download.html (accessed on 17 February 2023) and utilized to quantify the immunoblotting band images. The nuclear and cytoplasmic levels of LEF1 were normalized using lamin B1 and α-tubulin levels, respectively.The image processing software, ImageJ, was downloaded from A qPCR screening kit for Human Wnt Signaling Targets was purchased from Bioneer (SH-0000-10) . HEK293T cells transfected with either SFB-GFP or SFB-OTUD7B were subjected to total RNA extraction using the MagListo RNA extraction kit  and RNase-free DNase I , followed by reverse transcription with the iScript cDNA synthesis kit . A total of 20 ng of cDNA was mixed with SYBR Green reagent , and equal amounts were added to each well of a 96-well qPCR array plate containing gene-specific primer sets. Real-time PCR and data collection were performed using a CFX Connect instrument .t-test for comparing two independent groups. A p-value of less than 0.05 was considered significant.The data are presented as mean ± s.e.m., and statistical significance was determined using the two-tailed unpaired Previously, we conducted luciferase reporter assays to screen human DUBs involved in regulating the Wnt signaling pathway . In thisWe then examined which DUB has a greater effect on the transcriptional activity of LEF1. The luciferase reporter containing wildtype TCF/LEF binding sites (TOPflash) and mutated TCF/LEF binding sites (FOPflash) was transfected with SFB-USP36 or SFB-OTUD7B. Compared to FOPflash luciferase levels, both USP36 and OTUD7B increased the luciferase activities (TOPflash), but OTUD7B significantly enhanced LEF1 activity to a greater extent (6.64-fold) than USP36 did (2.04-fold) C. In addTo determine which domains are responsible for the interaction between LEF1 and OTUD7B, we generated three deletion mutants of LEF1: LEF1ΔHMG , LEF1ΔCRD , and LEF1ΔBCBD  A using tNext, we generated three deletion mutants of OTUD7B: OTUD7BΔZnF , OTUD7BΔOTU , and OTUD7BNt  using the SFB-OTUD7B plasmid C and attSince LEF1 is targeted for proteasomal degradation through ubiquitination by multiple E3 ligases, including NARF and Pja2 ,20, we iWe hypothesized that OTUD7B may be involved in the subcellular localization of LEF1 if OTUD7B does not affect protein stability. To investigate this, we co-transfected MYC-LEF1 with SFB-OTUD7B or SFB-GFP plasmids into HEK293T cells and separated the cytoplasmic and nuclear fractions of the cell lysates. The protein levels of LEF1 in both fractions were compared via immunoblotting using an anti-MYC antibody. Surprisingly, when OTUD7B was overexpressed, LEF1 was significantly more abundant in the nuclear fraction compared to the cytoplasmic fraction A. This iWe further investigated whether the increased nuclear translocation of LEF1 by OTUD7B leads to enhanced transcriptional activity by promoting the interaction between LEF1 and β-catenin. For this purpose, we performed a pulldown assay using nuclear and cytoplasmic fractions of cell lysates. HEK293T cells were transiently transfected with MYC-LEF1, SFB-β-catenin, and V5-tagged GFP or OTUD7B, and prior to cell lysis, the cells were treated with lithium chloride to increase the availability of nuclear β-catenin. β-catenin was pulled down using S-protein agarose beads, and the bound LEF1 was assessed in both fractions via immunoblotting. As shown in LEF1 is a crucial regulator of the canonical Wnt signaling pathway. Therefore, we investigated whether OTUD7B plays a role in Wnt signaling activation by promoting the nuclear localization of LEF1 and its interaction with β-catenin. To address this question, we utilized a quantitative PCR array kit that profiles the expression of 84 Wnt target genes and examined whether OTUD7B enhances the overall expression of these genes. We prepared cDNA samples from HEK293T cells transiently transfected with either SFB-GFP (control) or SFB-OTUD7B, and performed qPCR analysis. Our results revealed that 63 out of 84 genes were upregulated by OTUD7B , includiOTUD7B/Cezanne, a member of the ovarian tumor (OTU) domain-containing family of deubiquitinases (DUBs), is a critical regulator of various cellular processes, including DNA damage response and immune regulation ,28,29,30In our previous study, we discovered OTUD7B as one of the DUBs that activate the Wnt signaling pathway. While OTUD7B interacted with β-catenin, it did not deubiquitinate β-catenin . This fiCatalytic-independent functions are often observed in various kinases and DUBs. For example, the receptor tyrosine kinase EGFR interacts with the proapoptotic molecule PUMA in the cytoplasm, leading to tumor drug resistance. EGFR also interacts with certain transcription factors in the nucleus to regulate target gene expression ,39. RAF1LEF1 is known to be transported to the nucleus by importin α, which recognizes the nuclear localization sequence located in the HMG domain of LEF1 . Since OInterestingly, OTUD7B has been shown to deubiquitinate β-catenin in vascular smooth cells , althougStudies have exhibited that OTUD7B stimulates cancer progression in multiple human cancers ,36,48,49


In this work, the effect of cellulose nanofiber (CNF) on the mechanical properties of long pineapple leaf fiber (PALF)-reinforced epoxy composites was investigated. The content of PALF was fixed at 20 wt.% and the CNF content was varied at 1, 3, and 5 wt.% of the epoxy matrix. The composites were prepared by hand lay-up method. Comparison was conducted between CNF-, PALF- and CNF–PALF-reinforced composites. It was found that the introduction of these small amounts of CNF into epoxy resin caused very small effects on flexural modulus and strength of neat epoxy. However, impact strength of epoxy with 1 wt.% CNF increased to about 115% that of neat epoxy, and, as the content of CNF increased to 3 and 5 wt.%, the impact strength decreased to that of neat epoxy. Observation of the fractured surface under electron microscope revealed the change in failure mechanism from a smooth surface to a much rougher surface. For epoxy containing 20 wt.% PALF, both flexural modulus and strength increased significantly to about 300% and 240% that of neat epoxy. The composite impact strength increased to about 700% that of the neat epoxy. For hybrid systems containing both CNF and PALF, there were few changes observed in both flexural modulus and strength compared to the PALF epoxy system. However, much improvement in impact strength was obtained. By using epoxy containing 1 wt.% CNF as the matrix, the impact strength increased to about 220% that of 20 wt.% PALF epoxy or 1520% that of neat epoxy. It thus could be deduced that the spectacular improvement in impact strength was due to the synergistic effect of CNF and PALF. The failure mechanism leading to the improvement in impact strength will be discussed. As evuivalent . AlthougNumerous natural fibers, such as flax, hemp, sisal, and pineapple leaf fibers (PALF), have been evaluated as alternative materials for the construction of green buildings ,9. AgricMany studies have reported potential utilizations of PALF in various types of polymer composite , such asCurrently, nanofiller either from synthetic or natural sources draws a great deal of attention compared with bulk counterparts . As the Epoxy resins have been recognized as the most widely used thermoset for manufacturing fiber composite. Bio-based epoxy also provides an opportunity to originate lower-carbon-footprint materials . With thIn this work, we demonstrate that CNF and PALF could be combined, resulting in synergistic hybrid reinforcement for the epoxy resin, enhancing the impact strength of the composite materials. Fractography was used to understand the mechanism behind this synergistic toughening effect.Aqueous dispersion of CNF utilized in this work was prepared from wood pulp, and it is commercially available through Pheedoo Co., Ltd., Bangkok, Thailand. Long PALF was supplied by a local producer in Ratchaburi, Thailand. The long fiber was extracted from fresh pineapple leaf using a decorticating machine. Commercial epoxy resin (EP089) and hardener (HP089) were purchased from a local supplier . The matrix was prepared by mixing the resin and hardener at the ratio of 2:1 as recommended by the supplier. Sodium hydroxide  and commercial-grade acetone  were used without purification.w/v) in the aqueous dispersion to about 7.94% (w/v) after acetone exchange process. The prepared CNF acetone dispersion was diluted to 1.0 wt.% using ultrasonic treatment  with 70% power for 30 min. Then, epoxy resin HP089 was added into CNF solution. All acetone was eliminated using hot plate stirrer (60 °C) under air ventilation for 24 h and further evaporated in a hot air oven (80 °C) for 72 h. After that, the hardener was gently mixed with the previous epoxy resin and poured into silicone mold. The nanocomposites were left to cure at room temperature for 24 h. The whole process is shown in To prepare CNF-EP composite, water in the CNF dispersion was first exchanged with acetone to avoid nanofiller aggregation in the dry state. The procedure followed that reported by Tang, et al.  with sliLong pineapple leaf fiber (PALF), which could be regarded as continuous fiber for the specimen size used in this work, was prepared into a well-aligned sheet before incorporated into epoxy resin as shown in Transmission electron microscope  was used to observe the size and shape of cellulose nanofiber (CNF). To prepare the sample for the analysis, CNF suspension was diluted in distilled water to concentration of about 0.01 wt.% and sonicated for 30 min. A small drop of the suspension was placed on 300-mesh-Formvar-coated copper grids and allowed to dry at room temperature. The samples were stained with iodine vapor to improve image contrast before observation at an operating voltage of 100 kV.Field emission scanning electron microscope  was used to study the fracture surface of the composite after flexural and impact testing at room temperature. The samples were initially dried in the oven (80 °C) and mounted on SEM holder using double-sided carbon adhesive tape to achieve the appropriate conductivity. After that, the samples were coated with a thin layer of titanium  to prevent surface damaging during the operation at 5 kV.Glass transition temperature of the epoxy matrix was determined on a differential scanning calorimeter . The transition temperature was determined from the first heating cycle from 0 °C to 150 °C with a heating rate of 10 °C/min under nitrogen atmosphere.Flexural test: The test was carried out on a universal testing machine  following ASTM D790-3 using a crosshead speed of 1.28 mm/min, 5 kN of load cell, and a support span length of 48 mm. Specimens were cut from composite sheets with a scroll saw into strips of 12.7 mm wide and 120 mm long with the long axis parallel to the fiber direction. The average values of flexural strength and flexural modulus from 5 specimens were reported.Impact test: The test was carried out on a pendulum impact testing machine  in Izod configuration following ASTM D256. The composite samples were cut with a scroll saw from composite sheets along the fiber direction, providing a strip 12.7 mm wide and 60 mm long. The samples were notched with a Zwick/Roell manual notch cutting machine. Average values of 5 specimens were reported.t-test method, using Two-Sample Assuming Unequal Variances, was performed to analyze differences among the means at a confidence level of 95%.Statistical analysis was performed using analysis of variance (ANOVA) and Data Analysis tool in Microsoft Excel (Office16) program. The g and other properties. Therefore, DSC was investigated if CNF had such an effect in the system under investigation. g, for most systems . This can be measured as impact strength. When PALF was added to epoxy (0CNF/PALF), the impact strength increased (by 600%) from 27 J/m to 189 J/m compared to the neat epoxy. This increase in impact strength could be contributed by both crack deflection and de-bonding mechanisms at the fiber–matrix interface. When epoxy was replaced with 1CNF in a hybrid system (1CNF/PALF), the strength increased to 411 J/m, which is approximately 220% that of the system without CNF. However, when the amount of CNF was increased to 3 and 5 wt.%, the impact strengths decreased but were still higher than that of the system without CNF. This decrease was in good agreement with what was observed previously for CNF-filled epoxy composites. These results clearly reveal the synergistic toughening effect of hybrid CNF and PALF that provides higher toughness as compared to the single reinforcement system.In order to understand the mechanism regulating the improvement in impact strength of the hybrid CNF–PALF epoxy composites, impacted fracture surfaces were studied. The impact fracture surfaces of epoxy filled with CNF are shown in The impact fracture surfaces of hybrid composites are shown in In the previous section, the impact fracture surfaces of CNF-filled epoxy composites and those of hybrid composites were compared and not much could be deduced except that multiple crack deflections operate in both systems. One important point to note is that crack paths in CNF-filled and hybrid composites are very different, and the latter is much more complicated. Broken surfaces of hybrid composites are very rough and have a large number of protruding PALFs. This indicates that stress could be transferred along the fiber to a much greater volume of materials, causing a much greater number of crack deflection points within such volume and in so doing absorbing a substantial amount of energy. The mechanism can be schematically presented in In the case of the CNF-reinforced EP composite material (In the case of the hybrid (CNF/PALF-reinforced) epoxy composite material (Hybrid epoxy composites with improved mechanical properties were successfully prepared from CNF and PALF. The technique offers great enhancements in stiffness, strength, and toughness to the epoxy matrix. Being very small, incorporation of CNF is limited to very low loading, yet the amount of only 1 wt.% was found to be very effective in improving the impact strength while having very little effect on flexural properties. This is due to the ability of CNF to change the failure mechanism, at high speed, of the epoxy matrix. The PALF, on the other hand, allows much greater content to be incorporated with ease, and, hence, both flexural properties and impact could be significantly improved. By combining both CNF and PALF together, spectacular improvement in toughness is obtained. This is a result of the synergistic effect of CNF and PALF. This could lead to greener, more sustainable, and more cost-effective materials for demanding applications.


To review the antioxidant and anti-atherosclerotic potential of whole banana, banana flour, and its bioactive compounds.A non-systematic review of the literature covering the past 20 years, using the following databases and searching bases: PUBMED/MEDLINE: https://www.ncbi.nlm.nih.gov/pubmed/; Google Scholar: https://scholar.google.com.br/; and “Literatura Latinoamericana em Ciências da Saúde”/Latin American Literature in Life Sciences [LILACS]: http://lilacs.bvsalud.org/, was done. Studies with incomplete methodology and design were excluded.Bananas from different species are a good source of carbohydrates, dietary fiber, proteins, polyunsaturated fatty acids, potassium, carotenoids, flavonoids, vitamin C and E, phytosterols, gallocatechin, catechin, and other polyphenols. Some of these compounds play trigger important biological roles as antioxidants or anti-atherosclerotic and cardiovascular protective substances. This review summarizes and explains thirteen protective biological mechanisms of banana bioactive compounds and banana products. Including banana and its products in dietary menus, in food products and nutraceuticals should improve cardiovascular health of the populations. Musa spp) is one of the most eaten foods around the world. Banana pulp is rich in starch, fiber, vitamins C and A, thiamine, riboflavin, phosphorus, potassium, calcium, and iron . Banana peel also contains fiber, proteins, polyunsaturated fatty acids, potassium, carotenoids, vitamin C and E, gallocatechin, catechin, and other polyphenols . The most important banana plant species comprise Musa paradisiaca, M. cavendish, M. acuminata, M. balbisiana and M. sapientum .Banana .  Due to increased pharmaceutical interest in natural products as a source of both antioxidants and anti-atherosclerotic compounds , especially bioactive compounds with capacity to inhibit low density lipoprotein (LDL) cholesterol oxidation and/or molecules capable to decrease cholesterol absorption and/or down-regulate liver cholesterol , the present study reviewed the roles of banana bioactive compounds in prevention and protection against atherosclerosis. A non-systematic review of the literature covering the past 20 years (2000-2020), using the following databases and searching bases: PUBMED/MEDLINE: https://www.ncbi.nlm.nih.gov/pubmed/; Google Scholar: https://scholar.google.com.br/; and “Literatura Latinoamericana em Ciências da Saúde”/Latin American Literature in Life Sciences [LILACS]: http://lilacs.bvsalud.org/, was done. The major key words were “banana peel”, “banana pulp”, “banana products”, “antioxidant”, “antioxidant capacity”, “antioxidant activity”, “carotenoid”, “flavonoid”, “phenolics”, “cardiovascular prevention”, “cardiovascular disease”, “dyslipidemia” and “atherosclerosis”. Studies with incomplete methodology and design were excluded. Selected studies comprised theoretical as well as animal and laboratory studies and clinical trials.Reactive oxygen species and free radicalsReactive oxygen species comprise any chemical specie capable of independent existence exhibiting higher capacity to react with cell biomolecules, whereas free radicals are molecules with unpaired electrons in an orbital .The major source of free radicals is the respiratory mitochondrial chain, which releases the superoxide anion radical (O2-) that is converted by the enzyme superoxide dismutase (SOD) into hydrogen peroxide (H2O2), a highly reactive oxygen species . Since H2O2 is a very reactive oxygen species, capable to generate the most damaging hydroxyl radicals (OH°), two antioxidant enzymes, catalase (CAT) and glutathione peroxidase (GPx), can metabolize this substance to innocuous water and atomic oxygen . Other free radicals comprise lipoperoxyl radicals, formed by lipid peroxidation, and peroxynitrite formed by reaction of the nitric oxide radical (NO°) with superoxide anion. Other reactive species include molecules from oxygen, nitrogen and chlorine, such as hypochlorous acid (HOCl-), singlet oxygen (1O2), peroxyl radical (HO2°), and ozone .Free radicals, ROS and reactive nitrogen species (RNS) can cause lipid peroxidation of membrane phospholipids and of other cell lipids and they can oxidize proteins, enzymes, carbohydrates and nucleic acids .Reactive oxygen, nitrogen and chlorine species: role in atherosclerosisThe seminal paper that consolidated the cholesterol hypothesis of atherosclerosis was published by Steinberg et al. (1989). This paper emphasized the role of free radicals in the lipid peroxidation of blood lipids, especially the LDL cholesterol particles that, once oxidized (ox-LDL), are promptly engulfed by activated macrophages into the subendothelial space. Macrophages with intracytoplasmic accumulation of ox-LDL and other lipids become foam cells . With the death of foam cells, atherosclerotic plaque is formed  through many specific molecular mechanisms reviewed by specialists . The same study demonstrated that other cells from vascular walls  can engulf cholesterol and other lipids, becoming foam cells . It is important to observe that hyperlipidemia also induces neutrophil infiltration into the vascular wall, which can contribute to the genesis of the atherosclerotic plaque .Antioxidant activity of bananain vitro and in vivo.Due to their composition, bananas are recognized as a significant source of antioxidant activities In vitro antioxidant activity of bananas Many banana species have presented considerable antioxidant activity by scavenging of ROS and RNS, as well as inhibiting lipid peroxidation reactions.2O2 induced atherogenic cascades and amplified inflammatory signaling, worsening evolution and stability of atherosclerotic plaque .Baldi et al. (2012) reported that an alcoholic extract of banana had high antioxidant activity and considerable hydrogen peroxide-scavenging capacity. It is important to note that HMusa cavendish, it was observed that gallocatechin concentration was higher in banana peel (158 mg/100 gr dry weight) than pulp (29.6 mg/100 gr dry weight) and that the antioxidant activity of banana peel was higher compared to the pulp .Studying M. cavendish, imported by Japan, presented consistent antioxidant activity as measured by thiocyanate method, β-carotene bleaching assay, and the DPPH assay .Extracts of Musa spp in India, authors reported that banana extracts had potent antioxidant capacity, an effect due to its phenolic/flavonoid content .Studying 2O2, NO°, and O2- .Other research showed that banana extracts had antioxidant activity that was responsible to decrease lipid peroxidation and scavenge HBananas from Thailand also presented significant antioxidant activity measured by two different methods. In the ferric reducing antioxidant potential (FRAP) method, ripe samples had highest antioxidant activity compared to mature green and overripe samples, whereas when using DPPH assay ripe samples had lowest antioxidant activity compared to mature green and overripe group . In the same study, ripe and overripe had higher total phenolic content, whereas mature green had the higher flavonoid content .Ripe crude bananas from Philippines, imported by Oman, had total phenolic content ranging from 131.72 mg of gallic acid equivalent (GAE)/100 g to 386.22 mg GAE/100 g whereas the total flavonoid content ranging from 0.16 µg/quercetin equivalent (QE)/g of dry powder to 8.51µg of QE/g of dry powder . The same authors reported that banana had higher antioxidant activity measured by the DPPH assay.In this respect, it is important to note that banana flower and stem or pseudostem are rich in fiber and polyphenolics with significant antioxidant activity .Total phenolic content is associated with high antioxidant capacity of a banana industrial by-product from Malaysia .In vivo antioxidant activity of bananas Rats feeding banana extracts had decreased levels of both primary (hydroperoxides and conjugated dienes) and secondary ) lipid peroxidation products, whereas they had increased levels of antioxidant defense enzymes, such as SOD, CAT, and glutathione (GSH) .Mice fed with banana extract had decreased lipid peroxidation measured by MDA content and increased GSH content .Due to their antioxidant content as well as the levels of carotenoids, phenolic compounds, vitamins, microelements, fiber and other bioactive compounds, regular eating of bananas and their products can afford many health benefits as listed in Banana as a rich source of bioactive compounds with antioxidant activity Banana peel and pulp are rich in starch, total carotenoid, and antioxidant enzymes  . In the same study, β-carotene content varied from 28 µg/100 g to 117.2 µg/100 g in pulps and from 49 µg/100 g to 241.91 µg/100 g in peel .Another study reported a concentration of β-carotene from 45 µg/100 g to 7124 µg/100 g in banana cultivars from Solomon Islands .Banana flour, rich in resistant starch, phenolics, β-carotene and total carotenoids, presented also higher antioxidant activity by the DPPH method, by hydroxyl radical scavenging method and inhibition of lipid peroxidation assay . Anyasi et al. (2018) reported that banana flour contains substantial amounts of organic acids , potassium and many polyphenolics, especially myricetin-glycosides.Regarding carotenoid content, the following carotenoids were isolated from 36 banana varieties in China: violaxanthin, neoxanthin, antheraxanthin, α-cryptoxanthin, β-cryptoxanthin, lutein, α-carotene and β-carotene . Studying 15 Brazilian banana varieties, authors observed the occurrence of vitamin A, α-carotene, β-carotene, and lutein .It is also interesting to note that bananas are a source of lycopene. A study from Thailand reported that lycopene content of bananas represented 29.8% of the total carotenoid content observed in tomatoes Suwanaruang, 2016).M. cavendish) is also rich in dopamine, with 80-560 mg/100 g in the peel and 2.5-10 mg in the pulp . In the same study, authors found that dopamine had high antioxidant activity, similar to gallocatechin gallate and ascorbic acid, but superior to the antioxidant activity presented by GSH, quercetin, luteolin and catechin. Banana . Dopamine isolated from bananas was capable to improve LDL resistance to oxidation .Other authors emphasize that due to the higher content of L-dopa and dopamine, banana, banana peel and its products should be useful to the treatment of Parkinson’s disease patients . Another study also corroborated that Banana bioactive compounds and atheroprotectionEvidence from many biomedical studies as well as epidemiologic and clinical research showed that carotenoids, flavonoids, phenolics, tocopherols, and phytosterols, usually found in the different banana species, can display different antioxidant, anti-inflammatory and anti-atherosclerotic mechanisms, listed in In addition to several health benefits, bananas have different compounds with protective effects against cardiovascular diseases.Lutein and atherosclerosisin vitro and in vivo studies indicated that lutein intake or supplementation can substantially reduce the risk of atherosclerosis by inhibiting both LDL deposits in arterial macrophages and atherosclerotic plaque progression .Evidence from epidemiologic, It had been suggested that lutein as well as food carotenoids can inhibit macrophage uptake of oxidized LDL particles by decreasing cholesterol oxidation and reducing inflammation in the vascular domains .Lutein can also decrease disease progression in patients with early atherosclerosis, since it reduces serum LDL, triglycerides, interleukin-6 (IL-6), and macrophage chemo-attractant protein-1 .Studying 40 early atherosclerosis patients without clinical cardiac events, authors reported that serum lutein was inversely associated with IL-6 and positively associated with IFN-γ, whereas zeaxanthin was related to expression of vascular adhesion molecules and ApoE, and lycopene were negatively correlated with vascular adhesion molecules and LDL .Wang et al. (2013) observed that lutein supplementation improved total antioxidant capacity of plasma and decreased both MDA and C-reactive protein in non-smoking healthy people.Lutein is swallowed by blood mononuclear cells and it is capable to inhibit both gene expression and secretion of IL-6, IL-1β, and tumor necrosis factor (TNF) in stable coronary artery disease patients .Another two mechanisms through which lutein affords protection against atherosclerosis comprise PPAR expression and inhibition of NADPH oxidase .Lycopene and atherosclerosisIn a randomized clinical control trial, supplementation with lutein significantly decreased carotid artery intima-media thickness, whereas the degree of atherosclerosis inhibition was greater among those who received lutein plus lycopene supplementation . Daily supplementation with lycopene (5 mg/kg) during 4 weeks decreased by half the blood total cholesterol and the LDL cholesterol in rabbits . In the same study, lycopene did not improve aortic intima-media thickness or endothelial-dependent and independent aortic and carotid vasodilation.A meta-analysis of clinical studies reported that a daily dosage of 25 mg of lycopene effectively reduced total blood cholesterol, accounting for a 10% reduction on the LDL cholesterol .Lycopene can reduce carotid-intima thickness, LDL, LDL oxidation, vascular adhesion molecules, vascular inflammatory cytokines, proliferation of smooth muscle cells, and endothelial damage .Anti-atherosclerotic actions of catechins and quercetin2-, H2O2 and HOCl- can be scavenged by catechin and quercetin . This explains why dietary intake of polyphenolic-rich foods lower peroxynitrite formation, improve endothelial function, and decrease both LDL oxidation and atherogenesis .OIn this context, quercetin can enhance HDL cholesterol levels and reduce triglycerides levels, whereas it can diminish both blood pressure and LDL oxidation by ROS .Catechin is responsible to decrease the release of pro-inflammatory cytokines in the vasculature, to reduce vascular inflammation and atherosclerotic lesion size .Other dietary flavonols improved HDL levels and decreased total cholesterol levels in clinical trials .2O2 and the subsequent activation of phospholipase C and cicloxigenase-1, which also materially decrease thromboxane A2 synthesis and platelet aggregation . Dietary polyphenols also partially inhibit activation of NADPH-oxidase protecting the endothelium against excessive amounts of the powerful oxidizing peroxinitrite, contributing to decreased atherothrombotic damage and progression .Catechin, epicatechin and quercetin reduce endothelial platelet activation though enhancement of nitric oxide levels, whereas other polyphenols can decrease mitochondrial HThese results corroborate previous finding that quercetin and catechin, two banana polyphenols, inhibited phosphokinase C dependent activation of NADPH-oxidase, decreasing the production of oxygen and nitrogen free radicals and platelet aggregation . Quercetin and catechin are also responsible for increasing the nitric oxide production , which is known to have important endothelial vasodilator effects .Banana compounds such as gallocatechin gallate as well as campesterol and stigmasterol reduce cholesterol absorption, whereas dopamine and catechin improve resistance of LDL to oxidation, and quercetin, ferulic acid and catechins have arterial vasodilatory effects .Catechin and epicatechin also decreased the expression of vascular endothelial adhesion molecules and e-selectin, but sustainably increased endothelial-nitric oxide synthase and nitric oxide vascular releasing .Catechin, epicatechin and other two polyphenolics were able to decrease the expression of inflammatory cytokines , to inhibit NFκB, to induce the expression of the nuclear releasing factor-2 (Nrf-2), to substantially decrease both the production of nitric oxide and peroxynitrite (by inhibition of nitric oxide synthase) and the amount of oxygen free radicals due to inhibition of myeloperoxidase and direct free radical-scavenging mechanisms .Banana phytosterols, tocopherols and chlorogenic acid as anti-atherosclerotic compoundsM. acuminata, M. balbisiana and M. cavendish are rich in β-sitosterol, ω-3 and ω-6 fatty acids, and contain substantial amounts of α-tocopherol .Bananas of Tocopherols can scavenge ROS protecting LDL from oxidation and decreasing inflammation on the endothelial surfaces what was associated with reduced risk of atherosclerosis .Also found in bananas, ß-sitosterol can potently lower inflammation, fat accumulation and decreased atherosclerosis, lowering both plasma and liver cholesterol .Chlorogenic acid, found in coffee and bananas, inhibited the oxidative enzymes cycloxigenase-1 and inducible form of nitric oxide synthase (iNOS), and partially suppressed the release of inflammatory cytokines , effects in part mediated by the NFκB .A literature review pointed out that chlorogenic acid has potential benefits against atherosclerosis because it decreases endothelial inflammatory biomarkers, thromboxane A2 synthesis and oxidative/nitrosative stresses, and it improves blood pressure and endothelial functions .In vivo studies with banana feeding and atherosclerotic markers evaluationM. paradisiaca) had decreased liver and heart levels of triglycerides, phospholipids, free fatty acids, cholesterol and MDA .Rats feeding a flavonoid rich extract from banana .After administration of indomethacin rats presented increased levels of oxidative stress as measured by increased levels of lipid peroxidation (MDA content) and decreased glutathione content. In the same study, after feeding rats with M. sapientum) peel extract completely restored the HDL levels and partially recovered total cholesterol, triglycerides and LDL levels towards the normal levels .In a Nigerian study, Wistar rats were separated in groups with a control, a hypercholesterolemic control, and subgroups feeding different banana species. It was found that banana .M. sapientum) partially restored total cholesterol, LDL, VLDL, and HDL, decreasing MDA and increasing SOD, CAT, and GSH .In Wistar rats submitted to a cholesterol rich diet, feeding a methanolic sterol-rich extract from banana .Dietary intake of both flower and banana inflorescence from M. cavendish and M. acuminata had a significant hypoglycemic effect in Wistar rats, and antioxidant capacity in vitro as measured by scavenging of DPPH radical, ferric reducing power, and hydrogen peroxide assay .Extracts of , , it is important to note that hypoglycemic effect of bananas can contribute to decreased coronary artery disease risk.Since hyperglycemia is a risk factor for atherosclerosisConsidering some functional properties of banana, Kandasamy et al. (2016) found that banana crude extracts inhibited platelet aggregation. Since atherosclerosis origin and progression depend on inflammatory leukocyte recruiting and macrophage activation, banana peel compounds were found to inhibit both the expression of NFkB and chemoattractant and adhesion molecules; they were also associated with enhanced production of endothelial isoform of nitric oxide synthase (e-NOS), favoring vasodilation .Human clinical studies with banana feeding and atherosclerotic markers evaluationA clinical study with hypercholesterolemic subjects revealed only a small beneficial effect of eating bananas on glycemic and LDL/HDL ratio .In a randomized controlled clinical trial, the experimental group received nutritional counseling and 40 g of green banana biomass, whereas the control group received only a diet. After six months, there was a decrease in body mass index, fasting glucose, and systolic blood pressure only in the experimental group, whereas in both groups there were a decrement in waist and hip circumferences, fat mass (%), and fat intake .More clinical studies are needed in order to evaluate possible anti-atherosclerotic benefits of eating bananas.Based on research previously described regarding banana bioactive compounds, the suggested anti-atherosclerotic biological mechanisms are listed in Considering the major protective antioxidant, anti-atherosclerotic and cardiovascular mechanisms, It is important to note that until today, there is lower quantity of experimental animal studies and only a few clinical studies that evaluated possible anti-atherosclerotic effects of banana supplementation. More research on antioxidant and cardioprotective effects is necessary considering the different banana species, varieties, postharvest conditions, and the diverse banana food preparations and products. Knowledge of the mechanisms suggested here can stimulate more research on the antioxidant and cardiovascular protective mechanism of banana and its bioactive compounds.The authors have declared that there is no conflict of interest.


Polyurethane is widely used to toughen epoxy resins due to its excellent comprehensive properties and compatibility. However, some demerits of polyurethanes limit their applications, such as the harsh storage condition of isocyanate-terminated polyurethane (ITPU), the limited amount of ITPU in epoxy resin, and using solvents during the preparation of polyurethane-modified epoxy resins. To address these issues, in this study, we reported a facile and green approach for preparing epoxy-terminated polyurethane (EPU)-modified epoxy resins with different EPU contents. It was found that the toughness of the epoxy resin was significantly improved after the addition of EPU. When the EPU content was 30 wt%, the elongation at break and toughness were improved by 358.36% and 73.56%, respectively. In comparison, the toughening effect of EPU outperformed that of ITPU. Moreover, the high content of EPU did not significantly decrease the glass transition temperature and had little effect on the thermal stability of the epoxy resin. Epoxy resin (EP) has been extensively utilized in various fields, such as machinery manufacturing, aerospace, electronics, and civil construction, due to its exceptional mechanical properties, high adhesion strength, corrosion resistance, electrical insulation, and favorable processing ability ,2,3,4. TGenerally, the toughness of epoxy resin can be improved by adding toughening agents, such as thermoplastic materials , hyperbrPolyurethane (PU) is a common polymer for constructing IPN or SIPN structures in epoxy matrix. In 1974, Frisch first reported the modification of EP with PU to form an IPN structure . Since tIn this study, an EPU was synthesized by the solventless method, and no solvent was used during the preparation of EPU-modified epoxy resin , which i−1) was supplied by Jiangsu Sanmu Group Co., Ltd. . Hydroxyl-terminated polyether (DL−220 Mw: ~2000 g mol−1) was purchased from Guangzhou Baining New Materials Co., Ltd. . Glycidol (purity 96%) was obtained from Shanghai Aladdin Bio−Chem Technology Co., Ltd. . Toluene diisocyanate (TDI) (purity 99%), Dibutyl tin dilaurate (DBTDL) (purity 95%), and polyetheramine  were purchased from Shanghai Macklin Biochemical Co., Ltd. . Hydrochloric acid (HCl) (purity 37%) and acetone (purity 99%) were purchased from Nanjing Chemical Reagent Co., Ltd. . Sodium hydroxide (NaOH) (purity 96%) was received from Xilong Scientific Co., Ltd. . The chemical structures of DGEBA and polyetheramine D230 are shown in the Diglycidyl ether of bisphenol A  disappeared in the FT−IR spectra  at 120 °C for 2 h. The dried DL−220 (300 g) and TDI (52.20 g) were heated to 80 °C. After 1 h of reaction, two drops of catalyst DBTDL were added and the reaction was continued until the NCO content decreased to half of the initial (about 2 h) to obtain ITPU. The NCO content was detected in the reaction by using the di N-butylamine technique . Then, 1 spectra . A samplFirstly, DGEBA/EPU mixtures with different EPU contents were prepared by mechanically mixing DGEBA with EPU via IKA RW20  at around 1000 rpm for 10 min. Due to the fact that EPU was used to modify epoxy resin, the highest EPU content was 50 wt%. When the EPU content exceeded 50 wt%, DGEBA was employed to modify the EPU instead, which was contrary to the original goals. Consequently, we set 50 wt% as the maximum EPU content. Based on the EPU content, these samples were named EPU10, EPU20, EPU30, and EPU50. Secondly, according to the epoxy value of different DGEBA/EPU mixtures , a prede−1. FT-IR analysis was conducted with a scanning of 16 and a resolution of 4 cm−1. The dynamic mechanical analysis (DMA) was performed using the experimental instrument Q800  in dual cantilever mode from −50 to 150 °C at a heating rate of 3 °C min−1 and an oscillating frequency of 1 Hz. The size of samples for the DMA test was 60 mm × 10 mm × 4 mm. The thermostability of materials was analyzed by a thermogravimetric analyzer (TGA), which was performed on a TG209F1 TGA . The TGA test was operated in a nitrogen atmosphere from room temperature to 800 °C at a heating rate of 20 °C min−1. The surface morphology of the samples was investigated by a scanning electron microscope . Surfaces prepared by tensile test fracture samples were used to study the effect of toughening agents on the phase morphology. In order to obtain clearer phase morphology information of the fracture surface, all fracture surfaces were gold-coated with an E-1010 ion sputter , and the thickness of the coating was 10−20 nm before examination.Fourier-transform infrared (FT−IR) spectroscopy was performed using the Nicolet iS50 , with a range of 4000−500 cm−1. According to the international ISO 527−3:2018, dumbbell-shaped samples were prepared. From the viewpoint of energy, the integral area of the stress−strain curve can be used to represent the fracture absorption energy of the tensile test [−1 according to ISO 178:2019. Three−point bending specimens (100 mm × 10 mm × 4 mm) were used to determine flexural strength. The equipment for the bending test was the same as that for the tensile test. All mechanical tests were performed at 25 °C. At least five replicates were tested for each group of samples and average values were reported.The tensile test was carried out on the microcomputer-controlled electronic universal testing machine  at a crosshead speed of 5 mm minile test . The tou−1 in DL−220 disappears after the reaction and a new carbamate group  appears. Secondly, the isocyanate group at the end of ITPU reacts with glycidol to obtain EPU. Therefore, after the reaction, the isocyanate absorption peak of ITPU at 2271 cm−1 disappears and an epoxy group (910 cm−1) appears. The results of structural characterization prove the successful synthesis of EPU.The structures of TDI, DL−220, and EPU were analyzed by FT−IR. Tg). From the tan delta versus temperature curves , which is calculated from the rubber elasticity equation as follows [R is the gas constant, and T and E′ are the absolute temperature and storage modulus at Tg + 30 °C, respectively. The cross-linking densities for the neat epoxy resin and the modified epoxy resin are shown in Tg. Consistent with the variation trend of ν, Tg declines with increasing EPU content.The viscoelastic properties of the modified epoxy resins were determined by DMA . Similare curves b, it is  follows :(1)ν=E′3The storage modulus refers to the energy stored due to elastic deformation when the material is deformed, which can be used to reflect the elasticity of the material and characterize the stiffness of the material. As shown in Tg in the tan delta curve can reflect the dispersion of chain segment motion. As shown in Although the introduction of EPU into DGEBA does not cause phase separation b, it sti−3, which is 28.27% higher than that of the neat epoxy resin (3.29 ± 0.24 MJ m−3). To highlight the effect of EPU on toughness, ITPU10 was set as the control group, which contains 10 wt% ITPU. ITPU is linked to the internal epoxy network by pre-reaction with the hydroxyl group of DGEBA (−3) does not have a toughening effect at such a low dosage. Comparing the same amount of EPU and ITPU, the elongation at break of ITPU-modified epoxy resin is only half of that of EPU-modified epoxy resin and the toughness of ITPU-modified epoxy resin is much lower than that of EPU-modified epoxy resin. Unlike the change trend of elongation at break and toughness, other properties such as tensile strength . The specific parameters from TGA and DTG are listed in T5% decreases with increasing EPU content. This is because EPU has many C−C and C−O bonds, which are less stable than the aromatic structure. Consequently, epoxy resins with EPU will decompose at low temperatures. However, compared to neat epoxy, the decrease is not significant. It can be seen that the T5% of modified epoxy resin is above 330 °C, which is much higher than the processing temperature of the polymer. Therefore, the modified epoxy is stable during processing.The thermostability of epoxy resins with the addition of the EPU was also examined, as shown in Tm). The Tm of all samples is similar, and the content of EPU has little effect on Tm. The relatively poor stability of the polyether chain in EPU results in its decomposition at a low temperature. However, since the decomposition temperature of the polyether chain and the aromatic structure in DGEBA are not significantly different, the decomposition of both EPU and DGEBA co-occurs with increasing temperature, resulting in no significant change in Tm [The peak of the DTG curve corresponds to the maximum degradation rate temperature (ge in Tm . Additioge in Tm . HoweverDm) exhibits a decline trend. The polyether chain in EPU has more alkyl side chains than DGEBA and D230, and these side chains begin to break down at low temperatures. The Dm relates to the decomposition of the epoxy network, which starts to break down as the temperature rises. As a result, there is a declining trend of the Dm.From In this study, a solventless epoxy-terminated polyurethane (EPU) was synthesized and the preparation of EPU-modified epoxy resins was also environmentally friendly. It was found that the epoxy resins can be modified by any amount of EPU. In this study, the EPU content reached its highest content (50 wt%). For ITPU, liquid epoxy resin without hardener quickly formed a solid after the addition of 50 wt% ITPU. The comprehensive properties of EPU-modified epoxy resins were studied systematically and compared to the epoxy resins modified by ITPU with the same molecular structure as EPU. It was found that the toughness of epoxy resin significantly improved after the addition of EPU and the toughening effect of EPU was superior to that of ITPU. After the addition 10 wt% EPU, the elongation at break and toughness increased by 67.5% and 28.3%, respectively. In comparison, the elongation at break and toughness of epoxy resins after the addition of 10 wt% ITPU reduced by 15.81% and 7.29%, respectively. For thermal properties, they decreased after the addition of the EPU but remained at a high temperature. In addition, the thermal properties of EPU-modified epoxy resins were comparable with ITPU-modified epoxy resins at the same modifier content. These results demonstrate that the terminal epoxidation of polyurethane can solve the problems of conventional polyurethane-modified epoxy resins.