Kluyveromyces marxianus is a potentially excellent host for microbial cell factories using lignocellulosic biomass, due to its thermotolerance, high growth rate, and wide substrate spectrum. However, its tolerance to inhibitors derived from lignocellulosic biomass pretreatment needs to be improved. The prefoldin complex assists the folding of cytoskeleton which relates to the stress tolerance, moreover, several subunits of prefoldin have been verified to be involved in gene expression regulation. With the presence of inhibitors, the expression of a gene coding the subunit 4 of prefoldin (KmPFD4), a possible transcription factor, was significantly changed. Therefore, KmPFD4 was selected to evaluate its functions in inhibitors tolerance.KmPFD4) led to increased concentration of intracellular reactive oxygen species (ROS) and disturbed the assembly of actin and tubulin in the presence of inhibitors, resulting in reduced inhibitor tolerance. Nuclear localization of KmPFD4 indicated that it could regulate gene expression. Transcriptomic analysis showed that upregulated gene expression related to ROS elimination, ATP production, and NAD+ synthesis, which is a response to the presence of inhibitors, disappeared in KmPFD4-disrupted cells. Thus, KmPFD4 impacts inhibitor tolerance by maintaining integration of the cytoskeleton and directly or indirectly affecting the expression of genes in response to inhibitors. Finally, overexpression of KmPFD4 enhanced ethanol fermentation with a 46.27% improvement in productivity in presence of the inhibitors.In this study, the disruption of the prefoldin subunit 4 gene (KmPFD4 plays a positive role in the inhibitor tolerance and can be applied for the development of inhibitor-tolerant platform strains.This study demonstrated that The online version contains supplementary material available at 10.1186/s12934-021-01715-y. Though Cu/Zn superoxide dismutase (SOD1), nicotinamide-nucleotide adenylyltransferase 1 (NMNAT), NADH pyrophosphatase (NUDC), and multicopy suppressor of SNF1 mutation (MSN2) were up-regulated in both YSY007 and YSY003, the changes in YSY003 was less than that in YSY007. Peroxisomal catalase A(CTA1) and citrate synthase (CIT1) were up-regulated in YSY007, and down-regulated in YSY003. ATP synthase subunit 4 (ATP4) was down-regulated in both YSY003 and YSY007. Glucose-6-phosphate isomerase (PGI1) and glyceraldehyde-3-phosphate dehydrogenase 3 (TDH3) were up-regulated in both YSY003 and YSY007. Though the expression changes of the analyzed genes were not the same as RNA-seq results, their trends of the changes in real-time PCR were similar to those in RNA-seq , especially those DEGs related to ROS detoxification, central carbon metabolism, mitochondrial respiratory chain, NAD(P)+ biosynthetic enzymes, and nicotinate metabolism. There were some changes of the gene expression with the presence of inhibitors when KmPFD4 was disrupted. Though it was still difficult to make a conclusion that KmPFD4 regulated the gene expression, KmPFD4 did affect the gene expression directly or indirectly.Based on these results, we focused on the DEGs in KmPFD4-disrupted strain in the presence of inhibitors. Since ROS is produced during the metabolism of inhibitors [SOD1, SOD2, PRX1, GPX2, peroxiredoxin (HYR1), thioredoxin reductase(TRR1), glutamate–cysteine ligase (GSH1), superoxide dismutase 1 copper chaperone (CCS1), glutaredoxin-1 (GRX1), monothiol glutaredoxin-5 (GRX5), peptide methionine sulfoxide reductase (PMSR), NADPH-dependent methylglyoxal reductase (GRE2), thioredoxin-2 (TRX2), thioredoxin-3 (TRX3), and putative uncharacterized oxidoreductase gene (WCAG), while cytosolic catalase T (CTT1), peroxiredoxin (DOT5), glutathione S-transferase 1 (GST1), and Cys-Gly metallodipeptidase (DUG1) were downregulated in the YSY007-I vs. YSY007-C group, as previously reported [KmPFD4-disrupted YSY003-I vs. YSY003-C group, although the expression of GPX2, HYR1, TRR1, TRX1, and GRE2 were upregulated as in the YSY007-C vs. YSY007-I group, most genes were not significantly regulated in the YSY003-C vs. YSY003-I group , external NADH-ubiquinone oxidoreductase 1 (NDH1), SDH1/2/3/4, and ubiquinol cytochrome-c reductase complex (QCR9) were upregulated and there was no significant change in QCR1, cytochrome b-c1 complex subunit Rieske (RIP1), QCR6, and ATP4 in the control (YSY007-I vs YSY007-C) pairwise comparison. Whereas in the YSY003-I vs. YSY003-C group, there was no significant change in NDI1 or SDH1/2/3/4. Furthermore, QCR1, RIP1, QCR6, QCR9, and ATP4 were downregulated. The expression of the counterpart genes in the YSY003-I vs. YSY003-C group was downregulated compared with those in the YSY007-I vs. YSY007-C group. These results suggested that with the stress of multiple inhibitors, disruption of KmPFD4 reduced the expression of the genes of the respiratory chain and may hinder ATP generation further. In previous studies, in the presence of inhibitors, the expression of genes related to ATP production was upregulated due to increased ATP required for inhibitors degradation and removal [+. The shortage of ATP is expected to reduce inhibitor tolerance.The disruption of  removal , 27. How removal , disrupt removal , non-spe removal , and dec removal . In addi+ biosynthetic enzymes and nicotinate metabolism were not upregulated in the KmPFD4-disrupted strain compared to the control strain. The essential coenzymes nicotinamide adenine dinucleotides, NAD(P)+ and NAD(P)H, participate in key redox reactions and contribute to maintaining cells fitness and genome stability [+ is important in lignocellulosic biomass-derived inhibitor tolerance and is the source of total NAD(P)+ and NAD(P)H [+ biosynthetic enzymes and related proteins were upregulated, except PNC1 encoding nicotinamidase in the YSY007-I vs. YSY007-C group. However, in the YSY003-I vs. YSY003-C group, these genes were not significantly regulated except for nucleoside transporter FUN26 which was downregulated. This may imply that the NAD+ biosynthesis in the KmPFD4-disrupted strain was not enhanced as those in the wild-type strain in response to the presence of inhibitors. Therefore, the supply of NAD+ in the KmPFD4-disrupted strain was not improved with the increased demand for inhibitor tolerance, and the tolerance ability was decreased.The genes related to NADtability . NAD+ is NAD(P)H . Under tKmPFD4-disrupted strain. In contrast, most of the genes related to glycolysis were down-regulated or no significant change in the non-disrupted strain which was also reported in S. cerevisiae [PGI1, 6-phosphofructokinase subunit (PFK1/2), triosephosphate isomerase (TPI), TDH3, phosphoglycerate kinase (PGK), enolase (ENO), pyruvate kinase (PYK), Pyruvate decarboxylase (PDC) etc., were upregulated in the YSY003-I vs. YSY003-C group, while there was no significant change in YSY007-I vs. YSY007-C pairwise comparison.In the presence of inhibitors, the genes related to glycolysis were up-regulated, in the revisiae . CentralSDH1/2/3/4 and malate dehydrogenase (MDH1/2) were upregulated and there was no significant change to IDP2 in YSY007-I vs. YSY007-C pairwise comparison. On the other hand, in the YSY003-I vs. YSY003-C group, there was no significant change in the expression level of SDH1/2/3/4 and MDH1. Furthermore, MDH2 and IDP2 were downregulated. Likewise, isocitrate lyase (ICL1) was downregulated in YSY003-I vs. YSY003-C pairwise comparison, but there was no significant change in YSY007-I vs. YSY007-C pairwise comparison H, which are necessary for inhibitors removal and conversion. Our previous study have shown that the intracellular concentrations of ATP and NAD(P)H decreased due to the consumption of inhibitors [KmPFD4 led to a severe shortage of ATP and NAD(P)H and finally reduced the inhibitor tolerance.On the other hand, among those DEGs in the TCA cycle, hibitors . The disKmPFD4 decreased the respiration. On the plates, the YSY007 colonies were red, whereas, the color of YSY003 colonies was lighter than that of YSY007. And the white colonies indicated much weaker respiration. These results illustrated that the respiration of K. marxianus was interfered after KmPFD4 disruption  is intricate. Transcriptional activator MSN2 in S. cerevisiae regulates the transcription of the genes associated with oxidative stress, heat shock, and high concentration of ethanol [K. marxianus were also analyzed through RNA-seq. MSN4 was not found in the genome of K. marxianus, and the KmMSN2 expression was downregulated in YSY003 with the presence of inhibitors, whereas in YSY007, its expression was upregulated , glycogen phosphorylase (GPH1), heat shock protein SSA3, aldehyde dehydrogenase 5 (ALD5), heat shock protein 26 (HSP26) and heat shock protein 31 (HSP31) etc. were similar in both pairwise comparisons, most regulations of DEGs were different between the pairwise comparisons, and these genes also presented in ROS or central carbon metabolism groups H synthesis, and the TCA cycle H occurred, and led to decreased detoxication ability to inhibitors and reduced ROS elimination ability. Thus, the inhibitor tolerance of yeast cells was decreased.The transcriptome analysis results indicated that the disruption of KmPFD4 disruption on the tolerance of K. marxianus to lignocellulosic biomass-derived inhibitors was determined, its effect on ethanol, temperature, and osmotic stress was evaluated by measuring their growth , whereas YSY007 only produced 32.23 g/L ethanol in 48 h with a productivity of 0.67 g/(L·h) . These results were consistent with other reports that the inhibitors delayed yeast fermentation and did not significantly reduce the ethanol yield [KmPFD4.As overexpression of ol yield , 65. TheKmPFD4 affects the tolerance of K. marxianus to lignocellulosic biomass-derived inhibitors by maintaining the structure of the cytoskeleton and regulating the genes expression in response to ROS elimination and inhibitors degradation. Disruption of KmPFD4 led to a more severely disordered cytoskeleton and reduced the viability of the cells in the presence of inhibitors. Furthermore, the disruption of KmPFD4 led to a decrease in genes expression in response to the presence of inhibitors though the mechanism of KmPFD4 affecting gene expression needs further study. Thus, the supply of ATP and NAD(P)H required for inhibitor tolerance is decreased. Overexpression of KmPFD4 enhanced the restoration of the disturbed cytoskeleton and improved inhibitor tolerance. Therefore, KmPFD4 affects the tolerance to inhibitors in improving the cytoskeleton assembly and related gene expression. The study on KmPFD4 is related to a wide spectrum of stress tolerance, which provides a potential route to improve the tolerance of yeast to multiple lignocellulosic biomass-derived inhibitors.Additional file 1. Functional categories and fold changes of comparative expression (in form of log2FC) of differentially expressed genes in tolerance to multiple inhibitors.Additional file 2. Additional figures and tables.


Background: C-X-C chemokine receptor type 4 (CXCR4) plays a crucial role in mediating podocyte dysfunction, proteinuria and glomerulosclerosis. However, the underlying mechanism remains poorly understood. Here we studied the role of β-catenin in mediating CXCR4-triggered podocyte injury.Methods: Mouse models of proteinuric kidney diseases were used to assess CXCR4 and β-catenin expression. We utilized cultured podocytes and glomeruli to delineate the signal pathways involved. Conditional knockout mice with podocyte-specific deletion of CXCR4 were generated and used to corroborate a role of CXCR4/β-catenin in podocyte injury and proteinuria.Results: Both CXCR4 and β-catenin were induced and colocalized in the glomerular podocytes in several models of proteinuric kidney diseases. Activation of CXCR4 by its ligand SDF-1α stimulated β-catenin activation but did not affect the expression of Wnt ligands in vitro. Blockade of β-catenin signaling by ICG-001 preserved podocyte signature proteins and inhibited Snail1 and MMP-7 expression in vitro and ex vivo. Mechanistically, activation of CXCR4 by SDF-1α caused the formation of CXCR4/β-arrestin-1/Src signalosome in podocytes, which led to sequential phosphorylation of Src, EGFR, ERK1/2 and GSK-3β and ultimately β-catenin stabilization and activation. Silencing β-arrestin-1 abolished this cascade of events and inhibited β-catenin in response to CXCR4 stimulation. Podocyte-specific knockout of CXCR4 in mice abolished β-catenin activation, preserved podocyte integrity, reduced proteinuria and ameliorated glomerulosclerosis after Adriamycin injury.Conclusion: These results suggest that CXCR4 promotes podocyte dysfunction and proteinuria by assembling CXCR4/β-arrestin-1/Src signalosome, which triggers a cascade of signal events leading to β-catenin activation. Podocyte injury and proteinuria are the major pathological features of glomerular disease, which accounts for the vast majority of chronic kidney disease (CKD) leading to end-stage renal failure. As a key component of the glomerular filtration apparatus, the integrity of podocytes and their foot processes and slit diaphragm is essential for preventing proteinuria Although many cues can cause podocyte injury, increasing evidence demonstrates that oxidative stress is an important common culprit of podocyte damage and proteinuria Wnt/β-catenin is evolutionarily conserved signal pathway that plays an important role in regulating embryo development, tissue homeostasis, stem cell self-renewal, and tumorigenesis In this study, we report that CXCR4 in glomerular podocytes activates β-catenin signaling via forming a CXCR4/β-arrestin-1/Src signalosome. Podocyte-specific deletion of CXCR4 inhibits β-catenin activation, reduces proteinuria, ameliorates podocyte injury and glomerulosclerosis. Our results suggest that targeting CXCR4/β-arrestin-1/β-catenin may be a new strategy for the treatment of proteinuric CKD.NPHS2) promoter (Podo-Cre) was also obtained from Jackson Laboratories (Stock No. 008205). By mating CXCR4-floxed mice with Podo-Cre transgenic mice, conditional knockout mice (Podo-CXCR4-/-) in which CXCR4 gene was specifically disrupted in podocyte  were created. These mice were crossbred with homozygous CXCR4-floxed mice to generate offspring with 50% Podo-CXCR4-/- mice and 50% control mice  within the same litters. A routine PCR protocol was used for genotyping of tail DNA samples with the primer pairs as described in Homozygous CXCR4-floxed mice in C57BL/6J background were obtained from the Jackson Laboratories . Transgenic mice expressing Cre recombinase under the control of human podocin . For ADR nephropathy model, BALB/c mice were administered by a single intravenous injection of ADR  at 10 mg/kg body weight. Groups of mice (n=6) were euthanized at 1 and 3 weeks after ADR injection, and kidney tissues collected for various analyses. Because mice with C57BL/6J background are resistant to ADR nephropathy, podo-CXCR4-/- and CXCR4-floxed (control) mice were administered with two intravenous injections of ADR at 15 mg/kg body weight, one week apart. Groups of mice (n=7) were euthanized at 2 weeks after ADR injection, and kidney tissues collected for various analyses. In addition, mouse models of proteinuric kidney diseases including angiotensin II infusion (520 ng/min/kg for 4 weeks), db/db mice (5 months) and remnant kidney model after 5/6 nephrectomy (5/6NX) (8 weeks) were used, as described previously The enzyme-linked immunosorbent assay (ELISA) kit for mouse SDF-1α was purchased from the Cusabio Company . Serum SDF-1α level was measured according to the assay procedures specified by the manufacturer. The enzymatic reaction products were quantified in an automated microplate reader. Serum SDF-1α levels were expressed as nanogram per milliliter.The conditionally immortalized mouse podocyte cell line (MPC5) was provided by Peter Mundel . To propagate podocytes, cells were cultured at 33 °C in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 10 units/ml mouse recombinant IFN-γ  to enhance the expression of a thermosensitive T antigen. To induce differentiation, podocytes were grown under nonpermissive conditions at 37° C in the absence of IFN-γ. MPC5 cells were treated with SDF-1α  with different doses for various periods of time as indicated. For some experiments, MPC5 cells were pretreated with CXCR4 inhibitor AMD3100  at 5 µg/mL, ICG-001  at 5 µM and MEK1/2 inhibitor U0126  at 10 µM. For some studies, podocytes were transiently transfected with ON-TARGET plus SMART pool siRNA (400 pM) specific for mouse β-arrestin-1 or control nontarget siRNA using Lipofectamine 2000 reagent according to the manufacturer's instruction (Invitrogen). Forty-eight hours after transfection, podocytes were exposed to 100 ng/mL SDF-1α for 6 h and harvested for various analyses.Glomeruli were isolated by differential sieving technique from male Sprague Dawley rats (Harlan Sprague Dawley), as previously reported Urinary albumin was measured by using a mouse Albumin ELISA Quantitation kit, according to the manufacturer's protocol . Urinary and serum creatinine levels were determined by use of a QuantiChrom creatinine assay kit , according to the manufacturer's protocols. Urinary albumin was standardized to urine creatinine and expressed as mg/mg Ucr.Paraffin-embedded mouse kidney sections (3 µm thickness) were prepared and stained with periodic acid-Schiff (PAS) and Masson's trichrome staining (MTS) reagents. Immunohistochemical staining was performed using established protocols, as described previously Human kidney specimens were collected from diagnostic kidney biopsies performed at Nanfang Hospital, Southern Medical University. Non-tumor renal tissues from patients who had renal cell carcinoma and underwent nephrectomy were used as a normal control. Human kidney cryosections (3 μm thickness) were prepared by a routine method. The studies involving human samples were approved by the Medical Ethics Committee at the Nanfang Hospital, Southern Medical University.Kidney cryosections or cells cultured on coverslips were fixed with 4% paraformaldehyde for 15 min at room temperature. After blocking with 10% donkey serum for 1 h, the slides were immunostained with various primary antibodies as described in Protein expression was analyzed by Western blot analysis, as described previously The interaction of CXCR4 or Src with β-arrestin-1 in mouse podocytes treated with SDF-1α for 5 min was determined by co-IP as previously described Total RNA isolation and real-time quantitative qRT-PCR were carried out by procedures described previously P < 0.05 was considered significant.All data examined were expressed as mean ± standard error of the mean. Statistical analysis of the data was carried out using SPSS 19.0, . Comparison between groups was made using one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls test or Dunnett's T3 procedure. ADR nephropathy, a model of human FSGS, is characterized by podocyte injury, proteinuria and glomerulosclerosis Immunohistochemical staining confirmed that both SDF-1α and CXCR4 were upregulated in glomerular podocytes and, to a less extent, tubular epithelial cells after ADR injection  were treated with various doses of SDF-1α, the ligand of CXCR4. As illustrated in Figure To investigate the relationship between β-catenin and CXCR4, we examined the regulation of β-catenin after CXCR4 activation in cultured podocytes We then investigated the expression of the Wnt ligands in mouse podocytes. As shown in Figure We further investigated β-catenin localization and its functional consequence after SDF-1α stimulation. As shown in Figure We sought to delineate how CXCR4 activation by SDF-1α leads to an increased β-catenin signaling in podocytes. Earlier studies suggest that β-arrestin-1, a scaffold protein, plays a crucial role in mediating the signal transduction of G protein-coupled receptor We next investigated the potential role of β-arrestin-1 in SDF-1α/CXCR4-induced β-catenin activation. Co-immunoprecipitation (co-IP) studies showed that SDF-1α promoted the association between CXCR4 and β-arrestin-1 Figure A, which We further studied the downstream signaling of CXCR4/β-arrestin-1/Src signalosome after SDF-1α stimulation in podocytes. As activation of Src leads to epidermal growth factor receptor (EGFR) transactivation in vivo, we used glomerular mini-organ culture, an ex vivo model system that largely preserves the sophisticated three dimensional architecture of podocytes in the kidney To closely model podocytes in vivo. To this end, we generated conditional knockout mice in which CXCR4 gene is specifically disrupted in podocytes. Homozygous CXCR4-floxed mice were mated with podo-Cre transgenic mice expressing Cre recombinase under the control of podocin promoter . Age- and sex-matched CXCR4-floxed mice (genotype: CXCR4fl/fl) from the same litters were used as controls (We sought to determine the potential role of CXCR4 in podocyte physiology and pathology r Figure A. As shocontrols A.in vivo.Mice with podocyte-specific deletion of CXCR4 were phenotypically normal. There was no appreciable abnormality in body weight B and urith and 13th day, respectively.We next investigated the role of CXCR4 in podocyte injury by challenging podo-CXCR4-/- mice with ADR. As shown in Figure We examined CXCR4 expression in the kidneys of podo-CXCR4+/+ and podo-CXCR4-/- mice at 2 weeks after ADR injection. As shown in Figure in vivo, we examined β-catenin activation and its downstream target proteins in podo-CXCR4+/+ and podo-CXCR4-/- mice after ADR injury. Immunohistochemical staining revealed that total β-catenin, active β-catenin and its downstream MMP-7 were highly expressed in glomerular podocytes of podo-CXCR4+/+ mice at 2 weeks after ADR injection (Figure in vivo.To establish the connection between podocyte injury induced by CXCR4 and β-catenin signaling n Figure . These rWe finally investigated the effect of podocyte-specific ablation of CXCR4 on glomerulosclerotic lesions in ADR nephropathy. As illustrated in Figure in vitro or ADR in vivo recruits β-arrestin-1 and Src to form the CXCR4/β-arrestin-1/Src signalosome, which transactivates EGFR leading to subsequent phosphorylation of ERK1/2 and GSK-3β. The phosphorylation of GSK-3β leads to its inactivation, resulting in the dephosphorylation and stabilization of β-catenin (Figure In this study, we show that activation of CXCR4 by SDF-1α An interesting finding of the present study is that the SDF-1α/CXCR4 induces podocyte injury by activating β-catenin through a Wnt-independent pathway. Although increasing evidence indicates that Wnt/β-catenin is implicated in mediating proteinuric CKD including ADR nephropathy, most previous studies have focused on the canonical Wnt signaling The present study also delineates the mechanism how CXCR4 activates β-catenin in a Wnt-independent manner in podocytes. As a G protein-coupled receptor, activation of CXCR4 renders it to recruit scaffold protein β-arrestin-1, which can regulate CXCR4-mediated signaling ex vivo (Figure This study has also uncovered how the formation of CXCR4/β-arrestin-1/Src signalosome leads to β-catenin activation. It appears that the formation of CXCR4/β-arrestin-1/Src signalosome triggers a sequential phosphorylation of Src, EGFR, ERK1/2 and GSK-3β (Figure o Figure .in vivo. Consistent with podocyte-specific deletion of β-catenin The important role of CXCR4 in podocyte injury and proteinuria is unambiguously confirmed in mice with podocyte-specific ablation of CXCR4 The present study has some limitations, in which only ADR model of podocyte injury and proteinuria was used. In view of that CXCR4 and β-catenin are upregulated in the glomeruli of several CKD models Figure , future In summary, we show herein that CXCR4 mediates podocyte injury and proteinuria by activating β-catenin via a cascade of events involving β-arrestin-1, Src, EGFR, ERK1/2, GSK-3β. Podocyte-specific deletion of CXCR4 blunts β-catenin activation, ameliorates podocyte injury, reduces proteinuria and glomerulosclerosis, thereby halting the progression of CKD. This study provides significant insights into the mechanism by which CXCR4 mediates podocyte injury. Targeting any key step in the cascade of this signaling may open new avenues for developing the treatment of proteinuric kidney diseases.Supplementary figures and tables.Click here for additional data file.


CCK8 assay was used to detect the proliferation of glioma cells. Flow cytometry was used to test cell apoptosis and distribution of cell cycle. To detect the migration and invasion of glioma cells, wound healing assay and Transwell were performed. It was found that downregulation of TET1 could promote the proliferation migration and invasion of glioma cells and the concomitant upregulation of β-catenin, and its downstream targets like cyclinD1 and c-myc were observed. The further rescue experiments were performed, wherein downregulation of β-catenin markedly decreases glioma cell proliferation in vitro and in vivo. This study confirmed the tumor suppressive function of TET1 and illustrated the underlying molecular mechanisms regulated by TET1 in glioma.Glioma is the most common malignant tumor in adult brain characteristic with poor prognosis and low survival rate. Despite the application of advanced surgery, chemotherapy, and radiotherapy, the patients with glioma suffer poor treatment effects due to the complex molecular mechanisms of pathological process. In this paper, we conducted the experiments to prove the critical roles TET1 played in glioma and explored the downstream targets of TET1 in order to provide a novel theoretical basis for clinical glioma therapy. RT-qPCR was adopted to detect the RNA level of TET1 and  As shown in previous studies, modification of DNA plays critical roles in cell proliferation, apoptosis, tumorigenesis, and metastasis , 2. Meth5hmC is abundant in nervous system, and it has been found in a variety of nerve cells such as neural stem cells, neurons, astrocytes, and Purkinje cells , 8. Exprβ-catenin pathway [Amount of studies suggest that 5hmC and TET1 play significant roles in cancer process. It is proved that TET1 can act as a tumor suppressor in colon cancer because downregulation of TET1 can promote the expression of oncogene axis inhibition protein 1 and cell proliferation in colon cells by targeting  pathway . In oste pathway . In addi pathway .β-catenin which is one of the key regulators of Wnt/β-catenin pathway, and the further rescue experiments confirm the regulatory function of TET1 on Wnt/β-catenin signaling pathway.In accordance with previous studies, our study demonstrates that TET1 is downregulated in malignant glioma (WHO grade IV glioma) specimens, and knock down of TET1 promotes proliferation, migration, and invasion of malignant glioma cells. Results of xenografting prove that downregulation of TET1 inhibits tumor growth in vivo. Furthermore, we also conduct experiments to explore molecular mechanisms mediated by TET1 in tumor growth. It is found that downregulation of TET1 inhibits the expression of Tumor tissues and the adjacent normal tissues were obtained from 24 patients with malignant glioma who received surgery in The Third Affiliated Hospital of Chongqing Medical University . All patients did not received any other therapy before surgery. Informed consent was provided by all patients or their family. This study is in line with the Declaration of Helsinki and approved by the Ethics Review Committee of The Second Affiliated Hospital of Army Medical University .2.Human glioma cell lines U251 and SW1783 cells were purchased from American Type Culture Collection . U251 and SW1783 cells were cultured with Roswell Park Memorial Institute 1640 medium  with 10% fetal bovine serum  in incubator at 37°C, 5% COβ-catenin shRNA were transfected into U251 and SW1783 cells with Lipofectine 2000  according to manufacturer's instruction.Negative control shRNA, TET1 shRNA, and β-catenin, cyclinD1, and c-myc. GAPDH was used as internal control. Primers used for PCR are shown as follows: TET1-forward: 5′-TCTGTCCTGGGGAGACACTG-3′, TET1-reverse: 5′-GAGTCGGTTCCCAAAGGTCC-3′; β-catenin-forward: 5′-GAAGGGGCTGACGCTATTGA-3′, β-catenin-reverse: 5′-GCTCCCAAGGAACGTCATCA-3′. cyclinD1-forward: 5′-ACATGGTGGATGAAGTGGACA-3′, cyclinD1-reverse: 5′-CAAGTGTCCAGAAGGTGTGAC-3′; c-myc-forward: 5′-TTTGCGTCGCCAGGTGAAGA-3′, c-myc-reverse: 5′-GTGTGACCTTGTTTCACTTCCG-3′. GAPDH-forward: 5′-TTTGCGTCGCCAGGTGAAGA-3′, GAPDH-reverse: 5′-AGTTAAAAGCAGCCCTGGTGA-3′.Total RNA of U251 and SW1783 cells was extracted with Trizol regent . RNA was reverse trancripted into cDNA with QuantiTect Reverse Transcription Kit . SuperRT One Step RT-PCR Kit  was taken to detect the expression of TET1, β-catenin , mouse anti-GS3Kβ , mouse anti-p-GSK3β , GAPDH , and the membrane was detected by chemiluminescent ECL reagent .U251 and SW1783 cells were lysed by lysis buffer with RIPA on ice for 30 min and centrifuge at 4°C for 20 min at 12000 g/min. The supernatant was transferred into another microphage tube and quantitated with BCA Protein Assay Kit . Protein samples were separated with SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PDVF) membranes . After being incubated with 5% BSA block buffer, the primary antibody was used as follows: rabbit anti-TET1 , rabbit anti-cyclinD1 , rabbit anti-c-myc , rabbit anti-μl cell culture medium. After culturing for 48 hours, 10 μl CCK8 was added into cell culture medium and incubated at 37°C for 2 h. Calculate the absorbance at 450 nm with a plate reader .Cells transfected with TET1 shRNA were seed on 96-well plates and cultured with 100 β-catenin shRNA were seeded into 6-well plates in density of 1 × 105 cells/well and cultured for 48 h; the wound was scratch with 20 μl pipette. Images were taken at 0 h and 24 h; the width was measured with ImageJ.Cells transfected with TET1 shRNA and β-catenin shRNA were seeded in the density of 1 × 105 cells/well, and the upper chamber was added with 200 μl medium without serum. In the lower chamber, 600 μl cell culture medium with 15%FBS was added. Culture the cells at 37°C for 48 h, and the invasive cells were fixed with 4% paraformaldehyde (PFA) staining with crystal violet.The upper chambers were coated with Matrigel, and the cells transfected with NC shRNA, TET1 shRNA, and Cells transfected with NC shRNA and TET1 shRNA were seeded into 6-well plates in a density of 100 cells/ml. After 14 d culturing, the cells were fixed with 4% PFA and stained with crystal violet.Cells transfected with NC shRNA and TET1 shRNA were collected with 0.25% trypsin and washed with phosphate buffer saline for three times. After that, cells were costained with Annexin V-FITC/PI and detected by flow cytometry.Cells transfected with NC shRNA and TET1 shRNA were collected with 0.25% trypsin and washed with phosphate buffer saline for three times. After that, fix the cells with 70% ethanol for 3 h. PI was used to stain the cells, and flow cytometry was performed to detected the distribution of cell cycle.6 cells stably transfected with NC shRNA, TET1 shRNA, and β-catenin shRNA were injected subcutaneously in flanks of mice with 6 mice each group. After 30 days, mice were euthanized, and the weight of tumors was measured.Animal study was performed in accordance with institutional guidelines of Research Ethics Committee of The Third Affiliated Hospital of Chongqing Medical University  (CQ-023-201601142). During the experiment, the pain of the animals was reduced as much as possible without affecting the results of the experiment. 4-week male BALB/c athymic nude mice bought from Shanghai Slac Laboratory Animal Company were used for in vivo tumor xenograft model. 5 × 10To investigate the expression of TET1 in malignant glioma patients, we detected the relative RNA level of TET1 in WHO grade IV glioma specimens and adjacent normal tissues by RT-qPCR. The results showed the lower expression of TET1 in tumor tissues compared with adjacent normal tissues . AdditioTo explore the roles TET1 played in tumor growth of glioma, we downregulated TET1 by transfecting glioma cells LN-229 and SW1783 with TET1 shRNA. Western blot was adopt to detect the efficiency of TET1 shRNAs . ResultsTo test whether TET1 participates in the migration and invasion of U251 and SW1783 cells, wound healing assay and Transwell were performed. As shown Figures To identify the function of TET1 in cell cycle distribution and cell apoptosis, we downregulated TET1 in U251 and SW1783 cells and performed flow cytometry. As shown in results, deficiency of TET1 induced the G1 cell cycle arrest and enhanced the ratio of G2 cells Figures . And theKnockdown of TET1 could promote the cell proliferation, migration, and invasion in vitro. To detect its function on tumor growth in vivo, we stably transfected the U251 and SW1783 cells with negative control shRNA and TET1 shRNA and injected these cells subcutaneously into nude mice. As shown in Figures β-catenin pathway [β-catenin including β-catenin and phosphorylated GSK3β and the underlying targets of Wnt pathway such as cyclinD1 and c-myc  family which could convert 5mC to 5hmC. Numerous studies have demonstrated the participation of TET1 in the pathogenesis of cancers, and most of the researches are in keeping with our results. Orr et al. testify the association between TET proteins and malignant glioma. It is found that 5hmC and TET genes are significantly reduced in human glioblastoma, and the low expression of TET gene is associated with poor prognosis . In addiβ-catenin pathway is involved in a lot of biological process like cell proliferation, cell cycle distribution, and metabolism [β-catenin inhibits the glioma growth [β-catenin and markedly reduction of cell proliferation and migration caused by cycling 1 and c-myc which were downstream targets of β-catenin [β-catenin activation [β-catenin is decreased, and cell proliferation is inhibited [β-catenin [β-catenin and its downstream target protein cyclinD1 and c-myc are observed in TET1 knockdown experiments. In the rescue experiments, we downregulate TET1 and β-catenin at the same time. It is found that downregulation of β-catenin inhibits migration and invasion of glioma cells which are promoted by downregulation of TET1. The further experiments in vivo confirmed that TET1 inhibits glioma cell growth by targeting Wnt/β-catenin pathway.It well known that Wnt/tabolism . A mounttabolism , 29, anda growth , 31. For-catenin . Upregultivation . In YAP nhibited . Additio-catenin . In thisβ-catenin. In addition, overexpression of β-catenin leads to markedly reduction of glioma cell growth which is enhanced by overexpression of TET1. Our work provides theoretical foundation for serving TET1/Wnt axis as a new target for treatments of malignant glioma patients.In conclusion, our work demonstrates that deficiency of TET1 can promote the tumorgenesis of glioma by upregulating the expression of


A hermetic Micro-Electro-Mechanical Systems (MEMS) package with a metal lid is investigated to prevent lid-off failure and improve its reliability during the precondition test. While the MEMS package benefits from miniaturization and low cost, a hermetic version is highly sensitive to internal pressure caused by moisture penetration and the reflow process, thus affecting its reliability. In this research, the finite element method is applied to analyze the contact stress between the metal lid and the silver epoxy by applying the cohesive zone model (CZM). Moreover, the red dye penetration test is applied, revealing a microcrack at the metal lid/silver epoxy interface. Further analyses indicate that the crack is caused by internal pressure. According to the experimental testing and simulation results, the silver epoxy material, the curing process, the metal lid geometry, and the bonding layer contact area can enhance the bonding strength between the metal lid and the substrate. Huang et al. [The MEMS package is widely used in automotive, medical, and consumer electronic devices for measuring the mechanical, thermal, optical, and magnetic phenomena. Therefore, it needs to be highly reliable to maintain its functionality and safety for several years. A metal lid is included in the MEMS package to provide protection from the external environment, while also reducing the cost, weight, and size, thus improving production efficiency. While the hermetic MEMS package offers considerable benefits, it also causes reliability issues for the lid bonding technology and hermeticity. Hsu et al.  investigg et al.  proposedg et al.  developeg et al.  used a lg et al.  analyzedIn the present research, the reliability of a hermetic MEMS package with a metal lid is evaluated through the precondition test. The hermetic MEMS package adopted for this purpose comprises of a ceramic substrate, two dies, and a metal lid. The metal lid and the ceramic substrate are bonded together with silver epoxy under atmospheric pressure, as the aim is to reduce cost and improve the fabrication process efficiency. However, as moisture inside the MEMS package cavity is a potential risk, during the reliability test, the aim is to prevent the lid-off and improve the bonding strength between the metal lid and the ceramic. Thus, in the analyses, focus is given to the curing process, the silver epoxy material, the metal lid geometry, and the bonding layer contact area.The hermetic MEMS package with the metal lid used in this study was fabricated as shown in (a)Lid attachment: The metal lid is attached to the ceramic substrate with silver epoxy, which is applied between the metal lid and the ceramic substrate of the peripheral MEMS package. To fully cure silver epoxy, it is exposed to the 175 °C temperature for 2 h. Subsequent evaluations confirm that silver epoxy fully adheres with the metal lid and the ceramic substrate.(b)Moisture sensitivity level (MSL) 1 test: The MSL 1 test is carried out to determine the sensitivity level of the hermetic MEMS package under humid conditions. For this purpose, the hermetic MEMS package is exposed to high humidity and high temperature (85 °C/85% RH). When the moisture penetrates into the MEMS package cavity via silver epoxy, it weakens the metal lid/silver epoxy and the ceramic substrate/silver epoxy bonding strength.(c)Reflow process: During the reflow process, the moisture concentration inside the MEMS package causes damage to the metal lid/silver epoxy interface. When the MEMS package is exposed to the maximum temperature of 265 °C for three cycles, vapor pressure and thermal pressure are induced by the residual moisture in the hermetic cavity. Furthermore, thermal stress is generated at the metal lid/silver epoxy and the ceramic substrate/silver epoxy interface due to the coefficient of thermal expansion (CTE) mismatch.As shown in The shear test and the red dye penetration test results are presented in In the shear test, the thrust force is applied on the bottom side of the metal lid to remove it from the MEMS package. During this process, the maximum shear force is measured to determine the shear strength of both the metal lid and the silver epoxy. To investigate the influence of the internal pressure on the likelihood of lid-off failure, a hermetic MEMS package with vent hole was designed, as shown in To improve the maximum shear force of the hermetic MEMS package, additional tests were performed while controlling for the degree of curing and the pre-heat conditions, as these factors affect the material characteristics and the interfacial contact strength of the silver epoxy. To obtain fully cured and incompletely cured epoxy, the following conditions were respectively applied: 175 °C/1 h + 190 °C/1 h and 175 °C/1 h. As fully cured silver epoxy is harder and has a higher Young’s modulus, its maximum shear force is higher than that of the partially cured epoxy. Our analyses further indicate that when the fully cured epoxy is used in the hermetic MEMS package with a vent hole and the pre-heat (110 °C/0.5 h) step is performed, the maximum shear force increases by about 60% relative to the partially cured epoxy. In addition, when the fully cured epoxy is used in the hermetic MEMS package without a vent hole, the maximum shear force increases by about 48.5% after pre-heating. By observing the experimental testing results, the pre-heat does not have an effect on the hermetic MEMS package with a vent hole. The pre-heat condition can relieve the internal pressure applied on the metal lid without a vent hole in the curing process.The metal lid detaches from the ceramic substrate because of internal pressure during the reflow process. To analyze the stress and the deformation of the hermetic MEMS package with a metal lid under the reflow process, a finite element model was adopted by using ANSYS APDL. Specifically, the CZM method was used to calculate the contact stress at the metal lid/silver epoxy interface, which were denoted as contact and target elements. The MEMS package structure comprised of stacked dies, a die attach, a ceramic substrate, a metal ring, a silver epoxy, and lid metal, as shown in P is the pressure, V is the volume, n is the number of moles of gas, R is the idea gas constant (8.317 T is the absolute temperature, InternalP is the internal pressure, AirP is the air pressure, VaporP is the vapor pressure, Air_25P°C is the air pressure at 25 °C, 85P°C,SAT is the saturated vapor pressure at 85 °C, and RH is the relative humidity.The internal pressure inside the cavity can be obtained by using the ideal gas equation, as the following equations:The findings pertaining to the hermetic MEMS package with and without a vent hole were once again contrasted to investigate the contact stress and SEM observations, as shown in To decrease the contact stress at the metal lid/silver epoxy interface, one factor design was performed, considering lid thickness, connecting angle, epoxy height, lid height, lid size, and substrate height as factors, as presented in The one factor designs for the contact area, the lid geometry, and the material property effects are shown in The response surface method was also adopted to establish the relationship between the factors that are most influential on contact stress, as shown in In the research reported here, a hermetic MEMS package with a metal lid was designed and its reliability was tested. During the precondition test, lid-off failure occurred because moisture weakens the interfacial bonding strength and increases the internal pressure during the reflow process. The finite element method, which was adopted to simulate the contact stress of the metal lid/silver epoxy interface and the deformation of the metal lid, revealed that lid thickness, lid size, and the connecting angle are the critical factors for the contact stress. Although the Young’s modulus of the silver epoxy is not the critical factor, the moisture absorption, shear strength, and material curing characteristics are important for the contact stress. In the experimental testing, the pre-heat step and fully curing the epoxy can enhance the maximum shear force by 60% and 48.5% under the precondition test. The findings further indicate that increasing the lid thickness, and decreasing the lid size and the connecting angle can decrease the contact stress, thus reducing the likelihood of lid-off failure under the precondition test.


Staphylococcus aureus (MRSA) necessitate search for innovative strategies particularly, use of natural extracts such as lyophilized royal jelly (LRJ) and garlic extract (GE). Therefore, out study aimed to formulate emulgels containing different concentrations of both LRJ and GE and to evaluate their activities using a murine model infected with MRSA clinical isolate. Four plain emulgel formulas were prepared by mixing stearic acid/yellow soft paraffin-based O/W emulsion formulae based on Carbopol 940, Na alginate, Na carboxymethylcellulose or Hydroxypropyl methyl cellulose E4. Sodium alginate-based emulgel was selected for preparation of four medicated emulgel formulations combining LRJ and GE at four different concentrations. The selected medicated emulgels were used for the in vivo studies. The emulgel formulated with Na alginate and HPMC (MF3) exhibited optimum smooth homogeneous consistency, neutral pH, acceptable viscosity, spreadability, extrudability values and best storage stability properties. In vivo results revealed that, the wounds infected with MRSA isolate in rates were wet (oozing) and showed pus formation when compared to injured uninfected wounds. MF3 formula containing 4% LRJ and 50% GE showed the maximum wound healing properties, both in the apparent physical wound healing measurements and in the histopathological examination. In conclusion, the medicated emulgel formulation (MF3) prepared with Na alginate was found optimum for topical application. MF3 formula containing 4% LRJ and 50% GE has shown the highest in vivo wound healing capacities. Further clinical studies should be conducted to prove both its safety and efficacy and the potential use in human.The limited therapeutic options associated with methicillin-resistant  Four plain emulgel formulas were prepared and investigated for physiochemical and stability properties (F1–F4).3 exhibited optimum smooth homogeneous consistency, neutral pH, acceptable viscosity, spreadability, extrudability values and best storage stability properties.The emulgel FA murine skin model infected with clinical MRSA isolate was established and wounds were monitored via apparent physical wound healing measurements and histopathological examination.The MF3 containing 4% LRJ and 50% GE has shown the highest in vivo wound healing capacities. Staphylococcus aureus (MRSA) is one of the most common causes of nosocomial and community-acquired infections with limited therapeutic options  have been laboratory confirmed . Aqueous GE was prepared by filtering extracted chopped raw garlic in deionized water. Stearic acid, soft yellow paraffin, propylene glycol, and sodium carboxymethyl cellulose (Na CMC) were obtained from El-Nasr Pharmaceuticals . Butylated hydroxytoluene (BHT), methyl paraben, propyl paraben, triethanolamine (TEA), Carbopol 940 were gifted from ATCO® Pharma for Pharmaceutical Industries . Sodium alginate , S-allyl-l-cysteine (SAC), and Royal Jelly acid (RJA) were purchased from Merck®, Darmstadt, Germany. Hydroxypropyl methyl cellulose (HPMC E4) has been gotten from Memphis® Pharmaceutical Co. . Deionized water was produced from a Milli-Q Gradient A10 System .LRJ was kindly supplied from Egyptian International Pharmaceutical Industries (EIPICOhttp://ccinfo.wdcm.org/collection/by_id/1186).MRSA clinical isolate coded CCASU-MRSA3 was obtained from the Culture Collection Ain Shams University (CCASU-MRSA3) and was previously recovered from pus specimen of a patient suffering from deep wound infection who was admitted to Ain Shams specialized hospital. This isolate was identified via sequencing the 16S ribosomal RNA and was deposited in the NCBI GenBank  as well as in the Culture Collection Ain Shams University (CCASU) of the World Data Centre for Microorganisms (WDCM) (https://arriveguidelines.org).Female Wistar rats weighing about 110–120 g were used throughout the experiment. Animals were kept on a 12-h light-dark cycle and a persistent temperature of 22 ± 2 °C, housed (one per cage) and fed on standard diet pellets  comprised about 5% fiber, 20% protein, 3.5% fat, 6.5% ash and have free access to water. This study received an approval from the research ethical committee of Faculty of Pharmacy, Ain Shams University, Egypt . This study was carried out in accordance with Care and Use of Laboratory Animals recommendations and ARRIVE guidelines  in hot deionized water adjusted at 80 °C. Thereafter, the calculated amount of TEA was added. Next, the oily phase was added portion wise to the aqueous phase with continuous homogenization until cooled to room temperature.Different gel formers, Carbopol 940, Na alginate, Na CMC, and HPMC E4 were all investigated for the preparation of gel formulae. The least concentration forming gel of each agent was selected depending on tube inversion test  were incorporated by initially levigating the accurately weighed LRJ amounts in the o/w emulsion cream till a homogenous product was obtained. These medicated emulgels were all employed in the in-vivo studies.The emulgel formulas were examined for various physical parameters including, color, homogeneity, consistency, and phase separation.The pH values the prepared emulgels were determined by using a pH meter .The rheology of the emulgel formulations was examined by using a cone and plate viscometer , adjusted at 100 rpm and 25 ± 0.5 °C.About 100 mg of the emulgel was deposited in the center of a glass plate (5 × 5 cm). Another glass plate of the same dimension was dropped onto the first one from 5 cm. A constant weight (0.5 kg) was added on the glass plate surface and maintained in position for 1 min. The diameter of the spread circle in cm was measured after weight removal  of emulgel extruded from lacquered aluminum collapsible tube after application of a constant weight (500 g) in 10 s was determined. This examination was done in triplicates for each formula. The results of all the formulations were recorded in g/s  solvent system by sonication. RJA and SAC concentrations were measured after suitable dilution using high performance liquid chromatography (HPLC) technique, as previously reported by Zhou et al.  and Yoonhttps://www.agilent.com). Both RJA and SAC contents (%) were determined as % of the theoretical amounts and were calculated in triplicate.The UV detector was set at 210 nm. While for SAC analysis in the GE, the mobile phase was composed of eluent A consisted of 20 mmol/L sodium dihydrogen phosphate and 10 mmol/L heptane sulfonic acid with water adjusted to pH 2.1  containing 30% methanol in a closed glass jar. The whole assembly was maintained at 37 ± 0.5 °C in a shaking water bath. Two-milliliters samples were collected at intervals of, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5 and 6 h and immediately replaced with fresh dissolution medium. Samples were filtered with Millipore® disposable syringe filter 0.22 µm  and then analyzed for drug content by HPLC technique as previously described in “Emulgel (0.5 g) was evenly applied onto the surface of a dialysis cellulose membrane . The dialysis membrane was clamped on a glass cup with a cross-sectional area of 6.2 cmThe selected medicated emulgel formulation was stored in amber-glass jars at room temperature and under refrigeration at 5 ± 3 °C for 3 months. After storage, the formula was tested for its physical appearance, pH, rheological behavior, and drug contents.The MRSA skin infection rate model was performed according to El-Gayar et al.  with somGroup I: control, intact, non-infected, untreated.Group II: control, injured, infected, untreated.Group III: control, injured, infected, vehicle.Group IV: control, injured, non-infected, untreated.Group V: injured, infected, treated with Formula 1 (2% LRJ + 25% GE).Group VI: injured, infected, treated with Formula 2 (4% LRJ + 25% GE).Group VII: injured, infected, treated with Formula 3 (2% LRJ + 50% GE).Group VIII: injured, infected, treated with Formula 4 (4% LRJ + 50% GE).Four control and four test groups, six rats each, were included as follows:The follow up of the results was carried out by (I): measuring the wound diameter at D1, D3, D7, D10; (II): photographing the wound using digital camera at D9 of injury and, (III): Histopathological examination of the fixed samples at D10. Autopsy samples were taken from rats’ skin, in each group, and fixed in 10% formol for 24 h before examination .Data are displayed as mean ± S.D. Statistical analysis of wound diameter at different time intervals was performed by two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test at The emulgel was prepared by blending o/w emulsion cream with a gel at a ratio of 1:1 w/w. The compositions of different emulgel formulations are presented in Table 3 = F2 > F1 > F4, as shown in Table The characterization parameters  of the prepared plain and medicated emulgels were determined and the results are collected in Table The light-yellow medicated emulgels were prepared using the highest concentrations of both respective LRJ and aqueous GE, 4 and 50% w/w for characterization. After formulation, a phase separation and complete liquefaction occurred after the inclusion of GE into Na CMC-based emulgel. Moreover, medicated emulgel containing Carbopol exhibited uneven consistency with clumps formation. Hence these formulae were rejected, and their characterization tests were not performed. The medicated emulgels formulated with Na alginate and HPMC exhibited smooth homogeneous consistency with an almost neutral pH values, as obvious in Table RJA and SAC are of the main active constituents in LRJ and aqueous GE, respectively. The contents of RJA and SAC were thus traced in the optimized Na alginate emulgel formulation using a validated chromatographic assay by HPLC and found to range from 95.76 to 103.23% and from 91.67 to 94.32%, respectively  from emulgel.The results revealed that the total RJA content present in LRJ was released from emulgel after 5 h with an initial burst release of about 65 ± 9.95% after 30 min only. However, SAC began to release after starting the in-vitro diffusion experiment by 30 min. with a gradual release thereafter reaching 100% after 3 h. Mathematical kinetic modeling of the release profiles of both actives from the selected emulgel formulation was also determined, and their parameters were collected in Table 3) formulation was stored in amber-glass jars at room temperature and in a refrigerator adjusted at 5 ± 3 °C for 3 months. After storage in either condition, no significant change was observed in the physical appearance, pH, viscosity, and drug contents (P > 0.05) of the chosen emulgel. Stability data are presented in Table The selected medicated emulgel . As previously known, the concentration of the gel former is of great value regarding drug distribution and product handling  > F1 (Carbopol) > F3  = F2 (Na CMC). The spreadability and extrudability of plain formulations were inversely related to the viscosity data of each emulgel formulation  of the prepared plain and medicated emulgels were determined. The almost neutral pH of the different plain formulations confirmed the lack from any skin irritation potential, and hence skin compatibility. The viscosity of the emulgel formulations generally reflects its consistency  has powerful antioxidant and antiseptic effect . This implies that these emulgels could totally/partially eradicate MRSA. These results agree with Ejaz and coworkers  who founRegarding RJ, the results suggest that RJ may contain active components which have strong bactericidal or bacteriostatic effect as well as healing promoting effect. Some studies had reported that RJ contain active protein “royalisin” with bactericidal activity against Gram positive bacteria, while 10-hydroxy decanoic acid has healing promoting ability and broad-spectrum antibacterial activity (Hornitzky and Smith 3) prepared with Na alginate was found to be suitable for topical application. Based on the in-vivo testing and current laboratory findings, the emulgel formula containing 4% LRJ and 50% GE has shown the highest wound healing efficacy, followed by the formula containing 2% LRJ and 50% GE that showed also high activity. The other two formulas that have 25% GE were much less effective. Therefore, it could be concluded that GE has a more influence on wound healing than LRJ. In summary, through this study, we believe that the topical application of emulgel formula mentioned above, may be very effective in the treatment of MRSA skin infections and we advised for further clinical studies to confirm their safety and efficacy in human.At the end, it could be deduced that the emulgel formula containing (4% LRJ + 50% GE) has the highest wound healing efficacy, followed by the formula containing (2% LRJ + 50% GE) that showed also high activity. The other two formulas that have 25% GE were much less effective. emulgel formula containing (4% LRJ + 50% GE) has shown the highest wound healing efficacy, followed by the formula containing (2% LRJ + 50% GE) that showed also optimum activity. The other two formulas that have 25% GE were much less effective. Emulgel formula used for treating MRSA infections exhibited both anti-inflammatory activity in addition to their antimicrobial effects (Yang et al.


This research aimed to investigate the expression and function of FAM83A in the proliferation and metastasis in head and neck squamous cell carcinoma (HNSCC). FAM83A mRNA and protein expressions in HNSCC were detected in primary HNSCC samples and cell lines. The associations between FAM83A expression and clinicopathologic variables were evaluated through tissue microarrays. Besides, FAM83A knockdown and overexpression cell lines were constructed to assess cell growth and metastasis in vitro and the relationship between FAM83A and epithelial-mesenchymal transition (EMT). Furthermore, two models of xenograft tumors in nude mice were used to assess the tumorigenicity and metastasis ability of FAM83A in vivo. In the present study, overexpression of FAM83A in HNSCC samples was significantly associated with tumor size, lymph node status and clinical tumor stages. Mechanically, FAM83A could promote HNSCC cell growth and metastasis by inducing EMT via activating Wnt/β-catenin signaling pathway. Rescue experiment demonstrated the inhibition of β-catenin could counteract the function of FAM83A. Also, the FAM83A knockdown could suppress tumor growth and distant metastasis in the xenograft animal models of HNSCC. In conclusion, this study identifies FAM83A as an oncogene of HNSCC. This study provides new insights into the molecular pathways that contribute to EMT in HNSCC. We revealed a previously unknown FAM83A-Wnt–β-catenin signaling axis involved in the EMT of HNSCC. There may be a potential bi-directional signaling loop between FAM83A and Wnt/β-catenin signaling pathway in HNSCC.The online version contains supplementary material available at 10.1186/s12967-021-03089-6. In recent years, head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers worldwide, with more than 350,000 cancer-related deaths per year even though combined and multidisciplinary therapy has greatly advanced , 2. TherAs the smallest member of the FAM83 family (FAM83A to H), family with sequence similarity 83, member A (FAM83A), is located on chromosome 8q24. In 2005, FAM83A with the conserved DUF1669 domain at its N-terminus, was firstly reported as a potential cancer biomarker involved in tumor progression . It is aDUF1669 domain mediates the interaction with the α, δ or ε isoforms of the CK1 family of Ser/Thr protein kinases . The FAMIn brief, 242 patients including 148 males and 94 females, who underwent surgery and were diagnosed as primary HNSCC from 2009 to 2014 in the Affiliated Stomatological Hospital of Nanjing Medical University, were recruited. 242 HNSCC samples and 12 HNSCC adjacent normal tissues were used to make tissue microarrays [The procedure of immunohistochemistry (IHC) was carried out as we described previously . The staThe human normal oral keratinocytes (HOK) and the human HNSCC cell lines including CAL27, FADU, HN4, HN6 SCC-9, and SCC-25 cell, were purchased from China Center for Type Culture Collection , CAL27 and HOK were cultured in DMEM medium (Gibco). FADU, HN4, and HN6 were cultured in DMEM/F12 medium (Gibco). All culture medium contained 10% fetal bovine serum  and 1% penicillin/streptomycin. Cells were cultured in a humidified incubator containing 5% CO2 at 37 °C.The human FAM83A lentivirus targeting to upregulate FAM83A (LV-FAM83A), the negative control (LV-NC), the shRNA lentivirus targeting to knock down FAM83A (shFAM83A: 5’-GGAGUGUGGAAGGAGAGAUTT-3’), and the shRNA negative control lentivirus (shNC) were ordered from GeneChem Co., Ltd . Small interfere RNA (siRNA) of β-catenin (siβ-catenin: 5’-GGACACAGCAGCAAUUUGUTT-3’) purchased from GenePharma Co., Ltd  was used to silence β-catenin expression. A plasmid containing the β-catenin cDNA sequence  was used to overexpress β-catenin expression. XAV-939 , a small-molecule inhibitor, resolved with DMSO, was used to suppress exogenous β-catenin expression.Real-time RT-PCR and western blotting assays were carried out in accordance with standard process as we described previously . Nuclear4 HNSCC cells in 200ul complete medium were seeded into the upper compartment of a transwell insert with 8 µm pores . The lower chamber was filled with 700ul basal medium containing 10% fetal bovine serum (FBS). After 12 h (HN6 cell line) or 24 h , the invaded cells adhering to the lower compartment were fixed, stained, and counted under an inverted microscope . For the invasion assay, 2 × 105 cells/well were plated and the upper compartment was pre-coated with Matrigel .For the migration assay, 8 × 10Cell viability was detected by CCK-8 assay . Cells were seeded and cultured in 96-well plates at a density of 2000 cells/well. Every other day, CCK-8 reagent was added and the absorbance was determined at 450 nm.Cells were seeded into six-well plates and scratched with a 10ul pipette tip when cells grew to 90% confluence, and then cells were incubated with complete medium. At 0, 12, or 24 h, wound closure was photographed and its percentage was calculated.Cells were plated onto coverslips and fixed in 4% paraformaldehyde for 20 min when cells grew to 50% confluency. Then, the coverslips were permeabilized with or without 0.3% Triton, incubated with goat serum for 30 min and primary antibody overnight at 4 ℃. After it was rinsed thrice with PBS with Tween-20, cells were.incubated with fluorescent Cy3 secondary antibodies  for 1 h at 37 °C in the dark. After the slides were incubated with DAPI , cells were observed and photographed under an FV1000 laser confocal scanning microscope . Primary antibodies used were as follows: E-cadherin , Vimentin , and β-catenin .6 FAM83A knockdown HN6 cells and control cells were injected into the right armpit of mice. Tumor growth and body weight were determined every three days. After 24 days, mice were killed and tumors were stripped carefully. To explore the role of FAM83A on HNSCC tumor metastatic potential, a total of 1 × 106 HN6 cells were injected into the nude mice by tail intravenous. After 42 days, mice lungs were dissected carefully and the number of metastatic nodes were counted.All animal experiments were in accordance with the Animal Use and Care Committee of the Affiliated Hospital of Stomatology, Nanjing Medical University (IACUC-1906018). Female 4–6 weeks old BALB/c nude mice used were purchased from Vital River Laboratory Animal Technology Co.Ltd . To explore the role of FAM83A on HNSCC tumor growth, a total of 2 × 10P < 0.05 was considered significant in all experiments.The chi-square test, Fisher’s exact probability method, Mann–Whitney U test and Kruskal–Wallis test were applied to analyze the connection between FAM83A expression and clinicopathological characteristics. The Kolmogorov–Smirnov test and paired-samples T test were applied to compare FAM83A mRNA expression in HNSCC samples and adjacent normal tissues. Statistical significance in in vitro and in vivo experiments was assessed using one-way ANOVA and independent-samples T test. Data were presented as the mean ± SD from three independent experiments and all data were analyzed via GraphPad Prism 7  software. To explore the role of FAM83A in HNSCC, we examined FAM83A expression through 3 tissue microarrays consisting of 242 human HNSCC samples and 12 adjacent normal tissues through IHC analysis. The expression patterns of FAM83A in HNSCC and adjacent normal tissues were classified according to immunoreactive scores and the detailed demographic and clinicopathological parameters of these patients were listed in Table To further clarify the role of FAM83A in HNSCC, we collected 48 pairs of fresh tumor tissues which included HNSCC and adjacent normal tissues. Forty-eight pairs of tissues were used to extract mRNA and 10 pairs of tissues among them were used to extract protein. The qRT-PCR results in 48 HNSCC samples and paired normal tissues demonstrated that FAM83A mRNA was significantly increased in HNSCC than paired normal tissues  into the right flank of mice. Compared with the tumors produced by the negative control cells, the tumors separated from mice injected with the FAM83A-knockdown cells were smaller and showed a weaker ability in tumor growth  into the tail intravenous of nude mice. As described in Fig. To study the function of FAM83A on tumor growth in HNSCC, we used a subcutaneous xenograft tumor model and injected HN6 cells  plays a vital role in cancer metastasis , 27. TheIt is suggested that aberrant changes of Wnt/β-catenin signaling pathway are of decisive importance in EMT . Wnt canTo explore the underlying mechanism of FAM83A in HNSCC cells, we examined the effects of FAM83A on the Wnt signaling pathway and EMT. It is reported that c-myc elevated oncogenic activity provided an upregulation of EMT in HNSCC .Our study showed that FAM83A can enhance the expression of active β-catenin and downstream targets of the Wnt pathway, such as cyclin D1 and c-myc. Besides, western blotting assays and immunofluorescence staining assays demonstrated that β-catenin entered into the cell nucleus from the cytoplasm in CAL27 and HN6 FAM83A-upregulated cells, demonstrating the Wnt-pathway-mediated β-catenin activation. These results were further demonstrated by using the β-catenin inhibitor to pharmacologically block the Wnt signaling pathway. In the rescue experiment, the inhibitor, XAV-939, could reverse the promoting effect of FAM83A in HNSCC. It is known that FAM83F and FAM83G can regulate canonical Wnt signaling through an interaction with CK1α . PreviouIn this study, we found that β-catenin inhibitor could also downregulate the mRNA and protein level of FAM83A in CAL27 and HN6 cells. Furthermore, we found that the overexpression of β-catenin can result in the upregulation of mRNA and protein level of FAM83A. However, there is no β-catenin/TCF/LEF binding site in FAM83A promoter. The fact that the expressions of FAM83A and β-catenin are positively correlated could be explained by the fact that β-catenin can regulate FAM83A through other relative pathways. We think there are possible binding sites of the downstream genes of Wnt/β-catenin in FAM83A promoter. The underlying mechanism needs further investigation.In general, we presented evidence for the first time that overexpression of FAM83A in HNSCC tissues was positively correlated with advanced tumor size, lymph node metastasis and clinical tumor stages. Besides, the present study is the first to provide evidence that the Wnt-β-catenin-mediated EMT and metastasis by FAM83A in HNSCC, and there may be a potential bi-directional signaling loop between FAM83A and Wnt/β-catenin signaling pathway . B. Transwell assays of migration and invasion in CAL27 and HN6 cells after treated with XAV-939 (100×). C. CCK8 assays of the proliferation in CAL27 and HN6 cells after treated with XAV-939. Data represent the mean ± SD; *p< 0.05, **p< 0.01, ***p< 0.001.Additional file 2: Figure S2. β-catenin regulates FAM83A expression in HNSCC cells. A. Gene expressions of β-catenin and FAM83A were measured by RT-PCR in CAL27 cells after si-β-catenin transfection. B. Gene expressions of β-catenin and FAM83A were measured by RT-PCR in HN6 cells after si-β-catenin transfection. C. Gene expressions of β-catenin and FAM83A were measured by RT-PCR in CAL27 cells after β-catenin plasmid transfection. D. Gene expression of β-catenin and FAM83A were measured by RT-PCR in HN6 cells after β-catenin plasmid transfection. Data represent the mean ± SD; *p< 0.05, **p< 0.01, ***p< 0.001.Additional file 3: Figure S3. Hypothesized signaling mechanism involving FAM83A in the development of HNSCC.


However, the effects of ischemic stroke on endothelial canonical and non‐canonical Wnt signalings are unclear. Further, how non‐canonical Wnt signalings are influenced by upregulation of endothelial Wnt/β‐catenin signaling and subsequently affect BBB function following ischemic stroke have not been studied.Endothelial canonical (Wnt/β‐catenin) and non‐canonical Wnt signalings  with the deletion of Wnt receptor Frizzled (Fzd)4 or Fzd6, the two most abundant Fzds in brain ECs. Next, we observed the effect of ischemia/reperfusion injury on Wnt signalings in brain ECs and adult mice. Last, we assessed the changes of non‐canonical Wnt signalings and BBB injury in the early stage of ischemic stroke in mice with endothelial β‐catenin activation (β‐cat mice).First, we determined the levels of Wnt signaling markers including TCF/LEF1 transcription activity, Fzd4 or Fzd6 deletion dampened both Wnt/β‐catenin and Wnt/PCP signalings but enhanced Wnt/Ca2+ signaling in brain ECs. Both canonical and non‐canonical Wnt signalings in brain ECs were downregulated after ischemia/reperfusion injury in vitro and in vivo. Upregulating endothelial Wnt/β‐catenin signaling in β‐cat mice normalized the downregulated non‐canonical Wnt signalings, which did not compromise its protective effects on BBB integrity and endothelial tight junction following ischemic stroke.The BBB protection induced by upregulation of endothelial Wnt/β‐catenin signaling may be not interfered by the normalization of non‐canonical Wnt signalings in the early stage of ischemic stroke. The BBB protection induced by upregulation of Wnt/β‐catenin signaling may be not interfered by the normalization of non‐canonical Wnt signalings in the early stage of ischemic stroke. Both knockout cell lines had been authenticated by DNA sequencing  with 10% FBS and 4 μg/ml puromycin and plated into fibronectin‐precoated plates (10 μg/ml for 30 min at 37°C). 3 days later, medium was discarded and cells were washed with PBS twice and medium was replaced with EGM‐2MV/10% FBS. All cultures were maintained in a humidified 5% CO2 incubator at 37°C and routinely passaged when 80%–90% confluent. The optimal durations of Wnt5a for activating Wnt/PCP and Wnt/Ca2+ signalings by measuring the relative levels of phospho‐JNKThr183/Tyr185 and nuclear factor of activated T cells (NFAT) protein in bEnd.3 cell line had been determined in preliminary experiments  and cultured in Dulbecco modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The 2 and 99% N2, which was monitored with an O2 analyzer (HNZA). We assessed the effect of different durations of oxygen‐glucose recovery (OGR) after 6 hour (h)‐OGD for cell vitality and 3 h‐OGR was chosen in the following experiments . Briefly, cultured medium was replaced by Dulbecco's Modified of Eagle's Medium (Solarbio) and the cultured cells were put in a Modular Incubator Chamber (Billups‐Rothenberg) with 0.5%–1% O2.2−(ΔΔCt) method. Primer sequences of all genes are shown in Supplementary Table Total RNA was extracted using the Direct‐zol RNA MiniPrep kit (Zymo Research). RNA was reverse transcribed using iScript Reverse Transcription Supermix for real‐time quantitative PCR (RT‐qPCR) according to the manufacturer's instructions (Vazyme). RT‐qPCR was carried out on a StepOnePlus Real‐Time PCR System using the Power SYBR Green method (Roche). RNA expression was calculated using the comparative Ct method normalized to Actin. Data were expressed relative to a calibrator using the 22.3Fzd4−/−, and Fzd6−/− bEnd.3 cells were transfected by lentivirus with TCF/LEF  or NFAT firefly luciferase , and Renilla control  in 96‐well plates. Four replicate wells were prepared for each group. After 48 h, cells were stimulated with Wnt3a (R&D Systems) for 24 h, or Wnt5a (R&D Systems) for 30 min. Firefly and Renilla luciferase activities were measured using the Dual Stop and Glo system (Promega). Reporter activity was calculated as Firefly/Renilla activity in each well.For a typical dual luciferase assay, wild type (WT), 2.4Ctnnb1) constitutive activation, Cdh5‐CreER mice were crossed with mice bearing the lox(ex3)Ctnnb1 allele to generate lox(ex3)/+Ctnnb1; Cdh5‐CreER mice (termed β‐cat), and +/+Ctnnb1; Cdh5‐CreER control mice (termed WT). The recombination efficiency of Ctnnb1 gene in brain ECs of β‐cat mice has been confirmed in our previous studyCtnnb1 mRNA expression in β‐cat mice did not apparently decrease compared with WT mice, but the Wnt/β‐cat target genes Axin2, Apcdd1, and Nkd1 significantly increased (data not shown). Meanwhile, non‐canonical Wnt signaling target genes Pfn2, Vangl2, Celsr3, DAAM1, CamkII, and NFATc1/c3 expression in brain ECs of β‐cat mice had no significant changes  group. For the surgical procedures, anesthesia was induced with 4% isoflurane in an induction chamber; anesthesia was maintained with 2% isoflurane delivered through a face mask (RWD). A heating pad was used to maintain each mouse's core temperature at 37 ± 0.5°C throughout the surgical procedure. A modified intraluminal filament model was used to induce transient middle cerebral artery occlusion (MCAO) as previously described.2.5The infarct region in sections with H & E staining was defined as the area with reduced staining or areas containing eosinophilic‐necrotic cell bodies. The boundaries between regions of infarct and adjacent normal brain were clearly delineated. Tissue sections were photographed using a ZEISS microscope (Axio Imager 2 Pol), and the infarct area was measured using ImageJ software . To eliminate the contribution of postischemic edema to the area of injury, the infarct area (%) was calculated as follows: [(area of the non‐ischemic hemisphere – non‐infarct area of ischemic hemisphere)/area of the non‐ischemic hemisphere] × 100%.2.6Thr183/Tyr185 (CST), rabbit anti‐JNK (CST), rabbit anti‐active β‐catenin (CST), mouse anti‐NFAT (Santa Cruz), rabbit anti‐phospho‐CamkIIThr286 (CST), rabbit anti‐CamkII (CST), rabbit anti‐Laminin (Sigma‐Aldrich), rabbit anti‐zonula occludens‐1 (ZO‐1) , rabbit anti‐Desmin (Abcam), at a 1:1000 dilution. β‐actin  or GAPDH  served as the loading control for normalization. The nucleus NFAT is the marker of Wnt/Ca2+ signaling, so nucleus fraction was obtained using Nuclear and Cytoplasmic Protein Extraction Kit (TransGen biotech) according to the manufacturer's protocols, while PCNA  was used as the normalization control in nucleus protein analysis. The membranes were incubated with the respective secondary antibodies and blots were developed and imaged using the GelView 6000M system (BioLight).For the analyses of marker proteins of canonical and non‐canonical Wnt signaling or BBB components, ECs or ischemic hemisphere tissues of mice were obtained and analyzed by Western blot. The proteins of ECs or cerebral tissues were isolated and harvested by RIPA lysis buffer (Solarbio) supplemented with protease and phosphatase inhibitors and quantified by BCA assay according to the standard protocols. Equal amounts of protein lysates from each sample were fractionated on 10% SDS‐PAGE gels. The proteins were transferred onto polyvinylidene fluoride membranes. The membranes were then incubated with primary antibodies including rabbit anti‐phospho‐JNK2.7Frozen 7‐μm‐thickness sections were allowed to dry on Adhesion Microscope slides (CITOTEST) at room temperature before being rehydrated in PBS. Sections were blocked in 10% normal goat serum  in PBS +0.2% Triton X‐100 for 1 h at room temperature. Samples were incubated at 4°C with the following antibodies in PBS +5% goat serum +0.2% Triton X‐100: hamster anti‐mouse CD31 , rabbit anti‐ZO‐1 , rabbit anti‐Claudin‐5 , rabbit anti‐Laminin , rabbit anti‐Desmin , and donkey anti‐mouse IgG . Excess antibody was removed by rinsing in PBS for 5 min, 3 times. Samples were then incubated at room temperature for 1 h with the Cy3 goat anti‐hamster IgG (Jackson ImmunoResearch), Alexa Fluor 488 donkey anti‐rabbit IgG (Jackson ImmunoResearch) diluted 1:500 in PBS +5% BSA +0.2% Triton X‐100 for 1 h at room temperature. Excess antibody was removed by rinsing in PBS for 5 min, 3 times. Slides were mounted in antifading mounting medium with DAPI (Solarbio) and imaged with an Olympus microscope to obtain 20× or 40× images. The whole‐brain image was taken under low‐power microscope and adjusting white balance through healthy hemisphere. The optical density (OD) value was measured by an experimenter blinded to group allocation with ImageJ. Immunofluorescence signal area or density was quantified and normalized by CD31 signal area in 5 to 8 random ischemic areas of cortex. Pericyte coverage was quantified by measuring the staining signal length of pericytes or ECs with ImageJ. All tests were performed by an experimenter who was unaware of the identity of experimentations.2.82+ and Mg2+ (GIBCO) at 37°C for 60 min, with gentle trituration every 10 min. After that, the disaggregated cells were washed with cold PBS and filtered through a 40‐μm mesh. After centrifugation, cell pellets were resuspended in 10 ml 20% BSA and centrifuged at 1000 g at 4°C for 25 min to remove the myelin. The pellets were then resuspended in PBS/3% FBS/0.1% NaN3/2 mM EDTA and blocked rat anti‐mouse CD16/32  for 5 min on ice and labeled with antibodies according to our previous studyBrains of adult mice were minced and disaggregated in Type IV collagenase , Dispase , and DNase I  in PBS with Ca2.9p ≤ 0.05, the differentially expressed genes can be identified. Gene richness was analyzed by David database.For mouse brain ECs, the total RNA was extracted using the Arcturus picopure RNA Isolation Kit (Applied Biosystems). According to our previous study,2.10t test or the Dunnett T3 test, if the variance was heterogeneous, was used to further analyze differences. Two groups were analyzed by unpaired t tests. Statistical significance was defined as p < 0.05.Statistical analysis was performed with the SPSS 16.0 for Windows software package (SPSS). Data were expressed as means ± standard error (SE). One‐way analysis of variance (ANOVA) was used to determine statistical diﬀerences in all of the observed indicators from the diﬀerent groups. The least significant difference (LSD) 33.1Fzd4 and Fzd6 were abundantly expressed with their mRNA levels much higher than any of the other eight Fzd genes in bEnd.3 cell line and primary brain ECs of adult mice  and Axin2 mRNA level , two of the indicators of Wnt/β‐cat signaling activity, and the TCF/LEF TOP‐flash value in Fzd6−/− cells was lower than that in Fzd4−/− cells (p = 0.004), in a Wnt3a dose‐dependent manner, indicating that Fzd6, in addition to Fzd4, could also play a necessary role in mediating Wnt/β‐cat signaling.A plethora of evidences have suggested that Fzd4 serves as the principal receptor for Wnt/β‐cat signaling in brain ECs,Thr183/Tyr185 (P‐JNK) to JNK was significantly reduced in Fzd4−/− and Fzd6−/−cells, compared with WT cells , and Wnt5a failed to upregulate P‐JNK in Fzd4−/− and Fzd6−/−cells .Taken together, these data provided the following evidences: first, in addition to Fzd4, Fzd6 might be another key membrane receptor for Wnt/β‐cat signaling; second, although the role and importance might be different, Fzd4 and Fzd6 mediated both Wnt/PCP and Wnt/β‐cat signalings; third, the antagonism might exist between Wnt/Ca3.2Axin2 mRNA level , Nkd1  and Apcdd1  in primary brain ECs  and in primary brain ECs  was still significantly lower than cells in normal control  in primary brain ECs with Wnt5a stimulation  and Vangl2  significantly decreased after OGD/R  and P‐CamkII  protein, and the mRNA levels of Pfn2  and CamkII  in WT mice with I/R were apparently reduced, compared with WT mice with sham operation; after I/R, P‐JNK (p = 0.0310), P‐CamkII (p = 0.005), Pfn2 (p = 0.0127), and CamkII (p = 0.0231) mRNA levels in β‐cat mice were higher than that in WT mice. In order to confirm whether endothelial non‐canonical Wnt signalings were also normalized after the upregulation of endothelial Wnt/β‐cat signaling, we determined the changes of non‐canonical Wnt signaling markers in primary brain ECs with Wnt/β‐cat signaling activated by Wnt3a protein after OGD/R. We found that after Wnt3a upregulated Wnt/β‐cat signaling, only the relative P‐CamkII protein level increased significantly  than that in WT mice  One previous study showed that the Fzd4 deletion in ECs failed to completely abolish the physiological function of Wnt/β‐cat signaling,2+ signalings collectively contribute to the endothelial growth, angiogenesis, and BBB formation,2+) signalings in physiological status have been implicated.2+ signalings becomes a new question.Interacting with more than 15 receptors and co‐receptors in seven protein families, Wnt proteins‐elicited signalings are complex and involved in extensive physiological and pathological processes in cells.Axin2 mRNA levels were significantly reduced not only in Fzd4−/− cells, but also in Fzd6−/− cells, indicating that Fzd6 also had certain functions in Wnt/β‐cat signaling. Fzd6 has been shown to regulate Wnt/PCP signaling as its prevalent role,It has been shown that Fzd4 could activate Wnt/β‐cat signaling by specifically interacting with Wnt proteins and norrin, an atypical Fzd ligand, to play an essential role in barrier maintenance and plasticity in brain ECs.2+ signaling, Wnt5a could inhibit the interaction between Wnt3a and Fzd proteins to suppress Wnt/β‐cat signaling.2+ signaling still remains obscure. Our results showed that NFAT TOP‐flash values of Fzd4−/− and Fzd6−/− cells were significantly higher than those in WT cells. In consideration of the role of Fzd6 and Fzd4 in Wnt/PCP signaling, the result provided a clue of the possible antagonism between Wnt/Ca2+ and Wnt/PCP signaling in ECs.Canonical and non‐canonical Wnt signalings have been reported to antagonize each other. By competitively binding Fzd proteins to activate Wnt/PCP and Wnt/Ca2+ signalings in brain tissues could be normalized, while only Wnt/Ca2+ signaling was normalized in primary brain ECs. It must be mentioned that Wnt proteins in brain tissue are secreted principally by neurons and astrocytes,Previous studies showed BBB breakdown occurred rapidly following reperfusion within several hours and reached to a peak around at 24 to 48 h after ischemia.In conclusion, our findings demonstrate that: (1) After ischemic stroke, canonical and non‐canonical Wnt signalings were downregulated, while non‐canonical Wnt signalings could be normalized by upregulating endothelial canonical Wnt signaling. However, we did not investigate the effect of targeted manipulation of non‐canonical Wnt signalings on BBB injury after ischemic stroke, which may be worthy of further study. (2) The protection of the BBB by upregulation of Wnt/β‐cat signaling depends on protecting the endothelial tight junction in the early stage of ischemic stroke, which was not disturbed by normalization of non‐canonical Wnt signalings. (3) It is worth mentioning that except Fzd4, Fzd6 also plays an important role in endothelial Wnt/β‐cat signaling. Combined with previous studies,The authors declare that they have no conflict of interest.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Figure S5Click here for additional data file.Figure S6Click here for additional data file.SupinfoClick here for additional data file.Table S1Click here for additional data file.


The superior mechanical properties of multi-walled carbon nanotubes (MWCNTs) play a significant role in the improvement of the mechanical and thermal stability of an epoxy matrix. However, the agglomeration of carbon nanotubes (CNTs) in the epoxy is a common challenge and should be resolved to achieve the desired enhancement effect. The present paper investigated the thermal, mechanical, and water uptake properties of epoxy nanocomposites with surfactant-modified MWCNTs. The nanocomposites were prepared through the incorporation of different weight concentrations of MWCNTs into the epoxy matrix. Comparative analysis of neat epoxy and epoxy/CNT nanocomposites were conducted through thermal, mechanical, microscopic, and water uptake tests to reveal the improvement mechanism. The homogenous distribution of the CNTs in the epoxy was achieved by wrapping the surfactant onto the CNTs. The addition of surfactant-modified CNTs into the epoxy caused an obvious increase in the mechanical and thermal properties. This improvement mechanism could be attributed to the uniform dispersion of the CNTs in the epoxy matrix reducing the free volume between the polymer chains and restricting the chain segmental mobility, leading to strong interfacial bonding and an efficient load transfer capability between the CNTs and the epoxy matrix. However, the mechanical and thermal properties of the epoxy/CNT nanocomposite decreased owing to the agglomeration effect when the concentration of the CNTs exceeded the optimal percentage of 1.5%. Additionally, the CNTs could impart a reduction in the wettability of the surface of the epoxy/CNT nanocomposite, leading to the increase in the contact angle and a reduction in the water uptake, which was significant to improve the durability of the epoxy. Moreover, the higher weight concentration (2%) of the CNTs showed a greater water uptake owing to agglomeration, which may cause the formation of plenty of microcracks and microvoids in the nanocomposite. Considering Lijima’s report from 1991 and onwards, CNTs get much attention in science and engineering. The extraordinary physical, chemical, and mechanical properties of CNTs give them application potential in many fields. CNTs are excellent fillers due to their high strength and significant role in the nanocomposites used in civil engineering and aerospace applications . The mai2 groups, both the hydrogen and covalent bonds are supposed to form between the NH2-CNTs and the epoxy matrix. Therefore, the binding force of the interphase can be greatly enhanced. However, the agglomeration of CNTs is still a challenge for developing nanocomposites with a high performance for engineering applications.Added to the benefits of CNTs, the dispersion of CNTs in the epoxy matrix is still a challenge. Since CNTs tend to agglomerate due to their weak intermolecular interactions like Van der Waals forces, their pi–pi interactions between the pi clouds of the aromatic rings , their hWhile mixing CNTs into the polymer matrix, the uniform distribution of nanoparticles must be ensured without destroying the integrity of the particles. Generally, the modification of CNTs is one of the best methods for preventing the agglomeration of CNTs. Modification of CNTs can be composed mainly of two types: covalent bonding and non-covalent bonding. Regarding the non-covalent bonding, CNTs are wrapped or adsorbed onto the surface of polymers, while covalent bonding can be realized through a chemical interaction . Hence, 2). The presence of the polar amide group in the pyrrolidone ring and the polar methylene and methine groups can produce a strong steric influence and can control the CNTs agglomeration through the repulsive force from its hydrophobic carbon chains [There are many varieties of surfactants available, commonly in which Polyvinyl Pyrrolidone (PVP) is the most effective surfactant due to its unique properties. They were chosen here as the organic components to realize the uniform dispersion of nanoparticles in the polymer. PVPs are soluble in polar solvents  and can enhance the thermal and mechanical properties of the filler and polymer by increasing the cross-linking between the molecular chains. Moreover, the amorphous structure of PVPs decrease the scattering loss and also can be used in optical applications . PVP conn chains .There are different methods to introduce the CNTs into the epoxy matrix, such as high-speed shear mixing, mechanical mixing, sonication, etc. Montazeri and Chitsazzadeh observed that the ultrasonication process can improve the dispersion of the CNTs and increase the strength and modulus of the epoxy/CNT nanocomposites . Here, t7H16N2) was selected as the hardener as supplied by Sinopec Shanghai Petrochemical Company . The functionalized MWCNT-NH2, with the size of 50 μm (length) and 8–15 nm (outer diameter), was adopted through the chemical vapor deposition (CVD) method with a purity of 95 wt.%. Polyvinyl Pyrrolidone (PVP) was selected as the surfactant for dispersing the MWCNT and 99.9% denatured ethanol from Tianli Chemical Reagent Co. Ltd.,  was adopted as the dispersing media or solvent. The chemical structure of the epoxy, hardener, and surfactant PVP are shown in The commercially available diglycidyl ether of bisphenol-A (DGEBA)-based E51 epoxy was adopted in the present paper and 4-methyl-1,3 cyclohexane diamine  epoxy matrix was weighed and put into a beaker. Then, the CNT/PVP/ethanol solution was added slowly into the epoxy resin and blended using a mechanical mixer at the speed of 3000 rpm for 1 h. To evaporate the ethanol, the mixture was kept in a vacuum oven below the melting temperature of ethanol. After the ethanol was evaporated completely, the hardener was added into the mixture and stirred at the speed of 150 rpm for 10 min. The air bubbles were removed by placing the mixture into the vacuum chamber. The bubble-free mixture was then poured into a mold, cured at room temperature for 24 h, and post cured at 120 °C for 2 h. The schematic representation of the preparation procedure of the epoxy/CNT nanocomposite is shown in Owing to the strong Van der Waals intermolecular interaction, the CNTs were found to be bundle- or rope-like structures. Moreover, the CNTs could again combine and form tangled networks or agglomerates with lower mechanical properties than the individual CNTs. Researchers were seeking the methods to achieve a better dispersion of CNTs in various solvents using surfactants, such as water-soluble polymers and ionic and non-ionic solvents. To prevent agglomeration, the surfactants (such as PVP) were observed to develop micelles around the CNTs ,20,21,22The dispersion of the CNTs in the epoxy matrix was achieved through the wrapping of the sidewalls onto the surface of the CNTs. Generally, the surfactants interacted with carbon via the hydrophobic segments, meanwhile the hydrophilic segments reacted with the epoxy via hydrogen bonding . TherefoThe flexural tests of the epoxy/CNT nanocomposites were conducted with a universal testing machine  according to the standards ASTM D790. The ratio of the span length to the thickness of the specimens was 16:1. The test parameters included the support span and maximum deflection. The loading rate was set as 2 mm/min. Five repeated samples were tested and the average result was obtained for each weight percentage of the CNTs.The tensile tests of the specimens were conducted to obtain the tensile strength, tensile modulus, and elongation at break. The dumbbell shaped samples were prepared and tested using a universal testing machine  based on ASTM D638. The extensometer of the gauge length of 10 mm was adopted to obtain the strain variation during loading. The loading rate was 1 mm/min. To obtain accurate results, five samples of each condition were tested, and the average was recorded.Dynamic mechanical analysis represents an effective non-destructive technique to investigate the viscoelastic characteristics of polymeric materials, such as the storage modulus, loss modulus, and tan delta. The tests were conducted using the Dynamic Mechanical Analyzer . The dual cantilever mode was adopted for a rectangular specimen. Temperature scans from 20 °C to 250 °C at a 1 Hz frequency strain mode with a heating rate of 5 °C/min were performed.The surface morphology analysis of the specimens was conducted using scanning electron microscopy  at room temperature. Before the tests, all samples were coated with gold by a Gatan Model 682 precision etching coating system for about 15 min to improve the electron conductivity of the samples. The cryogenic fracture surface analysis was examined at a voltage of 10.0 kV.The transmission electron microscopy of the specimens was examined through JOEL JEM 2100. The specimens were prepared by dispersing the CNTs into the ethanol solution with and without PVP. A single drop of this dispersion from both specimens was placed on a carbon-coated copper grid. Then the specimens were dried at room temperature.The contact angle tests of the samples were conducted at room temperature using a goniometer . The contact angle test was realized by placing a droplet of a liquid at various spots on the sample surface and recording the images. The image acquisition and the determination of the contact angle were obtained using Drop Meter Software. More than ten readings of each sample were taken, and the average was obtained.Water uptake tests were performed through placing the specimens into water for 60 days at room temperature in accordance with ASTM D570. The weight of the samples was recorded before the immersion in deionized water at room temperature. The weighing of the samples at regular intervals used a high-precision four-digit electronic balance. Based on the above analysis, the increment of the flexural strength and modulus mainly depended on the added content of the CNTs. The flexural strength and modulus were augmented remarkably with the increase in CNTs. However, the saturation or reduction in the flexural properties occurred when the content of the CNTs exceeded the optimal content. This was because the dispersion of moderate CNTs in the epoxy matrix may boost the flexural properties . ExcessiAdditionally, the increase in the tensile modulus of the epoxy/CNT nanocomposites was ascribed to the strong bonding of the functional group formed between the CNTs and the epoxy matrix which restricted the mobility of the main chain of the epoxy matrix. Similar improvement accompanying the increase in CNT concentration was observed in the study . FurtherAccording to the above analysis, the tensile strength and modulus enlarged with the incorporation of the CNTs in the epoxy. A similar improvement effect on the tensile properties was summarized as follows. Montazeri et al.  observedAdditionally, it can be observed that the peak of the tan delta curves of the nanocomposites gradually decreased with the added content of the CNTs and reached the minimum at the optimal addition ratio of the CNTs. The tan delta represents the energy dissipation of materials and the lower peak of the tan delta indicates the better interfacial bonding between the CNTs and the matrix. Furthermore, by introducing CNTs into the epoxy, the rise of the glass transition temperature occurred due to the higher crosslinking density. Also, this can be ascribed to the free space reduction happening in the macromolecules or the reduction in the molecular motion within the polymer chain segments.The contact angle of the neat epoxy and the epoxy/CNT nanocomposites were measured with deionized water droplets as shown in The effect of the CNTs on the water uptake of the epoxy was investigated. (1)The addition of CNTs into the epoxy caused a maximum increase percentage of 52.9%  and 25.5 % , 29.5% (tensile strength) and 48.1% (tensile modulus) for the 1.5 wt.% of the CNTs. The improvement mechanism was attributed to strong interfacial bonding and an efficient load transfer between the epoxy and the CNTs. However, the mechanical properties of the epoxy/CNT nanocomposite decreased or saturated owing to the agglomeration effect when the weight concentration of the CNTs was 2.0%.(2)The maximum increase percentage (~9.5%) of the glass transition temperature (Tg) and the lower height of the tan delta peak of the nanocomposites were achieved for the 1.5 wt.% of the CNTs compared to the neat epoxy matrix. This can be attributed to the uniform dispersion of the CNTs in the epoxy effectively improving the crosslinking between the carbon nanotubes and the epoxy, and a reduced free space of the macromolecular segmental motion. The higher addition ratio (2%) of CNTs caused agglomeration, which acted as a plasticizer and decreased the crosslinking and interfacial bonding between the epoxy matrix and the CNTs. (3)The incorporation of the CNTs into the epoxy could impart a reduction in the wettability on the surface of the epoxy/CNT nanocomposite, leading to an increase in the contact angle and a reduction in the water uptake, which was significant to improve the durability of the epoxy. Moreover, a higher concentration (2%) of the CNTs showed a larger water uptake owing to the agglomeration possibly causing the formation of microcracks and microvoids in the epoxy. (4)The wrapping of PVP onto the surface of the CNTs was confirmed by TEM analysis. A surface morphology analysis showed a uniform distribution of the surfactant-modified CNTs in the epoxy matrix formed at the weight concentration of 1.5%. Meanwhile, the agglomeration of the CNTs in the epoxy matrix was observed at a higher weight concentration of 2%. It is recommended that the weight concentration of PVP/CNTs in epoxy does not exceed the optimal concentration of 1.5% to ensure enhanced mechanical properties and a longer service life. A further investigation is needed to analyze the effect of PVP modified CNTs on the epoxy nanocomposite at a higher concentration (> 2%).Here, the mechanical and water uptake properties of epoxy nanocomposites with surfactant modified MWCNTs were investigated. The homogenous distribution of the CNTs in the epoxy matrix was achieved by wrapping the surfactant onto the CNTs. Thermal, mechanical, morphological, contact angle, and water uptake tests were performed to reveal the improvement mechanism of the CNTs on the epoxy matrix. The following conclusions can be drawn: