And these phenotypes were attenuated by simultaneous knockdown of β-catenin with RNAi. Furthermore, we identified that FXR could bind with β-Catenin through AF1 domain, and disrupt the assembly of the core β-Catenin/TCF4 complex. Activation of FXR attenuated the DNA-binding activity of β-Catenin/TCF4, and subsequently, its targeting gene-cyclin D1 expression. Importantly, FXR expression was markedly reduced in human HCC, an event which correlated with aberrant activation of β-Catenin. These data identified FXR as a negative regulator of HCC development through direct suppression of Wnt/β-catenin pathway.The association between the temporal activation of Wnt/β-catenin pathway and the spontaneous hepatocellular carcinoma (HCC) development in Farnesoid X receptor (FXR) knockout mice is not well understood. We found that Huh7 cells depleted with FXR by RNAi showed enhanced cell growth, migration and invasion  Primary liver cancer is the fifth most common cancer in men and the seventh in women, and the third most common cause of cancer-related death worldwide. Hepatocellular carcinoma (HCC) accounts for about 85% of primary liver cancer. And its incidence increased dramatically during the last 20 years . The mol−/−) mice showed high incidence of spontaneous HCC development at the age of 12 to 15 months [et al. found that, compared with age-matched WT control, Fxr−/− mice showed ~30% increase of β-catenin gene expression at 3 month, and ~100% increase at 12 month [et al. also reported β-catenin activation in the HCC liver from 12 to 14 month old FXR−/− mice [Farnesoid X receptor , a member of the nuclear receptor superfamily, is mainly expressed in liver, intestine, kidney and, to a less extent, adipose tissue . FXR reg5 months , 9. Furt12 month . Wolfe ecyclin D1 expression. Moreover, FXR expression was inversely correlated with β-Catenin activity in HCC.In the present study, we found that down-regulation of FXR promoted hepatocytes proliferation and carcinogenesis through β-catenin activation. FXR directly bound with β-Catenin through AF1 domain, and negatively regulated the transcriptional activity of Wnt signaling by disrupting the assembly of the core β-Catenin/TCF4 complex. Subsequently, this interaction attenuated β-catenin DNA-binding activity and reduced its targeting gene Protein expression level of FXR, and β-Catenin was determined in nine different hepatocarcinoma cell lines. As shown in Figure Cylin D1 and c-Myc, overexpression. And the induced overexpression was partially reversed by down-regulation of β-Catenin by shRNA , a hinge domain, a ligand binding domain (LBD) and a C-terminal AF2 domain. To identify the specific domain that mediates the interaction with β-Catenin, a series of deletion mutants, including FXR(Δ125), FXR(Δ186), FXR(Δ240), FXR(ΔAF2) and FXR(ΔC-ter), were generated Figure . Myc tagWe then tested the effect of FXR on β-Catenin mediated transcriptional activity using a cell-based TOPflash reporter assay. TOPflash reporter, pRL-TK, β-Catenin and FXR were co-transfected into HEK293 cells. As shown in Figure We further studied whether the N-terminal AF1 domain of FXR, which is required in mediating FXR and β-Catenin interaction, is also involved in its inhibitory effect on Wnt/β-Catenin transcriptional activity. A series of FXR deletion mutants were co-transfected with β-Catenin and TOPflash reporter into HEK293 cells. We found that the full length FXR and FXR deletion mutants containing the N-terminal AF1 domain, including FXR(FL), FXR(ΔAF2) and FXR(ΔC-ter), inhibited β-Catenin mediated transcription activity. Whereas, the other FXR mutants without N-terminal AF1 domain, including FXR(Δ125), FXR(Δ186), FXR(Δ240) had no effect on β-Catenin mediated TOPflash reporter activity Figure . These rNext, we investigated whether FXR also interacts with the other core transcription factor, TCF4. HEK293 cells were transiently transfected with Myc-tagged FXR and HA-tagged TCF4. The interaction between FXR and TCF4 was tested with co-immunoprecipitation assay. We found that HA-TCF4 was not presented in the cell lysate immunoprecipitated with Myc agarose Figure . β-CatenCyclin D1 gene transcriptional activity was examined using a TOPflash reporter assay. Wild type Cyclin D1 promoter , or a mutated Cyclin D1 loss of TCF binding site  and pRL-TK plasmid were transfected into Huh7 cells, upon FXR activation by GW4064, relative Cyclin D1-WT promoter activation was reduced, while the CyclinD1-mTCF promoter activation was not altered  and matched adjacent tissues (N). Out of 12 paired samples, 8 pairs showed decreased FXR expression and increased active-β-Catenin expression in tumor tissue when compared with matched adjacent tissue Figure . AssociaTaken together, these data provided the evidence that, in human hepatocarcinoma, FXR expression is frequently reduced, along with elevated β-Catenin activation.In this study, we identified a crosstalk between FXR and canonical Wnt signaling pathway. The expression of FXR was significantly downregulated and was inversely correlated with β-Catenin activation in HCC. FXR directly bound with β-Catenin and inhibited the formation of β-Catenin/TCF4 complex, and subsequent transcriptional activity. Our data demonstrated a novel mechanism through which FXR exhibited tumor suppressive activity.−/− mice were more prone to develop hepatocellular carcinoma, along with elevated β-Catenin expression. We found that FXR negatively correlated with β-Catenin activation in both hepatoma carcinoma cell lines and human hepatocarcinoma tissue. And reduced FXR promoted tumorigenic behaviors was partially reversed by simultaneous knockdown of β-catenin. These data suggests that β-Catenin is a critical component in this process. Oncogenic Wnt/β-Catenin signaling is likely to be one of the important factors that contributes to the development and maintenance of HCC during loss of FXR function.We showed that FXR expression level was significantly downregulated in human HCC, and that loss of FXR promoted tumorigenic behaviors in hepatoma carcinoma cell line Huh7; whereas, stably over-expressing FXR inhibits tumor growth in HCCLM3 cells. Consistent with our observation, previous works also discovered reduced FXR expression in cholangiocarcinoma, biliary tract carcinoma and tumor sample from HCC patients –14. In aWnt/β-Catenin signaling is highly conserved from nematodes to humans, and is essential in various normal cellular processes such as development, growth, survival, regeneration and self-renewal . Mutatio2 vitamin D3 induced tumorigenesis [in vitro and in vivo, to disrupt the formation of β-Catenin/TCF4 complex and subsequently the binding ability with corresponding promoter. Interestingly, FXR can bind with and inhibit the corresponding transcriptional activity of both wild type β-Catenin and two oncogenic β-Catenin hot spot mutants (S33A/S37A/T41A/S45A and ΔN90)  family has been considered as a clinically and developmentally important research area of cancer biology . For exaigenesis . In the igenesis . We idennd ΔN90) . These f−/− mice developed HCC in a time-dependent fashion [Among 19 Wnt ligands, Wnt3a is classified as the canonical element, leading to canonical Wnt/FZD signaling pathway activation . Our stu fashion , 29. FXR fashion . Another fashion . Moreove fashion . Up-reguOur data provided a novel mechanism for FXR-mediated inhibition of tumor progression. Through competing with other co-activators for binding to β-Catenin, FXR suppressed the β-Catenin-mediated activation of the Wnt target genes, which are involved in tumor development. FXR, a new modulator of the Wnt signaling pathway, is a potential candidate as a molecular target of the Wnt signaling cascade to achieve anti-tumor effects.HCC tissues and the paired adjacent non-tumorous tissues were obtained from Zhongshan Hospital of Fudan University. Ethical approval for human subjects was obtained from the Research Ethics Committee of Zhongshan Hospital. And informed consent was obtained from each patient. For immunohistochemistry analysis of FXR and β-Catenin expression in human liver cancer tissue specimens, 8 paired fresh frozen liver tissues and matched normal tissues were used. 19 paired fresh resections were collected for western blot analysis.2. FXR agonists  treatment was carried out in charcoal stripped fetal bovine serum (Gibco).The SMMC-7721, HL-7702, BEL-7404, HepG2, Hep3B, Huh7, Sk-hep-1, MHCC-97L and HCCLM3 cell lines were obtained from the Type Culture Collection of Chinese Academy of Sciences , and were maintained in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) at 37°C in a humidified incubator containing 5% COXho1 and BamH1, and then ligated into pcDNA3-Myc vector. All constructs were verified by sequencing.pLKO.1-shβ-Catenin and shNC (negative control), pT3-ΔN90-β-Catenin, pcDNA3-β-Catenin-MutS33A/S37A/T41A/S45A, pGL3-Cyclin D1 WT, pGL3-Cyclin D1 mTCF, SuperTOPflash/FOPflash-TCF4 luciferase reporter (under the control of eight TCF4 consensus sites) were purchased from Addgene. pReceiver-Lv105-Wnt3a was purchased from GeneCopoeia. Human FXR (NM_005123) coding region was amplified and cloned into recombinant lentivirus vector pLKO.1. The deletion mutants of Myc tagged FXR 126–472, 187–472, 241–472, 1–393, or 1–263 were amplified by PCR using primers corresponding to each mutant. The PCR products were digested by We employed different approaches for transient or stable knocking down gene expression with small interfering RNA: 1) for transient suppression, synthesized siRNA oligo duplexes were transiently introduced into the cells using Lipofectamine 2000; 2) for stable suppression, shRNA was introduced into lentiviral vectors to produce lentiviral particles in HEK293T cells, and then infected Huh7 cells to generate stable gene silencing. Four independent piLenti-FXR shRNA-GFP were purchased from abm Inc (Cat. i015336). Among them, piLenti-FXR/shRNA-GFP#2, which was more efficient in knocking down FXR expression, was selected from establishing stable cell lines. Specific siRNA oligo duplexes targeting FXR  and the negative control siRNA were synthesized by GenePharma , and transiently introduced into the cells using Lipofectamine 2000. pLKO.1 shRNA FXR construct was derived from FXR siRNA#2 sequence and used to generate the Huh7 stable cells expressing FXR silencing in Cylin D1 promoter reporter and 10 ng of pRL-TK with or without 100nM FXR siRNA were co-transfected into Huh7 cells using Lipofectamine 2000 kit  according to the manufacturer's instructions. 48 h after transfection, luciferase activity was measured from harvested cell lysate using luminometer. And the result was normalized by Renilla activity.Luciferase assay was performed using a Dual-Luciferase® Reporter Assay System (Promega). Briefly, 200 ng of TOPflash or His-FXR  was purified according to the manufacturer's instructions , and then incubated with glutathione-Sepharose 4B beads bound GST or GST-β-Catenin in binding buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.2% Nonidet P40 and 10% glycerol). After incubation at 4°C overnight, the beads were washed 6 times for 10 min in binding buffer. And proteins bound to the beads were eluted into sample buffer, followed by SDS-PAGE, and Western blots.The 6 μM thick paraffin-embedded tissues were deparaffinized and rehydrated. Antigen retrieval was performed in a steamer for 15 minutes. The section was stained with a rabbit monoclonal anti-active-β-Catenin  and a mouse monoclonal anti-FXR  respectively overnight at 4°C. After washing with PBST, biotinylated secondary antibodies  was incubated for 2 hours at room temperature. Reactions were developed using a peroxidase-based streptavidin detection method and 3, 3′-diaminobenzidine tetrahydrochloride (DAB)  as chromogenic substrate. The tissue sections were mounted and examined under the CKX31 microscope .6 cells was incubated with anti-TCF4, anti-β-Catenin antibody , or IgG, in 300 μl ChIP buffer. DNA fragments were purified from immunopriciptates. PCR was performed with forward CCGGGCTTTGATCTTTGCTTA and reverse TCTGCTGCTCGCTGCTACTG primers, for 35 cycles (15 sec 95°C and 60 sec 60°C) to amplify a 120bp DNA fragment harboring the WRE sequence in the Cyclin D1 promoter.Chromatin immunoprecipitation analysis was performed using EZ ChIP kit (Millipore) according to the manufacturer's instructions. The sheared material was used for isolating input DNA. Aliquots of sheared chromatin corresponding to 1 × 102, 0.5% (v/v) Nonidet P-40). Equal amount of protein (10 ~ 80 μg) was electrophoresed by SDS-PAGE and then transferred to nitrocellulose membranes. After blocking with PBS containing 5% nonfat milk and 0.1% Tween 20 for 2 hours at room temperature, the membrane was incubated with primary antibody at 4°C overnight. After washing with PBS containing 0.1% Tween-20 3 times, 5 minutes each, the membrane was then incubated with horseradish peroxidase labeled secondary antibody for another 2 hours at room temperature. The membrane was then developed using Pierce ECL Western Blotting Substrate  and exposing to KODAK X-OMAT BT Film.Western blot were performed according to standard procedures. Briefly, the liver tissues or cultured cells were lysed in RIPA buffer . After centrifugation at 16,000 g for 15 minutes, the lysates were immunoprecipitated with primary antibodies followed by protein A/G-Sepharose (Santa Cruz) or tag conjugated beads. After washing 5 times with lysis buffer, proteins were resolved by SDS-PAGE gels and transferred to nitrocellulose membranes for Western blot.The primary antibodies used were purchased from Cell Signaling Technology, CA , Santa Cruz Biotechnology, CA , Roche Applied Science Indianapolis, IN, , and Abmart, China  respectively.For immunofluorescence analysis, cells were fixed with 4% paraformaldehyde in PBS at 4°C for 30 minutes, washed 3 times with PBS, and permeabilized by incubation in PBS with 0.1% Triton X-100 for 10 minutes. After blocking in PBS with 10% goat serum and 0.1% Triton X-100 for 1 hour at room temperature, cells were incubated with the primary antibodies  monoclonal mouse antibody and anti-β-Catenin rabbit antibody  at 4°C overnight, followed by incubation with the secondary antibodies  and Alexa 594-conjugated anti-mouse IgG  at room temperature for 2 hours. Coverslips were mounted onto glass slides with anti-fade mounting medium with DAPI (Vector Laboratories) and visualized with an Olympus FluoView 500 IX71 confocal microscope.3 cells) were seeded in triplicate in 96-well plates and incubated for 4 days. At indicated time point, 20 μl of MTT -2, 5-diphenyltetrazolium bromide ) solution (5 mg/ml) was added to each well. The plates were incubated at 37°C for 5 hours, followed by removing the medium, and then adding 150 μl DMSO to each well to dissolve crystals. The plates were then measured at 590 nm with a reference filter of 620 nm on a multi-well plate reader. Absorbance readings reflect the number of viable cells. To calculate relative cell proliferation, the following equation was used: relative cell proliferation= (Experiment group ODday(n)-Blank ODday(n))/(Experiment group ODday 0-Blank ODday 0). n = 0, 1, 2, 3, 4 respectively; experiment group includes, (1) cells transfected with control shRNA, (2) cells transfected with FXR shRNA, and (3) cells transfected with FXR shRNA +β-catenin shRNA; blank is cell culture medium only. For all the experiment group, including cells transfected with control shRNA, the relative value is 1 only on Day 0. While on Day 1 to 4, the value for experiment group, including cells transfected with control shRNA, reflects relative proliferation compared with Day 0.Cells (5 × 105 cells/well) were seeded on top of the 8.0 μm pore size transwell insert containing 600 μl DMEM medium in the lower chamber. Cells migrated to the other slide of membrane were stained with crystal violet and counted after 24 hours incubation.To assess cell migration, Huh7 or HCCLM3 cells (5 × 105) were plated in 8.0 μm pore size Matrigel coated transwell insert. Culture medium was changed twice a day for 3 days. After 48 hours, cells that had migrated through the Matrigel to the filter were stained with crystal violet and counted.To assess cell invasion, cells  =(ab2)/2, where “a” indicates the length and “b” indicates the width of the tumor.All animal procedures were performed in accordance with protocols approved by the Animal Use Committees at Fudan University. Huh7 or HCCLM3 cells were maintained in in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum (Gibco). Cells were infected with concentrated lentivirus (multiplicity of infection = 25). Stably transfected cell clones were selected. Cells were grown at 37°C in the tissue culture incubator for two days and dissociated into single cells using dispense. Following established procedures, 2.5 × 10r) analysis was used to determine the correlation between FXR and active-β-Catenin protein level in paired HCC tissues. Data were expressed as means ± SEM. Statistical analysis was performed either by Student's t test for unpaired data or one-way ANOVA for three groups or more. *, p < 0.05; and **, p < 0.001 was considered as significant.Pearson's correlation coefficients (


Elevated basal, ligand-independent, Wnt signaling in some canine breast cancer cells is not caused by classical mutations in APC, β-Catenin or GSK3β but, at least partially, by enhanced LEF1 expression. We examined the expression and function of EGFR/HER-regulated pathways on the ligand-independent Wnt signaling.Twelve canine mammary tumor cell lines with previously reported differential basal Wnt activity were used. The expression levels of genes related to EGF-signaling were analyzed by cluster analysis. Cell lines with a combined overexpression of EGF-related genes and enhanced basal Wnt activity were treated with PI3K/mTor or cSRC inhibitors or transfected with a construct expressing wild-type PTEN. Subsequently, effects were measured on Wnt activity, cell proliferation, gene expression and protein level.HER2/3, ID1, ID2, RAC1 and HSP90 together with low to absent cMET and PTEN mRNA expression, suggesting a connection between Wnt- and HER-signaling pathways. Inhibition of the HER-regulated PI3K/mTor pathway using the dual PI3K/mTor inhibitor BEZ235 or the mTor inhibitor Everolimus® resulted in reduced cell proliferation. In the cell line with high basal Wnt activity, however, an unexpected further increased Wnt activity was found that could be greatly reduced after inhibition of the HER-regulated cSRC activity. Inhibition of the PI3K/mTor pathway was associated with enhanced expression of β-Catenin, Axin2, MUC1, cMET, EGFR and HER2 and a somewhat increased β-Catenin protein content, whereas cSRC inhibition was associated with slightly enhanced HER3 and SLUG mRNA expression. A high protein expression of HER3 was found only in a cell line with high basal Wnt activity.High basal Wnt/LEF1 activity was associated with overexpression of PTEN. Inhibition of the PI3K/mTor pathway further stimulated, however, canonical Wnt signaling, whereas the inhibitory effect with the cSRC inhibitor Src-I1 on the Wnt activity further suggested a connection between Wnt and HER2/3-signaling.High basal Wnt activity in some mammary cancer cell lines is associated with overexpression of HER-receptor related genes and HER3 protein, and the absence of  Progesterone-induced Wnt signaling plays an important role in the development of the mammary gland during puberty, but may also induce the development of mammary tumors from stem/progenitor cells . There iWithout a Wnt signal, the β-Catenin destruction complex binds and phosphorylates non-cadherin-associated β-Catenin. This targets it for destruction by the proteasome, thereby allowing TCF family of transcriptional factors to bind to transcriptional repressors in the nucleus. The destruction complex is disrupted when active Wnt signaling is stimulated by binding of Wnt proteins to its Frizzled receptor on the cell membrane. As a result β-Catenin accumulates in the cytoplasm, translocates to the nucleus and associates with TCF family of proteins, enhancing transcriptional activation of a program of genes –5.2+ leading to activation of nuclear factor activated T cells (NFAT) and consequently inhibition of the canonical signaling. The alternative Wnt receptor RYK additionally signals through Dishevelled to cSRC in axon guidance. Traditionally Wnt ligands do not signal through β-Catenin in the non-canonical pathway, but it seems that Wnt 5A had some inhibitory characteristics on the canonical signaling. Likely there is some crosstalk between the canonical and the non-canonical Wnt pathways  2,5-diphenyltetrazolium bromide assay (MTT) . Briefly, cells were seeded in 96 wells plates  and after 24 h incubation to attach and stretch, treated with the different compounds for 40 h. Cell viability was determined by incubating 20 μl 5 mg/ml MTT in 100 μl medium in each well. After a 2 h incubation, the media was removed by decanting and 100 μl DMSO was added to each well, incubated for 30 min and the absorbance was measured at 595 nm in a spectrophotometer Anthos Multimode Detector . IC50 curves were plotted with Sigma-plot version 12.5.2 bottles and after 24 h incubation to attach and stretch, treated with the different compounds. After 40 h cells were washed with cold HANK’s balanced salt solution and lysed and scraped with RIPA buffer [Cells were seeded in 75 cmhttp://www.R-project.org)) with a Pearson:Spearman test  to find a correlation between the TOP/FOP ratio, related target genes and the cell lines.Cluster analysis with qPCR results from all the cell lines was done in RStudio (version 3 software (R Core Team (2013) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria; n = 8 for MTT and n = 4 for each transfected/incubation compound). After background subtraction, the cell viability, the Wnt-signaling and the protein levels were calculated as a percentage of the non-treated cells. IC50 curves were done in a single experiment with n = 8 and the HER3 Western blots were done in triplicate. Differences in TOP/FOP activities, RNA, cell viability and protein expression levels were statistically assessed using unpaired, two tailed Student’s t test, a P value less than 0.01 was considered significant.Transfection and incubation studies, and also the MTT assays, were done in three independent experiments , HER2/3, HSP90 and RAC1 mRNA and a down regulation of SLUG, NCOA3, MUC1, CYCLIN D1, BCL2, cMET and PTEN mRNA. In these highly activated Wnt cell lines, PTEN expression is completely lost. PTEN deficiency occurs in 5 to 14 % of primary human breast cancers and PTEN is also lost in human T47D and MCF7 cell lines [LEF1 is overexpressed and acts as a contributing factor of the high canonical Wnt pathway [HER expression) as in the silencing of PTEN with high ligand independent Wnt signaling. Previously, it has been shown that binding of Wnt proteins to the Frizzled receptor may activate the EGFR. EGFR can be further activated by homo or hetero-dimerization with HER2, HSP90 as its chaperone, or HER3. These activated complexes functions as an oncogenic unit to drive the proliferation of breast cancer cells [HER2 expression [HER2 and HER3 in our canine mammary cell lines and the fact that the HER2/HER3 complex is the strongest activator for downstream signaling pathways makes this an interesting area for further research [In a continuation of our previous findings of a variation in basal Wnt/β-Catenin activity in canine mammary tumor cell lines  we searcll lines . The cluer cells . In humapression , somethipression . Combiniresearch , 27, 28.LEF1 levels, loss of PTEN and high HER2/3 mRNA levels all point to the activation of the PI3K/AKT/mTor pathway [We concluded that the association between high Wnt signaling (high TOP/FOP ratios), high  pathway –32. We tCell lines with high Wnt activity and a cell line lacking basal Wnt activity (CIPm) were incubated with Everolimus as a specific mTor inhibitor and BEZ235 as a dual inhibitor of PI3K and mTOR. Both BEZ235 and Everolimus equally inhibited the viability of the highly activated Wnt cell lines and have only a slightly inhibitory effect on the CIPm cells . In contrast with this BCL2 is not upregulated in all tested canine cell lines and HER3 is only, although significantly, 1.5 times upregulated in some cases [MUC1 expression. At the same time, in all tested cell lines, basal MUC1 protein concentrations showed no difference, even in the presence of considerable amounts of the cytoplasmic domain MUC1-CD (data not shown). Still, overexpression of MUC1 plays a role in tumor formation and progression in > 90 % of the breast cancers [EGFR and oncoprotein MUC1 mRNA concentrations in our experiment together with the upregulation of cMET. Enhanced cMET expression has been linked to poor prognosis in human breast cancer, since it influences extravasation/angiogenesis, enhances resistance to endocrine therapy and trastuzumab treatment and may influence the regulation of β-Catenin/TCF transcription [cMET in the cell lines with elevated Wnt activity is low, but stimulated when the PI3K/mTOR pathway is blocked; blocking this pathway also affects the EGFR and HER2 expression. Overexpression of MUC1 is associated with the induction of anchorage-independent growth and tumorigenicity in human carcinomas. MUC1 overexpression is also directly associated with stabilization of β-Catenin either via MUC1 binding to E-Cadherin, or by inhibition of the adenomatous polyposis coli (APC)/GSK3β/ MUC1-CD complex, whereas phosphorylation of MUC1-CD by EGFR and cSRC increases β-Catenin [Inhibition of the PI3K/mTor pathway with BEZ235  or with Everolimus (a single mTor inhibitor) in cell lines with high ligand-independent canonical Wnt activity resulted in comparable upregulation of a number of candidate genes  route. The role of MUC1 is less clear and needs further research. In mice, however, data are available showing that MUC1 activates cSRC signaling by influencing the association of PI3K and β-Catenin leading to a delay in mammary tumor progression . Prior rcSRC may play a crucial role in tumor growth and metastasis. Metastasis involves loss of cellular adhesion, increased motility, intravasation, invasion, extravasation, resistance to anoikis, and colonization at distant sites. High cSRC expression is found in various breast cancers in association with reduced survival. Recently the cSRC inhibitors Dasatinib and Bosutinib were studied as a monotherapy in humans, but showed no clear reduction of antitumor activity, indicating that before further studies are done, clear biomarkers should be found for proper patient selection . The seaIn summary, inhibition of the PI3K/mTor pathway in tumor cell lines with ligand-independent Wnt activity further increases canonical Wnt signaling. As we have shown, this Wnt activity can be effectively inhibited by a cSRC inhibitor. While very recently, loss of PTEN has been associated with worse outcomes in HER2 positive breast cancer patients, no data are available on their Wnt activity . AlthougFurther studies will need to identify optimal biomarkers for selecting breast cancer patients most likely to benefit from concerted treatment with combination therapies of mTor and cSRC inhibitors. Such research is very likely to include studies in dogs with spontaneous mammary carcinomas.


Parkinson disease (PD) is a progressive neurodegenerative disorder characterized by loss of dopamine neurons in the substantia nigra pars compacta (SNpc) and widespread aggregates of the protein alpha-synuclein (α-syn). Increasing evidence points to inflammation as a chief mediator; however, the role of α-syn in triggering and sustaining inflammation remains unclear. In models of Alzheimer’s disease (AD), multiple sclerosis (MS) and neurotoxin models of PD, the chemokine CX3CL1  and its receptor (CX3CR1) have important roles in modulating neuroinflammation.in vivo mouse model in which human α-syn is overexpressed by an adeno associated viral vector serotype 2 (AAV2) and in vitro phagocytosis and protein internalization assays with primary microglia treated with aggregated α-syn.To examine the role of fractalkine signaling in α-syn-induced-neuroinflammation and neurodegeneration, we used an We observed that loss of CX3CR1 expression led to a reduced inflammatory response, with reduced IgG deposition and expression of MHCII 4 weeks post-transduction. Six months post transduction, AAV2 mediated overexpression of α-syn leads to loss of dopaminergic neurons, and this loss was not exacerbated in animals with deletion of CX3CR1. To determine the mechanism by which CX3CR1affects inflammatory responses in α-syn-induced inflammation, phagocytosis was assessed using a fluorescent microsphere assay as well as by microglial uptake of aggregated α-syn. CX3CR1-/- microglia showed reduced uptake of fluorescent beads and aggregated α-syn.Our results suggest that one mechanism by which CX3CR1-/- attenuates inflammation is at the level of phagocytosis of aggregated α-syn by microglia. These data implicate fractalkine signaling as a potential therapeutic target for regulating inflammatory response in α-syn models PD. Parkinson disease (PD) is a common neurodegenerative movement disorder characterized by a progressive loss of dopamine producing neurons in the substantia nigra pars compacta (SNpc) and widespread intracellular aggregates of the protein alpha-synuclein (α-syn). This protein is the principal component of Lewy bodies and Lewy neurites, the pathological hallmark of PD. α-syn has been genetically implicated through studies of familial PD. Mutations and multiplications in the SNCA locus convey risk for sporadic PD. ,2. GenomIncreasing evidence points to a key role of neuroinflammation in the pathogenesis of PD–6. AnalyAll of the inflammatory features found in human PD are also observed in animal models. Viral overexpression, transgenic, and neurotoxin models of PD in rodents and non-human primates recapitulate the reactive microgliosis, elevated pro-inflammatory cytokine expression, lymphocyte infiltration, and loss of TH+ cells in the SNpc ,13. Our CX3CL1  and its specific receptor, CX3CR1 have important roles in modulating inflammation in the CNS . Found ain vivo and primary mouse microglia in vitro to study the role of fractalkine receptor in modulating α-syn-induced inflammation and neurodegeneration. In contrast to the effects observed in neurotoxin models, we find that knockout of CX3CR1 attenuates the inflammation and fails to exacerbate neurodegeneration observed with AAV2-SYN-mediated overexpression. In examining the microglia in vitro, we find that CX3CR1-/- impaired or reduced uptake of either synthetic fluorescent microspheres or aggregated α-syn. These data suggest that CX3CR1 is important in disease progression of synucleinopathies, and could potentially be a target for neuroprotective therapies for PD.In this study, we used adeno-associated virus (AAV) overexpression of α-syn Cx3cr1tm1Litt/J  Cardona AE et al., 2006) maintained on a congenic background were used for these studies and were obtained from Jackson Laboratories . The locus for CX3CR1 was disrupted via insertion of the sequence encoding green fluorescent protein (GFP), replacing the first 390 bp of the coding exon (exon 2). The deleted region includes an amino-terminal portion of the protein that is critical for interaction with endogenous fractalkine ligand, CX3CL1. RIKO mice show abnormal microglial cell physiology including microglial migration during development [C57BL/6  and CX3CR1-/- mice (B6.129P-elopment . These melopment .Construction and purification of the rAAV vectors rAAV-CBA-IRES-EGFP-WPRE (CIGW) and rAAV-CBΑ-SYN UCLEIN- IRES-EGFP-WPRE (CSIGW) are described in previous publications –26.12 viral genome/mL diluted in sterile PBS) into the right SNpc. Co-ordinates were anterior- posterior -3.2 mm from bregma, medio-lateral -1.2 mm from midline, and dorso-ventral -4.6 mm from dura. All research conducted on animals was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Alabama at Birmingham.Male C57BL/6 (WT) and CX3CR1 knockout mice (8–12 weeks of age) were anesthetized with isoflurane and unilaterally injected with 2uL of AAV2-SYN of AAV2-GFP  pH 7.4 followed by 4% paraformaldehyde and then cryoprotected in 30% sucrose solution in PBS. Brains were frozen on dry ice and cryosectioned coronally on a sliding microtome (cut thickness: 40um); sections were collected serially throughout the striatum and SNpc, placed into tissue collection solution (50% glycerol in 0.01M PBS), and stored at -20C for immunohistochemical analysis.Fluorescent analyses of free-floating sections were labeled as previously described.  SectionsFor diaminobenzadine (DAB) staining of free-floating sections, sections were washed and stained with Tris-buffered saline (TBS) at pH 7.4 and labeled as previously described . BrieflyFor TH neuron quantification using unbiased stereology, free floating sections were stained with anti-Tyrosine Hydroxylase (TH) , coded, and analyzed with an Olympus BX51 with MicroBrightfield software . A total of 5 sections covering the rostro-caudal extent of the SNpc of ipsilateral and contralateral to the injection site were quantified using the optical fractionator method and the StereoInvestigator software. Neurons that stained positive for TH were counted and weighted section thickness was used to correct for variations in tissue thickness.Images were captured using a Leica TCS-SP5 laser scanning confocal microscope. Fluorescent images for MHCII and IgG were exported and processed using Adobe Photoshop. For quantification of MHCII Ni-DAB staining, slides were observed using a Nikon Eclipse E800M microscope. Slides were coded and scored using a numerical scale of 0 (no staining) to 4 (most intense staining) by a single observer blind to the treatment paradigm. Only MHCII staining surrounding the SNpc via TH positive neurons (DAB-brown) were scored while non-specific staining surrounding the needle tract was ignored. Scores were obtained from 5–6 mice per group. The groups were statistically analyzed using Kruskal-Wallis and Dunn’s multiple comparisons test (p = 0.0146).Primary murine microglia were isolated from postnatal day 0–2 pups according to previously published protocols  with a fMicroglia were plated in chamber slides  at 100,000 cells per well with serum free media. Purified recombinant human α-syn  was resuspended and incubated at 37C with constant agitation (500rpm) for 7 days as previously described . Prior tMicroglia were cultured as previously described in the methods from both the WT and CX3CR1-/- mice. Microglia were plated in chamber slides  at 100,000 cells per well with serum free media. Following 24 hours, cells were treated with 500nM aggregated α-syn for 4 hours followed by 5μL DQ Ovalbumin Red BSA  was added for 90 minutes. Cells were then washed with PBS and fixed using 1% PFA. Imaging was performed using the Leica TCS-SP5 laser scanning confocal microscope and quantified using ImageJ software as previously described.We have previously shown that overexpression of full-length, human α-syn via AAV2 in the mouse SNpc elicits a neuroinflammatory response which includes an increase in MHCII expression as well as a robust deposition of IgG in the ipsilateral SNpc as early as 4 weeks post transduction ,24,26. TDeposition of IgG is a component of the inflammatory response to AAV2-SYN, and also observed in human PD . Four weUsing the AAV-SYN model, we as well as others have found that viral-mediated overexpression of α-syn in mice results in a loss of approximately 30% of TH-immunopositive cells in the ipsilateral SNpc at six months post transduction. In order to determine whether or not CX3CR1 was involved in the neurodegenerative process in our model, we injected 8–12 week-old male C57BL/6 mice, including both WT and CX3CR1-/-, with a single, unilateral, stereotactic injection of AAV2-SYN and AAV2-GFP control virus into the right SNpc . At 6 months post transduction, TH+ nigral cell number was assessed by unbiased stereology. Our results indicate that CX3CR1-/- fails to exacerbate α-syn-induced dopaminergic cell loss in our model where it has previously exacerbated TH+ cell loss in neurotoxin models of PD. A 23.5±4.6% decrease  in the number of TH+ neurons in the SNpc was found when WT animals were injected with AAV2-SYN . Statistin vitro, we isolated primary microglia from both WT and CX3CR1-/- mice and plated them at 100,000 cells per well in chamber slides. Using uptake of fluorescent beads, we observed a reduced phagocytic capacity in the CX3CR1-/- microglia compared to the WT microglia  and 6-hydroxydopamine neurotoxin models of PD, a robust neuroinflammatory response along with extensive neurodegeneration is observed. DeletioStudies involving fractalkine signaling in proteinopathy models, where aggregated or misfolded proteins are the triggering factor, have yielded different results. In models of Alzheimer disease produced by overexpression of APP/PS1 or human APP, CX3CR1 knockout results in decreased levels of pro-inflammatory cytokines Tumor Necrosis Factor (TNF) and monocyte chemotactic protein 1 (CCL2) but increased Interleukin 1-beta (IL1β) ,33,34. OPutting these various observations in diverse systems together begins to provide insight in to the complexity of fractalkine signaling and its role in neurodegenerative disease. In models mediated by cellular toxins such as MPTP and 6-OHDA, enhancing the expression of fractalkine ligand provides protection, while knockout of the CX3CR1 receptor worsens pathology,22. It sThe recent study of Nash et al. using fractalkine ligand in an AAV-SYN model may provide some additional insight, but there are issues which require these comparisons to be made cautiously. Most importantly, the Nash et al. study was conducted in rats, while all the other studies described were conducted in mice, and it is possible that species difference is a contributor. A second issue relates to the spatial distribution of the effects; while the knockout is global, affecting all monocytes both within and outside of the brain, the overexpression of fractalkine in the rat was induced locally. Lastly, and we believe most likely, fractalkine may have multiple effects on the pathway between protein overexpression and neurodegeneration. In animals with deletion of CX3CR1, the dominant effect may be impairment of phagocytosis, an upstream event which prevents inflammation. Overexpression of the ligand appears to act far downstream, as demonstrated by the persistence of enhanced MHCII expression despite reduced neurodegeneration when AAV-SYN and fractalkine ligand are co-expressed.In the field of PD research there is a need for the development of neuroprotective strategies to delay the onset or progression of the disease. Fractalkine signaling appears to have potent effects on PD inflammatory models, but these effects are complex and depend on the nature of the triggering event. Indeed, depending on model is possible to demonstrate both pro- and anti-inflammatory effects of fractalkine signaling. In the context of human disease, the most important issue may prove to be the timing of fractalkine signaling with respect to the degenerative process. Early in PD, blockade of fractalkine may slow disease initiation by interfering with phagocytosis. In established PD, the ability of fractalkine ligand to modulate the inflammatory state of monocytes may be the more important. It will be important to understand more about the state of fractalkine signaling in human disease in order to use this information effectively in the search for a neuroprotective treatment.We conclude that CX3CR1 knockout decreases phagocytosis in microglia resulting in decreased inflammation associated with α-syn internalization. The decreased internalization of aggregated α-syn results in attenuated antigen processing and presentation in microglia. In vivo, these deficits result in a decrease in inflammation marked by a decrease in MHCII expression and IgG deposition. As a result of the attenuated immune response, we also found that deletion of the fractalkine receptor, CX3CR1, did not exacerbate α-syn-induced TH+ neuron loss in the ipsilateral SNpc at 6 months post transduction as reported in neurotoxin models of PD. These data implicate fractalkine signaling as a potential therapeutic target for regulating the inflammatory response in α-syn models PD.S1 FigUnbiased Stereological counts for contralateral SNpc in WT and CX3CR1-/- mice.(TIF)Click here for additional data file.


In vitro data have shown that Mrap2 interaction with the melanocortin-4-receptor (Mc4r) affects receptor signaling. However, the mechanism by which Mrap2 regulates body weight in vivo is not fully understood and differences between the phenotypes of Mrap2 and Mc4r KO mice may point toward Mc4r independent mechanisms.Melanocortin receptor accessory proteins (MRAPs) are regulators of the melanocortin receptor family. MRAP is an essential accessory factor for the functional expression of the MC2R/ACTH receptor. The importance of MRAP in adrenal gland physiology is demonstrated by the clinical condition familial glucocorticoid deficiency type 2. The role of its paralog melanocortin-2-receptor accessory protein 2 (MRAP2), which is predominantly expressed in the hypothalamus including the paraventricular nucleus, has recently been linked to mammalian obesity. Whole body deletion and targeted brain specific deletion of the Mrap2 gene result in severe obesity in mice. Interestingly, Mrap2 complete knockout (KO) mice have increased body weight without detectable changes to food intake or energy expenditure. Rare heterozygous variants of MRAP2 have been found in humans with severe, early-onset obesity.  Melanocortin receptors (MCRs) comprise a family of five, class A, G protein-coupled receptors designated MC1R-MC5R with diverse physiological roles. MCRs are found in chordates and are regarded as having evolved from a single ancestral receptor, possibly corresponding most closely to MC4R Dores, . SignaliThe melanocortin agonist ligands for MCRs, adrenocorticotropic hormone (ACTH) and the melanocyte stimulating hormones αMSH, β MSH, and γMSH, are neuropeptides derived by enzymatic cleavage from proopiomelanocortin   of the hypothalamus (CNS), a region crucial in the control of food intake. Global homozygous deletion of MC4R in mice results in hyperphagia, increased fat and lean mass, increased body length, reduced activity, and reduced metabolic rate  in 2005 by Metherell et al. has provided insight into a novel aspect of MCR regulation, previously unknown  or 87%  of wild type intake) was required to normalize their weight gain to that of wild-type Mrap2+/+ mice. Faecal energy content was also indistinguishable between Mrap2−/− and Mrap2+/+ mice  when measured by indirect calorimetry. There was no difference in locomotor activity during the day or night and body temperature even when challenged by cold was not different  (Asai et al., There is no suggestion that Mrap2 plays a role in MC1R or MC2R function as Mrap2It is not yet known if MRAP is associated with mammalian obesity. There is a single case report describing a family with a splice site mutation of MRAP. Family members homozygous and heterozygous for the mutation were obese compared with normal weighted unaffected members (Rumie et al., −/− compared to wild-type littermates. This may represent the lack of sensitivity of the systems in place to detect the relatively small changes in food intake and energy expenditure. If so this demonstrates the fine balance in energy homeostasis, where small changes could tip the scale leading to significant weight gain. However, in light of the differences between Mc4r−/− and Mrap2−/− mice, it is likely that MC4R independent mechanisms as well as MCR independent pathways are at play. The complexity of these proteins and how they regulate MCR and GPCR function is only just beginning to be explored.The discovery of MRAP and MRAP2 has initiated a paradigm shift in our understanding of MCR and GPCR signaling Figure . For MRAThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The presence of an intonational phrase boundary is often marked by three major acoustic cues: pause, final lengthening, and pitch reset. The present study investigates how these three acoustic cues are weighted in the perception of intonational phrase boundaries in two experiments. Sentences that contained two intonational phrases with a critical boundary between them were used as the experimental stimuli. The roles of the three acoustic cues at the critical boundary were manipulated in five conditions. The first condition featured none of the acoustic cues. The following three conditions featured only one cue each: pause, final lengthening, and pitch reset, respectively. The fifth condition featured both pause duration and pre-final lengthening. A baseline condition was also included in which all three acoustic cues were preserved intact. Listeners were asked to detect the presence of the critical boundaries in Experiment 1 and judge the strength of the critical boundaries in Experiment 2. The results of both experiments showed that listeners used all three acoustic cues in the perception of prosodic boundaries. More importantly, these acoustic cues were weighted differently across the two experiments: Pause was a more powerful perceptual cue than both final lengthening and pitch reset, with the latter two cues perceptually equivalent; the effect of pause and the effects of the other two acoustic cues were not additive. These results suggest that the weighting of acoustic cues contributes significantly to the perceptual differences of intonational phrase boundary. Spoken language is hierarchically structured into prosodic units divided by prosodic breaks. While researchers disagree on the number and definitions of prosodic units, they generally agree that prosodic units include prosodic words, phonological phrases, and intonational phrases Previous studies have established three major acoustic correlates of IPBs: pause, final lengthening, and pitch reset Although it is relatively clear that IPBs often coincide with prosodic parameters such as pause, final lengthening, and pitch reset, which help listeners interpret prosodic boundaries, it is still unclear how these three cues are weighted on the perceptual side. Studies on how listeners weight these cues in the perception of IPBs have been scarce, and most findings are based on studies involving only two of the three major acoustic correlates The studies described above compared either the role of pause or the role of pitch change with that of final lengthening. Thus, there is, as yet, no clear picture of how these three correlates are weighted in the perception of IPBs. This issue has been partially resolved by Zhang As described above, whereas numerous studies have explored the acoustic correlates of IPBs in production, only a relatively small number have focused on how the acoustic correlates are weighted in the perception of IPBs, and no clear picture of it has emerged yet. In this study, we present two experiments explicitly testing the roles of pause, final lengthening, and pitch reset in Chinese. In line with Lin and Fon Forty-eight sentences that were originally used in Li and Yang 先/]IPB1 [花/时间/进行/练习/非常/重要/]IPB2.(1a) [想/保持/领xian1]IPB1 [hua1/shi2jian1/jin4xing2/lian4xi2/fei1chang2/bi4yao4]IPB2.If/you/want to/keep/ahead/, taking/time/to do/exercises/is very/necessary.‘If you want to keep ahead, it is very necessary to take time to do exercises.’IPB1.(1b) [商店里的/xian1 hua1/san4 fa1 chu1/zhen4 zhen4/nong2 yu4 de0/fang1xiang1]IPB1.In the store/the flowers/emit/bouts of/full-bodied/aroma.‘The flowers in the store emit bouts of full-bodied aroma.’[Shang1 dian4 li3 de0/These SB counterparts were used in the procedures of exchanging acoustic parameters. For the removal of final lengthening at IPBs, the duration patterns of the pre-boundary syllables in the IPB sentences were compressed to conform to the duration patterns of pre-boundary syllables in the SB sentences. For the removal of pitch reset, the fundamental frequency contours for the pre- and post-boundary syllables from the SB sentences were superimposed on the pre- and post-boundary syllables in the IPB sentences. The procedures used to create the experimental conditions are shown in Through the procedures described above, 48 sentence sets were created out of the original 48 sentences, with each set containing five manipulated sentences and one baseline sentence. Thus, altogether, 288 sentences were used for the perception experiment. The 48 sentence sets were counterbalanced according to a Latin square design and divided into six lists, with each sentence set presented only once within each list. Each list contained eight sentences per condition. To each list, 48 filler sentences with no sentence-internal IPB boundaries were also added. These filler sentences were added to balance the “Yes” and “No” responses of the task.All participants provided written informed consent in accordance with the Declaration of Helsinki. The ethics committee of the Institute of Psychology, Chinese Academy of Sciences approved this study, including its participant recruitment procedure and methodology.Twenty-four university students  participated in the experiment for cash. All were native speakers of Chinese. All of them reported having no hearing problems.The participants were tested individually in a sound-attenuating shielded chamber. They were seated in a comfortable chair approximately 60 cm in front of a monitor and were instructed to listen to the sentences attentively to detect speech boundaries. A trial started with a fixation cross, and 1000 ms later, a sentence was presented via headphones. At the end of each sentence, a question appeared on the screen which tested whether the participants had perceived the intended IPBs. For example, the question following sentence (1a) was “Do you perceive a boundary between “xian1” and “hua1”?” The participants were told to respond to this question by pressing “J” on the keyboard for a “Yes” response and “F” for a “No” response. The next trial began immediately after the participants gave their response. The experiment lasted about 15 minutes.A repeated-measures analysis of variance (ANOVA) was performed with the factor condition as the independent variable and the mean proportions of boundaries detected by the participants as the dependent variable. Greenhouse-Geisser adjustment was used to correct for violations of sphericity. Post hoc comparisons were adjusted using Bonferroni's correction.p<0.001, η2partial = 0.45. Post hoc comparisons revealed that the proportion of boundaries detected for the no-cue condition was significantly lower than those for all the other five conditions (ps<0.05). The proportion of boundaries detected for the pause condition was significantly higher than those for the final lengthening (p<0.05) and pitch reset condition (p<0.05). Similarly, the proportions of boundaries detected for the pause + final lengthening condition were also significantly higher than those for the final lengthening (p<0.01) and pitch reset condition (p<0.01). This pattern was the same for the baseline condition: A higher proportion was found for the baseline condition than for the final lengthening (p<0.05) and pitch reset condition (p<0.05). There was no significant difference between the pause, pause + final lengthening, and baseline conditions (ps>0.05). The final lengthening and pitch reset conditions did not differ from each other (p>0.05).Mean proportions of boundaries detected by the participants are displayed in The above results showed that participants' responses for boundary detection varied among the experimental conditions. However, note that except for the baseline condition, the five manipulated conditions were not natural speech but synthesized speech created in PRAAT. This could result in different degrees of naturalness, which may confound the effects of the acoustic parameters on the participants' responses for boundary detection. To examine whether the perceived boundary strength was influenced by the degrees of naturalness, we conducted a posttest by asking another 24 participants to rate the naturalness of the sentences on a scale of 1  to 7 . The results for the naturalness rating are given in p<0.001, η2partial = 0.53. Post hoc comparisons revealed that the no-cue condition was rated as less natural than all the other five conditions (ps<0.05). Furthermore, the pause condition was rated as more natural than the lengthening condition (p<0.01) and pitch reset condition (p<0.05). The pause + final lengthening condition was also rated as more natural than the lengthening condition (p<0.001) and pitch reset condition (p<0.01). Rating scores were higher for the baseline condition than for the lengthening condition (p<0.01) and pitch reset condition (p<0.01). No other differences were significant (ps>0.05). Thus, it appears that the naturalness of the sentences differed across conditions. To control for the influence of naturalness on the results of boundary detection, we ran a univariate analysis with condition as the independent variable, naturalness rating score as the covariate, and mean proportion of boundaries detected as the dependent variable. The results are shown in A repeated-measures ANOVA for the rating scores showed a main effect of condition, F  = 25.83, p<0.001, η2partial = 0.23. Post hoc comparisons revealed that the proportion of boundaries detected was significantly lower in the no-cue condition than in all the other five conditions (ps<0.01). The proportion of boundaries detected for the pause condition was significantly higher than those for the final lengthening condition (p<0.01) and pitch reset condition (p<0.01). Similarly, the proportion of boundaries detected for the pause + final lengthening condition was significantly higher than those for the final lengthening condition (p<0.01) and pitch reset condition (p<0.01). Finally, the proportion was also higher for the baseline condition than for the final lengthening condition (p<0.01) and pitch reset condition (p<0.01). No significant differences were found between the pause, pause + final lengthening, and baseline conditions (ps>0.05). There was also no significant difference between the final lengthening and pitch reset conditions (p>0.05). These results suggest that naturalness degree was not a confounding factor for the results of boundary detection.As shown in The above results showed that acoustic cues were weighted differently in the detection of IPBs: Pause was the strongest indicator of an IPB; final lengthening was perceptually equivalent to pitch reset; the effect of a pause and the effects of the other two acoustic cues were not additive. However, these results alone cannot provide us with a full picture of how the three acoustic cues are weighted in the perception of IPBs. It has been shown that listeners are sensitive not only to the presence or absence of a boundary, but also to how strong a boundary is The materials of Experiment 1 were used.All participants provided written informed consent in accordance with the Declaration of Helsinki. The ethics committee of the Institute of Psychology, Chinese Academy of Sciences approved this study, including its participant recruitment procedure and methodology.Twenty-four undergraduate students  participated for financial compensation. All were native speakers of Chinese with normal hearing. None of them had participated in Experiment 1.The procedures were the same as those of Experiment 1 except that the participants were asked to perform a different task in this experiment. Instead of judging the absence or presence of a boundary, they were asked to indicate how strong a boundary was on a 7-point scale from 1 “no boundary at all” to 7 “a very strong boundary.” For example, the question following sentence (1a) was “How strong is the boundary between “xian1” and “hua1?” The participants gave their answers by pressing the appropriate number keys on the keyboard.A repeated-measures ANOVA was performed with the factor condition as the independent variable and perceived boundary strength as the dependent variable. Greenhouse-Geisser adjustment was used to correct for violations of sphericity. Post hoc comparisons were adjusted using Bonferroni's correction.p<0.001, η2partial = 0.40. Post hoc comparisons revealed that the perceived strength for the no-cue condition was significantly weaker than for all the other five conditions (ps<0.05). Perceived strength for the pause condition was significantly stronger than that for the final lengthening condition (p<0.05) and pitch reset condition (p = 0.05). Stronger boundary strength was also found for the pause + final lengthening condition than for the final lengthening condition (p<0.01) and pitch reset condition (p<0.05). Finally, perceived strength was also stronger for the baseline condition than for the final lengthening condition (p = 0.06) and pitch reset condition (p = 0.07). There was no significant difference between the pause, pause + final lengthening, and baseline conditions (ps>0.05). The final lengthening and pitch reset conditions did not differ from each other (p>0.05).As shown in p<0.001, η2partial = 0.25. Post hoc comparisons revealed that the no-cue condition was perceived as less strong than all the other five conditions (ps<0.001). Perceived strength for the pause condition was significantly stronger than that for the final lengthening condition (p<0.001) and pitch reset condition (p<0.001). Stronger boundary strength was also found for the pause + final lengthening condition than for the final lengthening condition (p<0.001) and pitch reset condition (p<0.001). Finally, perceived strength was also stronger for the baseline condition than for the final lengthening condition (p<0.001) and pitch reset condition (p<0.001). No significant difference was found between the pause, pause + final lengthening, and baseline conditions (ps>0.05). There was also no significant difference between the final lengthening and pitch reset conditions (p>0.05). These results suggest that naturalness degree was not a confounding factor in our results. Thus, a strong pattern emerged whereby pause appeared to be a more powerful perceptual cue for the perceived strength of an IPB than final lengthening and pitch reset, with the latter two cues perceptually equivalent.These results showed that perceived boundary strength varied among experimental conditions. As with Experiment 1, to test whether the influence of naturalness degree was a confounding factor for the results of perceived boundary strength, we ran a univariate analysis with condition as the independent variable, naturalness rating score as the covariate, and perceived boundary strength as the dependent variable. The results shown in  again shThe aim of the present study was to test listeners' weighting of acoustic cues in their perception of IPB. The roles of three acoustic cues  were examined in two experiments. In Experiment 1, we examined how listeners weighted these acoustic cues in the detection of prosodic boundaries, while in Experiment 2, we examined how they weighted the same acoustic cues in the perceived strength of prosodic boundaries. The results of the two experiments consistently showed that the three acoustic cues played significant roles in the perception of IPB. More importantly, we found that they were weighted differently: Of the three cues, listeners relied most heavily on pause. Final lengthening and pitch reset were not heavily weighted, and these two cues were perceptually equivalent. Finally, the effect of pause and the effects of the other two acoustic parameters were not additive. These results suggest that acoustic cues are weighted differently not only in the detection of boundary presence, but also in the judgment of boundary strength.In Experiment 1, we found that the proportions of boundaries detected were significantly lower in conditions where none of the acoustic cues were present than in conditions where one or more of the acoustic cues were present. This suggests an important role of the acoustic cues in speech segmentation. Note that for the no-cue condition, the proportions of prosodic boundaries detected were around 40%. This might be because we used syntactically unambiguous sentences as the experimental material, and the explicit syntactic structure of the sentences probably has a predictive power on where a break is placed, as has been found previously In Experiment 2, we found that the no-cue condition was less natural than all the other five conditions. This suggests that IPBs were perceived as more natural when they were accompanied by acoustic cues. Consistent with Experiment 1, Experiment 2 revealed the same pattern of acoustic weighting in the perceived strength of IPBs: Pause appeared to be a more powerful perceptual cue for the perceived strength of an IPB than final lengthening and pitch reset, with the latter two cues perceptually equivalent. These results suggest that listeners' weighting of acoustic cues is not only displayed in their judgment of boundary presence, but also in their judgment of boundary strength.Previous research has found that pause is more responsible in cueing boundary size than final lengthening The results of both experiments showed that the pause condition was perceptually equivalent to the pause + final lengthening and baseline conditions. This suggests that perception of an IPB is heavily dependent on the presence of pauses, even to the extent that it may overrule the contribution of other parameters such as pre-boundary lengthening and pitch reset. The perceptual equivalence of a grammatical pause and a pause + final lengthening has been noted in Scott Across both experiments, no significant difference was found between final lengthening and pitch reset in terms of their perceptual effects on the listeners. This implies a discrepancy between production and perception: In the production of an IPB in Chinese, pitch reset was found to be a more reliable cue than final lengthening One thing that should be noted is that although we only used IPBs in the present study, it is difficult to know whether an IPB is still perceived as one when cues are removed or manipulated. In studies that have manipulated acoustic cues at IPBs, listeners showed the closure positive shift, a particular ERP component known to reflect the perception of IPBs, independent of the presence of a pause cue, suggesting that the remaining cues in combination were sufficient for successful perception of IPBs It should be noted that one reason listeners might be more affected by the removal of one cue compared to another may have to do with the salience of these variations for listeners. Previous study has suggested that only when the salience of two acoustic cues is comparable can their relative contributions be assessed In summary, the contribution of this research is that it is the first study to systematically manipulate the three acoustic correlates to examine cue weighting in the perception of IPBs. It is clear from the results that acoustic cues were weighted differently for the perception of IPBs: Pause was a more powerful perceptual cue than both final lengthening and pitch reset, with the latter two cues perceptually equivalent. Also, the effect of pause and the effects of the other two acoustic cues were not additive. However, this study is limited in that we only studied acoustic weighting in Chinese. Given that prosodic features vary across languages, which can result in different patterns of cue weighting across languages, future studies are needed to compare the perceptual weighting of the prosodic cues in different languages. Furthermore, note that we chose natural speech with well-formed structures to allow for a more natural acoustic realization of the boundaries. This is important in that it resembles what we encounter in everyday life. However, this could have reduced the effect of acoustic features in cueing boundaries. Thus, future studies are also needed to systematically explore the weighting of the three major acoustic correlates in syntactically ambiguous sentences.


HSPA1A, which encodes cognate heat shock protein 70, plays important roles in various cellular metabolic pathways. To investigate its effects on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), its expression level was compared between undifferentiated and differentiated BMSCs. Rat HSPA1A overexpression in BMSCs increased osteoblast-specific gene expression, alkaline phosphatase activity, and mineral deposition in vitro. Moreover, it upregulated β-catenin and downregulated DKK1 and SOST. The enhanced osteogenesis due to HSPA1A overexpression was partly rescued by a Wnt/β-catenin inhibitor. Additionally, using a rat tibial fracture model, a sheet of HSPA1A-overexpressing BMSCs improved bone fracture healing, as determined by imaging and histological analysis. Taken together, these findings suggest that HSPA1A overexpression enhances osteogenic differentiation of BMSCs, partly through Wnt/β-catenin. Mesenchymal stem cells (MSCs) retain their self-renewal capability and have the potential to differentiate into a variety of cell typesHSPA1A)38911HSPA1A might play a positive role in osteogenic differentiation of MSCs.Heat shock protein (HSP) 70 family is evolutionarily highly conserved molecular chaperone stabilizes existing proteins against aggregation and mediates the folding of newly translated proteins3et al. revealed crosstalk between HSC70 and Wnt/β-catenin signaling; that is, HSPA1A is upregulated in response to Wnt activation in stem cellsWnt/β-catenin signaling is a crucial regulator of MSCs1418HSPA1A increased during the process of osteogenic differentiation. Accordingly, we hypothesize that upregulated expression of HSPA1A could promote osteogenic differentiation of MSCs via Wnt/β-catenin signaling. by assessing the expression levels of specific genes and calcium deposition. We revealed that HSPA1A overexpression enhanced osteogenic differentiation of rat bone marrow MSCs (rBMSCs) partly via the Wnt/β-catenin signaling pathway in vitro. Moreover, using a rat tibial fracture model, we found that a sheet of rBMSCs overexpressing HSPA1A improved bone fracture healing in vivo.In this study, we found the endogenous expression of rBMSCs were purchased from Cyagen Biosciences . These cells can differentiate into osteoblasts, adipoblasts, and chondrocytes under specific inductive conditions. Adherent cells were trypsinized and passaged after reaching 80% confluence. Cells from passages 3–9 were used in subsequent experiments.Recombinant DKK1 was purchased from PeproTech . In accordance with a previous study, the applied concentration of DKK1 was 0.5 μg/mLHSPA1A  particles and lentiviral GFP particles  were prepared by Cyagen Biosciences. The overexpressing HSPA1A lentivirus particles details are specified in Lentivirus overexpressing HSPA1A was quantified by quantitative real-time polymerase chain reaction (qPCR) and Western blot analyses.For infections, rBMSCs were incubated with lentiviral particles and polybrene (5 μg/mL) in growth medium. After 5–6 h, the infection medium was discarded. After 3 days, the cells were screened using puromycin  and then passaged for use in subsequent experiments. The expression of HSPA1A overexpression on the proliferation of rBMSCs, the cells were seeded into a 96-well plate (5000/well) and allowed to adhere for 24 h. After 24 h, the medium was removed, and the cells were treated with 10% CCK-8  in 100 μL low-sugar Dulbecco’s modified Eagle’s medium (L-DMEM) without fetal bovine serum (FBS) for 2 h at 37 °C. Absorbance at 450 nm, which is directly proportional to cell proliferation, was measured using a microplate reader .To assess the effect of 4/cm2, and incubated for 48 h at 37 °C under 5% CO2. The cells were subsequently cultured in osteogenic induction medium . The cells were maintained by the addition of fresh osteogenic induction medium every 2–3 days.MSCs were cultured in growth medium  in 6- or 12-well cell culture plates , at a density of 3 × 10For the measurement of ALP activity, cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer , and the lysate (10 μL) was incubated with 90 μL fresh solution containing p-nitrophenyl phosphate substrate at 37 °C for 30–60 min. The reaction was stopped by the addition of 0.5 N NaOH (100 μL), and the absorbance was measured at 405 nm using a microplate reader . The total protein concentration was measured using a BCA protein assay kit . The relative ALP activity is expressed as the percentage change in optical density (OD) per unit time per milligram protein: (OD/15 min/mg protein) ×100.After the induction of osteogenic differentiation, mineral deposition was assessed by ARS (Cyagen Biosciences). Cells were fixed in 4% paraformaldehyde  for 15 min at room temperature and then washed with distilled water. A 1% solution of alizarin red was added and incubated for 30 min at room temperature, followed by rinsing with distilled water. The solution was collected, and 200 μL were plated on 96-well plates, which were read at 560 nm using a microplate reader . The readings were normalized to the total protein concentration.Cells were cultured in induction medium in a 12-well plate, and RUNX2, COL1A1, and β-catenin were detected using a fluorescence microscope . Briefly, cells were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized, and blocked for 30 min in 0.05% Triton X-100 and 2% bovine serum albumin. Fixed cells were washed and incubated overnight with anti-RUNX2 , COL1A1 , or β-catenin . Cells were incubated with a fluorescence-conjugated secondary antibody (Beyotime) for 120 min, and nuclei were stained with 4′,6-diamidino-2-phenylindole  for 2 min. Samples were observed under a fluorescence microscope (Leica).−△△Ct method.Total cellular RNA was isolated using RNAiso reagent  and quantified by measuring the absorbance at 260 nm . Total RNA (≤1000 ng) was reverse-transcribed into cDNA in a reaction volume of 20 μL using the Double-Strand cDNA Synthesis Kit . One microliter of cDNA was used as the template for the qPCR reaction. All gene transcripts were quantified by qPCR using the Power SYBR® Green PCR Master Mix (Takara) on the ABI StepOnePlus System . The mRNAs of the target genes and the housekeeping gene (18S) were quantified in separate tubes. All primers were synthesized by Sangon Biotech . The primer sequences used are shown in HSPA1A , Osteocalcin (OCN) , RUNX2 (1:1600), COL1A1 (1:1000), or β-catenin (1:1000). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG  was applied as a secondary antibody for 2 h at room temperature. The immunoreactive bands were detected using an enhanced chemiluminescent detection reagent . Signal intensity was measured using a Bio-Rad XRS chemiluminescence detection system .Cells were lysed in RIPA lysis buffer supplemented with a proteasome inhibitor (Beyotime). Total proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane . After blocking in 5% non-fat milk for 2 h, the membranes were incubated overnight at 4 °C with antibodies specific to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) , 5/cm2 were cultured in L-DMEM with the addition of vitamin C (20 μg/mL)A cell sheet was fabricated as we reported previouslyAll Sprague Dawley (SD) rats were supplied by the Academy of Medical Sciences of Zhejiang province. All animal experiments were performed in accordance with the Animal Care and Use Committee guidelines of Zhejiang province. All experimental procedures were approved by the Institutional Animal Care and Use Committee at Zhejiang university.HSPA1A group (n = 5), a sheet of rBMSCs overexpressing HSPA1A was wrapped around the fracture site.All surgical procedures were performed by two experienced traumatic orthopedic surgeons (Zhijun Pan and Deting Xue). Fractures were generated in 8-week-old male SD rats (weighing approximately 200 g). The rats were anesthetized with 0.3% pentobarbital sodium (Sigma) intraperitoneally at 30 mg/kg body weight. The fracture model was established as reported previously2425+ Mo target mammography machine  8 weeks after surgery to evaluate callus formation and bridging bone formation at the fracture sites. All samples were scanned for bone formation using a μCT 100 imaging system  with the following scan parameters: X-ray energy setting of 70 kVp, 1024 reconstruction matrix, slice thickness of 0.0148 mm, and integration time of 300 ms. The bone volume fraction (BV/TV) including the area of bone formation was calculated by three-dimensional standard microstructural analysisAnimals were euthanized 8 weeks after surgery, and samples were collected. Radiographs were taken using a dual-track molybdenum/rhodiumThe samples were fixed using 4% paraformaldehyde for 72 h at room temperature and then decalcified using 10% ethylenediaminetetraacetic acid (Sigma), with a solution change once a week for more than 8 weeks, before embedding in paraffin. Serial sections of 3 mm thickness were cut and mounted on polylysine-coated slides. Hematoxylin and eosin, Safranin O, Masson, and immunofluorescence staining of COL1A1 were performed separately on consecutive tissue sections in accordance with previous studies24t-test when comparing two groups, and one-way ANOVA followed by Bonferroni’s post hoc test when comparing more than two groups. A P value of 0.05 or less was considered to represent a statistically significant difference.Statistical analysis was performed using SPSS 17.0 software . All experiments were performed at least in triplicate, and the data are presented as means ± standard deviation. Statistical significance was determined using a two-tailed Student’s HSPA1A associated with osteogenic differentiation of MSCs, we examined HSPA1A endogenous expression in rBMSCs at days 0, 7, and 14 during the process of osteogenic differentiation. Compared with undifferentiated rBMSCs, the mRNA expression of HSPA1A increased significantly at days 7 and 14 during osteogenic differentiation -positive cells to the total cell number , HSPA1A were overexpressed 4.12-fold in the lenti-HSPA1A group , and COL1A1, were detected by qPCR and Western blot analyses. qPCR analysis revealed that ALP, RUNX2, OCN, and COL1A1 mRNA levels were significantly higher in rBMSCs overexpressing HSPA1A at days 3, 7, and 14 than in the non-overexpressing rBMSCs  than in the cells with HSPA1A overexpression alone.To verify the involvement of the Wnt/β-catenin signaling pathway, we evaluated the inhibitory effect of this pathway on osteogenesis in the HSPA1A than in the lenti-HSPA1A+DKK1 group  and late markers of osteogenic differentiation  were also increased due to HSPA1A overexpression. In addition, this overexpression significantly enhanced mineral deposition. Meanwhile, HSPA1A overexpression had no adverse effects on the metabolic activity or cell proliferation of rBMSCs. These results indicate that HSPA1A overexpression promoted the osteogenic differentiation of rBMSCs in vitro.Mounting evidence has revealed that increased expression of HSPA1A upregulation was closely related to activation of the Wnt/β-catenin signaling pathway14HSPA1A during osteogenesis. Moreover, higher β-catenin accumulation was observed, the majority of which was in the nucleus upon HSPA1A overexpression, suggesting that HSPA1A overexpression activates β-catenin-mediated transcription. Meanwhile, lower levels of DKK1 and SOST were observed, which inhibited Wnt/β-catenin by binding to the third β-propeller domain of LRP5/6 via interference of Wnt/LRP/Fz trimolecular complex formationHSPA1A overexpression was rescued partly by an inhibitor of Wnt/β-catenin (DKK1). These findings indicate that overexpression of HSPA1A regulates the Wnt/β-catenin signaling pathway during osteogenesis of MSCs.A previous study reported crosstalk between HSC70 and Wnt/β-catenin signaling in stem cells; that is, HSPA1A were present in the sheet. Additionally, these HSPA1A-overexpressing rBMSCs did not cause localized tumors or malignancies. However, there were only a few cells exhibiting GFP fluorescence in the rBMSC sheet groups, which suggested that only a fraction of the implanted rBMSCs directly differentiated into osteocytes and produced bone matrix. The majority of new bone formation arose from exogenous cells, which is consistent with previous studies28in vivo, when the sheet of rBMSCs overexpressing HSPA1A was implanted for fracture repair. Guang et al. revealed that HSC70 plays a significant role in endothelial cell development and angiogenesiset al. also reported that HSC70 could bind and stabilize endogenous AU-rich element-containing mRNAs encoding vascular endothelial growth factorIt has been demonstrated that MSC sheets can enhance bone regeneration2632HSPA1A overexpression mediates the Wnt/β-catenin signaling pathway to promote osteogenic differentiation of rBMSCs, it is probably involved in the activation of other signaling pathways as well. Second, the mechanisms of bone healing acceleration using a sheet of rBMSCs overexpressing HSPA1A have not been clarified completely, and thus further studies are needed.The present study has some limitations. First, although we indicated that HSPA1A overexpression enhanced osteogenic differentiation of rBMSCs, partly through activation of the Wnt/β-catenin signaling pathway. The use of HSPA1A-overexpressing MSCs to enhance fracture repair has potential as a new MSC-based therapy.Based on our data, we found that How to cite this article: Zhang, W. et al. Overexpression of HSPA1A enhances the osteogenic differentiation of bone marrow mesenchymal stem cells via activation of the Wnt/β-catenin signaling pathway. Sci. Rep.6, 27622; doi: 10.1038/srep27622 (2016).


Hydrophilic nature of epoxy polymers can lead to both reversible and irreversible/permanent changes in epoxy upon moisture absorption. The permanent changes leading to the degradation of mechanical properties due to combined effect of moisture and elevated temperature on EPON 862, Nanomer I.28E, and Somasif MAE clay-epoxy nanocomposites are investigated in this study. The extent of permanent degradation on fracture and flexural properties due to the hygrothermal aging is determined by drying the epoxy and their clay-epoxy nanocomposites after moisture absorption. Significant permanent damage is observed for fracture toughness and flexural modulus, while the extent of permanent damage is less significant for flexural strength. It is also observed that permanent degradation in Somasif MAE clay-epoxy nanocomposites is higher compared to Nanomer I.28E clay-epoxy nanocomposites. Fourier transform infrared (FTIR) spectroscopy revealed that both clays retained their original chemical structure after the absorption-desorption cycle without undergoing significant changes. Scanning electron microscopy (SEM) images of the fracture surfaces provide evidence that Somasif MAE clay particles offered very little resistance to crack propagation in case of redried specimens when compared to Nanomer I.28E counterpart. The reason for the observed higher extent of permanent degradation in Somasif MAE clay-epoxy system has been attributed to the weakening of the filler-matrix interface. Their main distinction from other types of polymers lies in their densely crosslinked molecular structure. This crosslinking leads to a number of favorable thermal and mechanical properties including high strength and modulus, high creep resistance, high glass transition temperature, low shrinkage, and better chemical resistance. These properties in conjunction with ease of processing have made epoxy resins an attractive choice for use in many engineering components and structures. They have found huge applications in aerospace, automotive, packaging, coating, and microelectric industries. In recent years, researchers have developed and investigated polymer nanocomposites based on a wide variety of micro-/nanoscale fillers including clay particles –6, alumiarticles , graphenarticles , 10, cararticles –13, and articles , 14 to iEpoxy polymers are characteristically hydrophilic, which means that they have strong affinity towards water. This nature of epoxy resins makes them susceptible to high moisture absorption; in general, depending on the nature of the epoxy resin, the equilibrium moisture uptake can be in the range of 1–7% . Absorbeg from 260°C to 130°C [g is reduced by 20°C/1 wt% of moisture intake [Water absorption into a polymer matrix leads to change in both chemical and physical characteristics and affects the mechanical properties through different mechanisms such as plasticization, crazing, hydrolysis, and swelling. Plasticization is the most important physical change that occurs through the interaction of the water molecules with polar groups in the matrix, which can severely depress the glass transition temperature , 21–24. to 130°C –27. In ge intake . The dece intake –32. Othee intake –35, where intake . This wae intake . The afoe intake , 36. PlaIn recent years, effect of moisture absorption on the mechanical properties of neat epoxy and clay-epoxy nanocomposite has been frequently studied. Zhao and Li reported that tensile strength and modulus decreased for both neat epoxy and nanocomposites upon moisture absorption, while the tensile strain increased significantly for moisture absorbed samples. Although failure occurred in brittle manner, effect of plasticization was found in SEM images, which showed shear yielding for both neat epoxy and nanocomposite samples after being exposed to moisture . Similarperature . It is eperature , 29, 33,perature , shear mperature , 31, fleperature , 43, yieperature  as waterMost of the research on polymer-clay nanocomposites has been mainly focused on investigating the effect of moisture absorption on mechanical properties such as elastic modulus and tensile strength. Although fracture toughness is a very important property for these nanocomposites as these are used in various structural applications, the effects of moisture absorption on fracture toughness of polymer-clay nanocomposites have not been studied extensively. Durability of polymer/clay nanocomposites is still needed to be studied in depth, particularly for hygrothermal aging in which the degradation of the mechanical properties and loss of integrity of these nanocomposites occur from the simultaneous action of moisture and temperature. This study on clay-epoxy nanocomposites was designed to investigate the effect of hygrothermal aging on mechanical properties of these nanocomposites. Two different clay particles were used to investigate the effect of clay structure on the permanent property changes due to hygrothermal aging. A drying cycle was employed to quantify the recovery of the properties after hygrothermal aging. This was helpful to understand the extent of permanent degradation that occurred by the combined application of elevated temperature and moisture. Mechanical properties in terms of fracture toughness, flexural strength, and flexural modulus are the properties that were studied. Scanning electron microscopy and Fourier transform spectroscopy were conducted to further elucidate the underlying fracture mechanisms of these preconditioned specimens.The epoxy resin used for this study is EPON 862, which is a diglycidyl ether of bisphenol F. The curing agent used for this resin system was a moderately reactive, low viscosity aliphatic amine curing agent, Epikure 3274. Both of these chemicals were supplied by Miller-Stephenson Chemical Company, Inc., Dunbury, Connecticut. Two structurally different clay particles were used as reinforcement. Nanomer I.28E is a surface modified montmorillonite based layered silicate  modified with a quaternary amine (trimethyl stearyl). Somasif MAE , which was the other clay particle used for this study, is a synthetic mica modified with dimethyl dihydrogenated tallow ammonium chloride. Epoxy was preheated to 65°C before desired amount of clay was introduced and mixed using mechanical mixer for 12 hours. To reduce the viscosity of the mixture and to facilitate mixing, temperature was maintained at 65°C using a hot plate for the entire duration of mixing. The mixture was then degassed for around 30 minutes to remove any entrapped air bubbles. Bubble-free mixture of clay and epoxy was then shear-mixed using a high-speed shear disperser  for 30 minutes. During this process, temperature was maintained at 65°C using an ice bath. Subsequently, the mixture was then degassed until it was completely bubble-free. Curing agent was added to the mixture at 100 : 40 weight ratio and carefully hand-mixed to avoid introduction of any air bubble. After it was properly mixed, the final slurry containing epoxy and clay was poured into an aluminum mold and cured at room temperature for 24 hours followed by postcuring at 121°C for 6 hours. The final sample had a nominal dimension of 177.80 mm × 152.50 mm × 12.70/6.35 mm. To study the effect of loading percentage, the weight fraction of the clay in the nanocomposite was varied from 0.5 to 2.0%.After specimens were cut into the final required dimension according to the ASTM standards D5045 and D790, they were subjected to degradation. Specimens from each nanocomposite were taken and submerged in purified deionized boiling water for 24 hours. Water saturated specimens were dried in an oven at 110°C for 6 hours to remove moisture from the samples leaving only permanent degradation in the form of bonded water.KIc, was determined by single edge notch bend (SENB) test as per the ASTM D5045 on universal testing machine  in a displacement-controlled mode with fixed crosshead speed of 10 mm/min. Nominal dimension for the SENB test samples was 67.30 mm × 15.20 mm × 6.35 mm. A notch was created using precision diamond saw  and, afterwards, a sharp precrack with ratio of 0.45 < a/W < 0.55 was created by tapping a fresh razor blade into the notch. At least 5 specimens were tested for every condition. Fracture toughness for the specimens was calculated in terms of critical stress intensity factor, KIc. The crack length, a, was measured using an optical microscope (Nikon L150) which has a traveling plate with graduations:P = maximum applied force (N), B = thickness of the specimen (mm), W = width of the specimen (mm), and f(a/W) = geometry factor, and it is given by the following equation:S = support span (mm) and a = length of the precrack (mm).Critical stress intensity factor, R = rate of crosshead motion (mm/min), L = support span (mm), d = depth of beam (mm), and  Z = rate of straining of the outer fiber (mm/mm/min) = 0.01.Flexural strength and flexural modulus were determined using three-point bend 3PB) test according to ASTM D790 on universal testing machine . The nominal dimension for the flexural test specimens was 55.90 mm × 12.70 mm × 6.35 mm. The crosshead speed for the test was calculated using , Eb = flexural modulus (MPa), Pmax = maximum load on the load-deflection curve (N), L = support span (mm), b = width of beam tested (mm), d = depth of beam tested (mm), and m = slope of the tangent to the initial straight-line portion of the load-deflection curve (N/mm of deflection).The flexural strength and flexural modulus were calculated using the following equations, respectively:Surface morphology of the fractured specimens from SENB tests was observed using scanning electron microscopy . As polymer materials are nonconductive to electrons, all fracture surfaces were sputtered with gold-palladium alloy before SEM imaging.−1. Each spectrum was recorded from 4000 to 500 cm−1 at room temperature. Spectra were analyzed using OriginPro 9.0 .FTIR spectroscopy measurements were performed using ATR-FTIR spectrometer  using 64 scans at a resolution of 2.0 cmKIc values for the as-prepared samples are also listed as a reference.The critical stress intensity factors as a function of clay loading percentage for Nanomer I.28E and Somasif MAE clay-epoxy nanocomposites are shown in KIc, increased 28% for the as-prepared 0.5 wt% Nanomer I.28E clay-epoxy nanocomposite compared to neat epoxy. The reason behind this observation can be attributed to the layered structure of the clay. Clay in a polymer material physically blocks, bifurcates, and deflects the crack path, compelling the crack to travel longer path, which in turn results in higher toughness in a clay-polymer nanocomposite. The toughening effect of clay on epoxy polymer started to decrease with any additional clay reinforcement. This is a common behavior for several types of epoxy-clay nanocomposites and has been reported in previous studies conducted on clay-epoxy nanocomposites [KIc was found to be lower compared to the as-prepared nanocomposite samples for all the Nanomer I.28E nanocomposites. As water molecules diffuse into the nanofiller-epoxy interface, debonding and weakening of the interface occur, resulting in poor stress transfer between the filler and the epoxy matrix [Critical stress intensity factor, mposites , 45, 46.y matrix , 47, 48.For the as-prepared and moisture saturated samples, Somasif MAE nanocomposites showed comparable trend in fracture toughness data; clay reinforcement successfully improved the baseline epoxy properties and moisture absorption degraded the mechanical properties for all clay percentages. However, the permanent degradation after absorption-desorption cycle was found to be more prominent in the case of Somasif MAE clay nanocomposites compared to Nanomer I.28E clay nanocomposites. The recovery of property after 6 hours of drying was negligible for Somasif MAE clay nanocomposites, whereas Nanomer I.28E nanocomposites showed significant recovery of property after drying. The difference in property recovery of these two clay-epoxy nanocomposites can be attributed to the structural differences of the two clay particles and has been further investigated through SEM and FTIR technique.Flexural modulus for the neat epoxy and clay-epoxy nanocomposites was determined from 3PB test and has been plotted against clay loading percentage in For the hygrothermally conditioned specimens, the modulus is lower compared to the as-prepared specimens. This behavior observed is mostly due to the presence of water inside the epoxy system, which increases the ductility of the epoxy system. Water acts as an effective plasticizer and can diffuse into the nanofiller-polymer interface and weaken the bonding between them , 43. PreFor Nanomer I.28E clay-epoxy samples conditioned at 110°C for 6 h, recovery of flexural modulus was observed. Once redried, free water residing in the microvoids was evaporated, and the effect of plasticization was not prominent anymore. As a result, the ductility of the polymer reduced and the recovery of mechanical properties from moisture absorbed state occurred. Nevertheless, in case of Somasif MAE clay-epoxy samples, modulus recovery was negligible after the desorption cycle. Due to the structural difference between the two clay particles, it is possible that the interface of Somasif MAE clay-epoxy is being more affected by the hygrothermal degradation than the Nanomer I.28E clay-epoxy interface.Flexural strengths for the epoxy and clay-epoxy nanocomposites were determined using three-point bend (3PB) test and are plotted against clay loading percentage in Almost similar trend was observed for Somasif MAE clay-epoxy nanocomposites, where addition of clay did not change the property significantly, and after 24 h of hygrothermal aging property decreased to a lower value compared to the as-prepared samples. However, the severity of degradation was much less in both clay-epoxy systems compared to neat epoxy system. Well dispersed high aspect ratio clay platelets have the capability of crack deflection and crack arresting, which can lead to the observed higher flexural strength in wet clay-epoxy samples in comparison to neat epoxy samples , 57. ForThe scanning electron micrographs of the fracture surface of tested neat epoxy and clay-epoxy nanocomposites are shown in Figures The SEM micrographs of clay incorporated epoxy systems showed significantly rougher fracture surface compared to the neat polymer. Clay, if present in a system, physically blocks and slows down the crack propagation and the resulting fracture surface of the nanocomposite shows river-markings instead of smooth fracture surface found in the neat polymer. These river-markings provide clear indication of the enhanced toughening mechanism in polymeric materials due to clay incorporation and support the observed higher fracture toughness for clay-epoxy nanocomposites compared to the neat epoxy polymer. Fracture surfaces of nanofiller reinforced polymer nanocomposites showing higher surface roughness have been reported in prior studies , 6, 59. Fracture surface of moisture absorbed Nanomer I.28E clay nanocomposites Figures  showed lFracture surface micrographs of redried Nanomer I.28E nanocomposites Figures  showed m−1, which is characteristic OH stretching of the hydroxyl group. However, significant difference was not observed for the bands at 3200–3400 cm−1.FTIR spectra of the as-prepared and redried neat epoxy are shown in −1 region. Nanomer I.28E clay showed another peak at 915 cm−1 region, which is characteristic Al–OH peak. In Somasif MAE, the OH groups present in the corner of the octahedral layer of Nanomer I.28E were substituted with F, which explains the absence of the peak at 915 cm−1. Both clays in as-is condition showed 3200–3400 cm−1 band for hydroxyl groups. However, the band was weaker in case of Somasif MAE compared to Nanomer I.28E clay because of the hydrophobic nature of the Somasif MAE clay. It was interesting to note that the overall changes the clay powders underwent as a result of absorption-desorption cycle are fairly small and can be considered as insignificant.Property deterioration due to moisture absorption has been one of the most important areas of interest in polymer research for the last few years. To be used as structural materials, it is of utmost importance to understand the reversible and irreversible changes occurring in polymeric materials as a result of moisture absorption. This study was conducted to elucidate the effect of moisture absorption on the mechanical properties of clay reinforced epoxy polymers. Fracture toughness, flexural strength, and flexural modulus were determined for two different clay-epoxy nanocomposites following the ASTM standards. The effects of hygrothermal aging and subsequent redrying on the mechanical properties of these polymer nanocomposites were investigated. After removing the free water by drying, the irreversible effect or the permanent damage due to hygrothermal aging on the clay-epoxy nanocomposite systems was determined. Irrespective of the clay reinforcement type, all the studied properties were degraded due to hygrothermal aging. Several physical and chemical changes, such as interface weakening, hydrolysis, and chain scission, are responsible for the observed effect. The permanent damage or degradation was severe in case of fracture toughness and flexural modulus. Flexural strength of both systems was relatively unaffected by the absorption-desorption cycle. Permanent damage was found to be the highest for Somasif MAE clay reinforced specimens between two clay-epoxy nanocomposite systems. After studying the SEM micrographs of the fracture surfaces, it was speculated that moisture absorption had higher negative impact on the interface of Somasif MAE clay and epoxy matrix compared to the other clay-epoxy system. The hydrophobic nature of the Somasif MAE clay due to the presence of –F (fluorine) in the structure may have created additional tension between the polymer crosslinks in presence of moisture. FTIR spectra of the clay particles treated with the same absorption-desorption cycle provided proof that both nanoparticles undergo minimal chemical change and retain their respective original chemistry. This observation makes the aforementioned speculation more plausible. Although incorporation of clay in epoxy matrix did not fully stop the degradation, it had positive effects to some extent. It was observed that the studied properties in general were less severely degraded for clay-epoxy nanocomposites compared to neat epoxy samples. Therefore, clay particles could be successfully used to reinforce polymer materials to reduce the severity of property deterioration caused by the moisture absorption. However, the chemistry between the clay particles and polymer matrix and more specifically the chemical structure of the clay particles should be carefully considered to attain the best possible resistance against the property deterioration.