The Wnt/β-catenin pathway is a major signaling cascade in bone biology, playing a key role in bone development and remodeling. The objectives of this study were firstly, to determine the effects of dexamethasone exposure on Wnt/β-catenin signaling at an intracellular and transcriptional level, and secondly, to assess the phenotypic effects of silencing the Wnt antagonist, Dickkopf-1 (Dkk1) in the setting of dexamethasone exposure.-8 M dexamethasone over a 72 h time course. The phenotypic marker of osteoblast differentiation was analyzed was alkaline phosphatase activity. Intracellular β-catenin trafficking was assessed using immunoflourescence staining and TCF/LEF mediated transcription was analyzed using a Wnt luciferase reporter assay. Dkk1 expression was silenced using small interfering RNA (siRNA).Primary human osteoblasts were exposed in vitro to 10Primary human osteoblasts exposed to dexamethasone displayed a significant reductions in alkaline phosphatase activity over a 72 h time course. Immunoflourescence analaysis of β-catenin localization demonstrated a significant reduction in intracytosolic and intranuclear β-catenin in response to dexamethasone exposure. These changes were associated with a reduction of TCF/LEF mediated transcription. Silencing Dkk1 expression in primary human osteoblasts exposed to dexamethasone resulted in an increase in alkaline phosphatase activity when compared to scrambled control.in vitro. Silencing Dkk1 expression rescues dexamethasone-induced suppression of primary human osteoblast differentiation. Targeting of the Wnt/β-catenin signaling pathway offers an exciting opportunity to develop novel anabolic bone agents to treat osteoporosis and disorders of bone mass.Wnt/β-catenin signaling plays a key role in regulating glucocorticoid-induced osteoporosis  Osteoporosis is a systemic skeletal disorder characterized by low bone mass and a progressive micro-architectural deterioration of bone tissue, leading to an increase in bone fragility and ultimately susceptibility to fracture . There hwingless and the murine int-1 proto-oncogene. Human and mouse genomes have been shown to encode 19 and 18 Wnt genes, respectively [Wnt proteins are a family of secreted cysteine-rich glycoproteins with functions relating to cell specification, formation of the body plan, cell growth, proliferation, differentiation and apoptosis -5. Wnts ectively . Wnt proThe role of canonical Wnt signaling in bone development and remodeling was first identified when the loss-of-function mutation of the LDL receptor-related protein (LRP5) was associated with osteoporosis psuedoglioma syndrome, a recessive disorder characterized by osteogenesis imperfecta with low bone mass and a predisposition to fractures as well as ocular defects ,8. SubseIn this study, we focused our attention on the role played by Wnt/β-catenin signaling in glucocorticoid-induced osteoporosis. We determined the effects of dexamethasone exposure on Wnt/β-catenin signaling in primary human osteoblasts. We also examined the phenotypic effects of attenuating the expression of Dkk1, a Wnt antagonist, in primary human osteoblasts exposed to dexamethasone.2. For all experiments primary human osteoblasts used were between 2th and 5th passage. Cells were treated over a 48 h time course with 10-8 M Dexamethasone (Dex) . Institutional Review Board approval for this study was obtained from the Mater Misericordiae University Hospital Research Ethics Committee.Primary human osteoblasts from normal human hip samples were . Cells were cultured in Osteoblast Growth Medium  containing 10% FCS and antibiotics (100 IU/ml penicillin and 100 μg/ml streptomycin) at 37°C, 5% COp-nitrophenol from p-nitrophenolphosphate (pNPP). Post-treatment cell lysates were collected by Cell Lytic MT  treatment and the extracted protein was frozen at -80°C for at least 2 h prior to ALP activity assay to disrupt cellular membranes. 10 μl of thawed cell lysates were incubated with 200 μl pNPP reagent  for 30 min at room temperature. ALP activity was measured at 405 nm and normalized to total protein extracted, which was measured using a bicinchoninic acid (BCA) protein assay kit . One unit of ALP activity was defined as the amount required for the liberation of 1.0 μmol p-nitrophenol/min/mg of cellular protein.Alkaline phosphatase (ALP) activity was assayed by the measurement of released plus research digital camera . The digital images were processed using the ProgRes CapturePro 2.1 image analysis softwear package .Beta-catenin was visualized by indirect immunocytochemistry using a mouse anti β-catenin  as the primary antibody. Primary human osteoblasts were plated on coverslips and treated with Dex-containing media. Cells were fixed in ice-cold methanol and permeabilized for ten minutes. Cells were then blocked with 10% goat serum for ten minutes at room temperature. Samples were incubated for 1 hr with primary antibody followed by a 30 min incubation with a goat anti-mouse TRITC-conjugate. Cells were viewed with a Zeiss Axioskop 40 fluorescence microscope using 20× objectives. Digital images were captured with a ProgRes C10Mouse Wnt3a overexpressing cells (L-Wnt3a) and control non-transfected L-cells were obtained from the American Type Culture Collection  and cultured in DMEM with glutamax, 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin. L-Wnt3a culture medium was supplemented with 400 μg/ml geneticin to maintain selective pressure. Conditioned medium from L-Wnt3a and control L-cells was collected according to the manufacturer's instructions. Briefly, cells were passaged 1:10 in 8 ml medium without geneticin and left to grow for 10 days. Medium was collected from each cell line and replaced with 8 ml fresh medium for a further 3 days. This second batch of medium was then collected and the cells discarded. First and second batches were combined, sterile filtered (0.2 μm) and stored at -20°C until required.4/cm2 were transiently transfected with the Wnt-luciferase reporter construct pBAR  and the control reporter pfuBAR  using GeneJuice . The β-catenin activated reporter (pBAR) contains 12 TCF binding sites (5'-AGATCAAAGG-3') separated by distinct 5 base linkers. These elements are directly upstream of a minimal thymidine kinase promoter which then drives the expression of firefly luciferase. The control reporter pfuBAR is identical to its sister reporter with the exception of a two base substitution in each TCF binding site. Both reporters contain a separate PGK promoter that constitutively drives the expression of a puromycin resistance gene, and both are in a lentiviral platform. Co-transfection with 0.5 μg of the internal control reporter pSL9EF1a(P)RLUC , driving constitutive expression of Renilla luciferase, was systematically performed to normalize for transfection efficiency. Sixteen hours after transfection, cells were washed and cultured for 48 hr in Dex-containing media with 2% FCS, in the presence or absence Wnt3a-conditioned media. Cells were lysated, and luciferase assays were performed with the Dual Luciferase Assay Kit  according to manufacturer's instructions. 10 μl of cell lysates were first assayed for firefly luciferase and then Renilla luciferase activity. Firefly luciferase activity was normalized to Renilla luciferase activity.Primary human osteoblasts plated in 24-well plates at 2 × 102, at which point the medium was changed and experimentation was commenced. Transfection efficiency was determined in three preliminary experiments in which a fluorescent control RNA-RNAiFect complex  was transfected into the cells instead of the siRNA-RNAiFect complex. The uptake of the fluorescent RNA as determined by fluorescence microscopy was in the range of 75-85%. The ratio of siRNA to the RNAiFect reagent was determined three preliminary experiments with a ratio of 1 μg siRNA:3 μl RNAiFect providing a maximal gene silencing of 75% knockdown as determined by qRT-PCR.Predesigned short interfering RNA (siRNA) targeting human Dkk1 (Hs_DKK1_1) and a control scrambled RNA targeting a sequence not sharing homology with the human genome (AllStars Negative Control) were purchased . Primary human osteoblasts were transfected with siRNAs and control scrambled RNA using the RNAiFect transfection reagent  as per manufacturer's protocol. Briefly, siRNA or scrambled RNA solutions were prepared 15-25 min before the cell transfection. The ratio of siRNA to the RNAiFect reagent was 1 μg siRNA to 3 μl transfection reagent. The siRNA-RNAiFect transfection complexes were incubated for 15 min at room temperature (15-25°C). Osteoblast growth medium was replaced with fresh medium and the siRNA-RNAiFect suspension was added drop-wise onto the cells. The cells with adherent complexes were incubated for 24 h at 37°C, 5% CODkk-1 mRNA regulation in primary human osteoblasts treated with dexamethasone was measured by quantitative Real Time PCR using human Dkk-1 QuantiTect assay . The QuantiProbe sequence for Dkk-1 was 5'-CACACCAAAGGACAAGA-3'. The forward primer sequence for Dkk-1 was 5'-GGGAATTACTGCAAAAATGGAATA-3', and the reverse primer sequence was 5'-ATGACCGGAGACAAACAGAAC-3'. Total RNA was extracted by TRI-reagent/chlorophorm method, then assayed in duplicate using a Rotorgene 3.0 Real Time PCR instrument  and the Real Time PCR amplification kit SYBR Green I . Using gene specific primer pairs, Dkk1 gene products were measured by Absolute Quantification and were reported as a function of crossing time (Ct), the cycle number at which PCR amplification becomes linear. mRNA expression was normalized to control and GAPDH expression resulting in Mean Fold Change values or ΔΔCt. Following cycling, to ensure specificity, melt curve analysis was carried out to verify the amplification of PCR products starting at 65°C and ramping to 95°C at 0.1°C/sec. One peak in the melt curve indicated no secondary, non-specific products were formed.Human Dkk1 ELISA kit  was obtained to analyse Dkk1 protein expression on cell supernatant. ELISA was performed in accordance with manufacturer's protocols.t-test or ANOVA for multiple comparisons. p values < 0.05 was considered statistically significant.All experimental data presented were obtained from three independent experiments, each in triplicate. Data were expressed as mean ± SE. Statistical differences were calculated using Student's -8 M dexamethasone over a 72 h time course. The marker of osteoblast differentiation assessed was alkaline phosphatase activity.To study the effects of dexamethasone, on markers of osteoblast differentiation, we exposed primary human osteoblasts to 10t-test) of dexamethasone exposure when compared to control primary human osteoblasts  and 48 h  of dexamethasone treatment in the pBAR reporter cells, and the luciferase activity in the pfuBAR reporter cells remained unchanged during treatment  and control L-cell conditioned media  over the 48 h time course, representing a reduction in Wnt/β-catenin signaling through the inhibition of TCF/LEF mediated transcription  and L-cell conditioned media (control) over a 48 h time course. Significant reductions in luciferase activity were identified when 10In order to explore the phenotypic effects that aberrations in Wnt/β-catenin signaling have on primary human osteoblasts exposed to dexamethasone, we silenced the expression of Dickkopf-1 (Dkk1), a powerful Wnt antagonist, using siRNA. Primary human osteoblasts were transfected with siRNA targeting Dkk1 expression or scrambled control RNA and Dkk1 gene expression was assessed using quantitative RT-PCR Figure  and ELISIn order to confirm that knockdown of Dkk1 was inhibiting Wnt/β-catenin signaling in primary human osteoblasts, we firstly examined β-catenin trafficking using immunoflourescence analysis. Primary human osteoblasts were grown to confluency and transfected with siRNA targeting Dkk1 expression or scrambled control RNA. siRNA transfected primary human osteoblasts Figure  demonstrt-test) in the pBAR reporter cells, whilst the luciferase activity in the pfuBAR reporter cells remained unchanged during treatment , 24 h (p < 0.05), 48 h (p < 0.01) and 72 h  of dexamethasone exposure . Dkk1 and Dkk2 are the best characterized members of this family. Dkk1 has been shown to inhibit canonical Wnt/β-catenin signaling pathway in several different organisms and cell types -25. The skeleton -30.in vitro display a reduction in alkaline phosphatase activity over a 72 h time course when compared to control osteoblasts. These phenotypic changes are driven by a Wnt/β-catenin-dependent mechanism as clearly demonstrated by both alterations in β-catenin trafficking and changes in TCF/LEF-mediated transcription.Our data emphasizes the key role of Wnt/β-catenin signaling in regulating bone development and remodeling, further strengthening its crucial role in glucocorticoid-induced osteoporosis -33. PrimDkk1 is a powerful antagonist of canonical Wnt/β-catenin signaling. Silencing Dkk1 expression rescues dexamethasone-induced suppression of primary human osteoblast function. Increased alkaline phosphatase activity was displayed by primary human osteoblasts treated with siRNA targeting Dkk1 expression when compared to scrambled control cells over a 72 h time course of dexamethasone exposure. The pharmacological targeting of the Wnt/β-catenin signaling pathway offers an exciting opportunity for the development of novel anabolic bone agents to treat osteoporosis and disorders of bone mass.in vitro. Silencing Dkk1 expression rescues dexamethasone-induced suppression of primary human osteoblast differentiation. Targeting of the Wnt/β-catenin signaling pathway offers an exciting opportunity to develop novel anabolic bone agents to treat osteoporosis and disorders of bone mass.Wnt/β-catenin signaling plays a key role in regulating glucocorticoid-induced osteoporosis The authors declare that they have no competing interests.JSB performed the majority of the research work. JMQ, BMD and DWM participated in study design, data interpretation and drafted the manuscript. PPD and JMO'B provided direction and oversight regarding all aspects of study design and interpretation of results. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/11/210/prepub


Intramyocellular lipid accumulation is strongly related to insulin resistance in humans, and we have shown that high glucose concentration induced de novo lipogenesis and insulin resistance in murin muscle cells. Alterations in Wnt signaling impact the balance between myogenic and adipogenic programs in myoblasts, partly due to the decrease of Wnt10b protein. As recent studies point towards a role for Wnt signaling in the pathogenesis of type 2 diabetes, we hypothesized that activation of Wnt signaling could play a crucial role in muscle insulin sensitivity.Here we demonstrate that SREBP-1c and Wnt10b display inverse expression patterns during muscle ontogenesis and regeneration, as well as during satellite cells differentiation. The Wnt/β-catenin pathway was reactivated in contracting myotubes using siRNA mediated SREBP-1 knockdown, Wnt10b over-expression or inhibition of GSK-3β, whereas Wnt signaling was inhibited in myoblasts through silencing of Wnt10b. SREBP-1 knockdown was sufficient to induce Wnt10b protein expression in contracting myotubes and to activate the Wnt/β-catenin pathway. Conversely, silencing Wnt10b in myoblasts induced SREBP-1c protein expression, suggesting a reciprocal regulation. Stimulation of the Wnt/β-catenin pathway i) drastically decreased SREBP-1c protein and intramyocellular lipid deposition in myotubes; ii) increased basal glucose transport in both insulin-sensitive and insulin-resistant myotubes through a differential activation of Akt and AMPK pathways; iii) restored insulin sensitivity in insulin-resistant myotubes.We conclude that activation of Wnt/β-catenin signaling in skeletal muscle cells improved insulin sensitivity by i) decreasing intramyocellular lipid deposition through downregulation of SREBP-1c; ii) increasing insulin effects through a differential activation of the Akt/PKB and AMPK pathways; iii) inhibiting the MAPK pathway. A crosstalk between these pathways and Wnt/β-catenin signaling in skeletal muscle opens the exciting possibility that organ-selective modulation of Wnt signaling might become an attractive therapeutic target in regenerative medicine and to treat obese and diabetic populations. The first suggestion for a role of Wnt signaling in the pathogenesis of type 2 diabetes came from a study which reported a single nucleotide polymorphism locus in the Wnt5b gene that confered susceptibility to type 2 diabetes in a Japonese population A central feature of the canonical Wnt/β-catenin pathway is the regulation of cytosolic β-catenin protein levels via a destruction complex containing glycogen synthase kinase-3β (GSK-3β), adenomatous polyposis coli (APC) and axin. In the absence of Wnt signals, β-catenin is targeted for ubiquitin-mediated degradation Several tissues, including skeletal muscle, display with ageing an adverse shift in lipid metabolism which contributes to insulin resistance and type 2 diabetes Down-regulation of Wnt signaling may alter myoblastic differentiation potential as a function of age, as it controls the balance between myogenic and adipogenic potential in myoblasts We have shown previously that high glucose concentration up-regulated SREBP-1c in cultured muscle satellite cells, leading to de novo lipogenesis and insulin resistance To challenge this hypothesis, we assessed the differential expression of Wnt10b and SREBP-1c proteins during skeletal muscle regeneration in adult EDL muscles. EDL muscle from the left hind limb was crushed using tweezers as previously described As muscle growth and regeneration occur through the activation of satellite cells, we studied the expression of Wnt10b protein in primary cultures of satellite cells isolated from hind limb muscles. Myoblasts were allowed to proliferate for 5 days, then fusion was induced and spontaneously contracting myotubes were obtained at day 9. Wnt10b protein was strongly expressed in proliferating myoblasts, then decreased during cell fusion and was no longer detectable in contracting myotubes , whereasTo determine whether Wnt10b knockdown could induce SREBP-1c expression, myoblasts were transfected with a mix of 3 specific Wnt10b siRNAs or with a control scrambled siRNA. In parallel, myotubes were transfected with a vector containing mouse Wnt10b cDNA. Total RNA was extracted 48 hours after transfection and semi-quantitative PCR was performed. Wnt10b knockdown was sufficient to induce SREBP-1c mRNA expression in myoblasts, whereas Wnt10b over-expression drastically decreased SREBP-1c mRNA expression in myotubes . FurtherY216 which is active when phosphorylated, but is inactive when dephosphorylated in response to Wnt/β-catenin signaling In order to determine whether SREBP-1c could inhibit Wnt10b expression, myotubes were transfected with a SREBP-1 siRNA duplex or with a control scrambled siRNA, and total proteins were extracted 48 hours later. SREBP-1c protein was hardly detectable in siRNA-transfected myotubes as compared with control myotubes, indicating a knockdown efficiency of more than 90%. Gene silencing was successful even in the presence of 10 nM insulin, a potent activator of SREBP-1c transcription. Surprisingly, SREBP-1c knockdown was sufficient to induce Wnt10b protein expression in contracting myotubes, particularly in the presence of insulin . To deteY216 phosphorylation led to the down-regulation of active β-catenin in transfected myoblasts, showing an inhibition of the Wnt/β-catenin pathway  drastically diminished the lipogenic factor SREBP-1c in contracting myotubes.Myotubes were treated for 48 hours with 6-BromoIndirubin-3′-Oxime (BIO), a selective inhibitor of GSK-3 activity that activates the Wnt/β-catenin pathway rylation , but hadIn myotubes cultured under physiological glucose concentration (5 mM), few lipid droplets were detected using Oil Red O staining , whereas3-2-Deoxyglucose (2-DG) uptake was measured in contracting myotubes. Myotubes cultured under physiological glucose concentration (G5) showed high insulin sensitivity, as 10 nM insulin induced within 30 minutes a 2-fold increase in 2-deoxyglucose uptake. Surprisingly, BIO treatment or Wnt10b over-expression increased basal 2-deoxyglucose uptake by 30% and 40% respectively, whereas uptake in the presence of BIO and insulin was similar to insulin-stimulated uptake  was observed after about 4 hours, then remained for 12 hours in both cases. In contrast, BIO had no significant effect on GSK-3βS9 phosphorylation  473 by mTor S241 autophosphorylation within 30 minutes, which induced Akt1T308 phosphorylation for 8 hours, but had no effect on Akt2S473 phosphorylation. Finally the Akt substrate AS160 (a Rab-GAPase involved in GLUT4 translocation) was activated by phosphorylation on S588 for at least 24 hours  pathway is involved in insulin signal transduction, we wonder whether BIO, which was designed as a specific GSK-3β inhibitor 9, or BIO-induced dephosphorylation of Y216  muscles of 2 month-old Sprague Dawley male rats according to Bassaglia and Gautron 3H]glucose (Amersham Pharmacia Biotech) as previously described Satellite cells from hind limb muscles were isolated and cultured as previously described 5′-GGAGCCATGGATTGCACATT-3′; antisense 5′-GCTTCCAGAGAGGAGCCCAG-3′. β-actin sense 5′-TCATGAAGTGTGACGTTGACATCC-3′; antisense 5′-GTAAAACGCAGCTCAGTAACAGTC-3′. MYHC-2 sense 5′GGTACTTGGCGAGAGTGGAG-3′; antisense 5′-AGGGCCAGTGTTTCACATTC-3′; PPAR-β sense 5′-CTCAACATGGAATGTCGGGTGTGC-3′; antisense 5′-CTGATCTCGTTGTAGGGCGGCAGC-3′; PPAR-γ sense 5′-ATGAAGACATTCCATTCACAAGAGC-3′; antisense 5′-ATAGTGGAAGCCAGATGCTTTATCC-3′. Results were obtained from three to four independent RNA samples from individual experiments, each tested in triplicate.Total RNA was extracted using TRIzol reagent (Invitrogen) according to the supplier's instructions. Gene expression was measured using semi-quantitative RT-PCR. Sense and antisense primers against mouse cDNAs were designed as follows: SREBP-1c sense Cytoplasmic and nuclear protein extracts were prepared using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents kit (Pierce) according to the supplier's instructions. Plasma membranes were isolated according to a protocol previously described Cells were immediately frozen in liquid nitrogen, scraped in ice-cold RIPA buffer containing 1 mM PMSF, 2 µg/ml pepstatin A, 2.5 µg/ml benzamidine, 2 µg/ml leupeptin and 5 µg/ml aprotinin, then lysed for 2 hours at 4°C under rotational agitation. Total cell lysates were recovered after centrifugation at 16,000×g for 30 minutes at 4°C to discard insoluble material. Proteins (30 µg) were subjected to immunoblot analysis.Ser473, P-Akt1Thr308 and polyclonal antibodies against P-GSK-3βSer9, P-AMPK-α1Ser485, P-AMPK-αThr172, P-PDK1Ser241 and Lamine A/C were from Cell Signaling. Rabbit monoclonal antibodies against AMPK-α1 and β-catenin were from Epitomics. Monoclonal antibody against active β-catenin was from Chemicon, as well as GLUT1 and AS160 antibodies. Antibody against P-AS160Ser588 was from Symansis. Monoclonal P-GSK-3βTyr216 antibody was from BD-Transduction Laboratories. Monoclonal antibodies against MyHC-Dev, MyHC-2 were purchased from Sigma and anti-myogenin from DAKO. Anti-FAS polyclonal antibody was a gift from Dr Dugail (Paris).SREBP-1c was detected using a monoclonal antibody raised against human SREBP-1 (NeoMarkers). Polyclonal antibodies against MyoD, GAPDH, Wnt10b, GLUT4 and β-tubulin were from Santa-Cruz Biotechnology. Rabbit monoclonal antibodies against GSK-3β, Akt, P-Akt2A target-specific 21-nt siRNA duplex against rat SREBP-1 was designed as previously described Myotubes were cultured in 6-well plates in DMEM without serum, and antibiotics were removed the day before transfection. SiRNA (60 pmoles/well) or pCAGGS plasmid containing the mouse Wnt10b cDNA were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Experiments were performed 48 hours later.−6 M BIO for 48 hours, or with Me-BIO, an inactive form, as a control, then 2-deoxyglucose uptake was performed, or total protein lysates and plasma membranes were prepared.BIO, a cell-permeable, highly potent, selective, reversible and ATP-competitive specific inhibitor of GSK-3α/β activity, was provided by Drs A. Brivanlou and L. Meijer . Myotubes were treated with 10Intramyocellular lipids were detected in myotubes using Oil Red O staining according to Koopman et al


Burkholderia mallei is a zoonotic Gram negative bacterium which primarily infects solipeds but can cause lethal disease in humans if left untreated. The effect of two antibiotics with different modes of action on Burkholderia mallei strain ATCC23344 was investigated by using in vitro and in vivo studies.in vitro was done by the agar diffusion method and the dilution method. The MICs of levofloxacin and ceftazidime were in the similar range, 2.5 and 5.0 μg/ml, respectively. Intracellular susceptibility of the bacterium to these two antibiotics in J774A.1 mouse macrophages in vitro was also investigated. Macrophages treated with antibiotics demonstrated uptake of the drugs and reduced bacterial loads in vitro. The efficacy of ceftazidime and levofloxacin were studied in BALB/c mice as post-exposure treatment following intranasal B. mallei infection. Intranasal infection with 5 × 105 CFUs of B. mallei resulted in 90% death in non-treated control mice. Antibiotic treatments 10 days post-infection proved to be effective in vivo with all antibiotic treated mice surviving to day 34 post-infection. The antibiotics did not result in complete clearance of the bacterial infection and presence of the bacteria was found in lungs and spleens of the survivors, although bacterial burden recovered from levofloxacin treated animals appeared reduced compared to ceftazidime.Determination of minimal inhibitory concentrations (MICs) in vitro.Both antibiotics demonstrated utility for the treatment of glanders, including the ability for intracellular penetration and clearance of organisms  Burkholderia mallei, the causative agent of glanders, a primary equine disease, is a Gram-negative, facultative intracellular bacterium which can be transmitted to humans with fatal consequences [B. mallei are also highly infectious organisms by aerosol and it is widely believed that it harbors the potential for use as a biological weapon [B. mallei can lead to septicemia, pulmonary or chronic infections of the muscle, liver and spleen. The disease has a 95% case fatality rate for untreated septicemia infections and a 50% case fatality rate in antibiotic-treated individuals [B. mallei to cause severe, rapidly fatal invasive infection initiated via aerosol in animals and humans, coupled with intrinsic resistance to antibiotics and diagnostic difficulty at early stage of disease make the bacterium a good candidate as a possible biological threat agent [B. mallei is minimal. The disease was eliminated from domestic animals in the United States during the 1940s and the last reported naturally acquired human case in the United States occurred in 1945. There is little data available on antibiotic treatment of glanders and human cases are treated with the same regimens used for melioidosis, an endemic disease in Southeast of Asia and Northern Australia, caused by Burkholderia pseudomallei. Only one case of laboratory-acquired human glanders was reported to CDC recently [in vitro data with in vivo efficacy for the B. mallei ATCC 23344 strain when a combination of intravenous doxycycline plus imipenem followed by oral doxycycline plus azithromycin successfully controlled a disseminated infection [B. mallei with antibiotic therapy is still not well established and no effective vaccines are available.equences . Human il weapon . In factl weapon . Inhalatividuals . The abiat agent . Our knorecently . This sinfection . Howeverin vitro antibiotic susceptibility studies for B. mallei have been performed. The antibiotic susceptibility of B. mallei is similar to that of B. pseudomallei, with resistance to a number of antibiotics [B. pseudomallei infections has shown that despite good in vitro activity, an antibiotic may be ineffective in vivo [Pseudomonas and other Gram-negative bacteria. The second antibiotic chosen, levofloxacin, belongs to the quinolone group which inhibits the bacterial DNA gyrase in Gram-negative bacteria, thereby inhibiting DNA replication and transcription. Quinolones can enter cells easily and therefore are often used to treat intracellular pathogens. As there is a need for effective treatment and post-exposure prophylaxis, the objective of this study was to assess the in vitro susceptibilities of these antibiotics with different modes of action and compare with efficacy in macrophages and mice infected with B. mallei.Few ibiotics . Both oribiotics . Unfortu in vivo ,11. We cB. mallei strain ATCC 23344 is susceptible to a concentration as low as 10 μg/ml of ceftazidime and 25 μg/ml of levofloxacin comparable to our E. coli control strain. The MICs were further evaluated by the dilution method for confirmation, resulting in 5 μg/ml of ceftazidime or 2.5 μg/ml of levofloxacin sufficient to inhibit the growth of B. mallei in LBG after 18–24 h incubation at 37°C under shaking conditions.MICs were determined by the agar diffusion method and dilution method. The results from the agar diffusion method are listed in Tables B. mallei was 4.7 × 105 CFU per animal delivered i.n. in 50 μl PBS (25 μl per nare). Non-treated control animals became sick within 48 h post-challenge indicated by non-specific signs such as piloerection and hypo-activity with trembling. The infection progressed with first deaths observed by day 4 post-challenge . At two hour intervals post-antibiotic exposure, intracellular CFU were determined resulting in a significant reduction of intracellular bacteria which continued throughout the assay . The lactate dehydrogenase (LDH) cytotoxicity assay was performed during bacterial invasion assays to monitor cytotoxic effects of bacteria on J774A.1 macrophages. Throughout the assay LDH levels were below 20%. Cytotoxicity was observed at 8 h in ceftazidime treated macrophages, reaching 25.7% which may have contributed to the decrease in recoverable intracellular bacteria in this treatment. Possible cytotoxic effects of antibiotics alone was tested in separate experiments for up to 24 h, including concentrations higher than that tested, showing no significant LDH levels (data not shown).For the determination of intracellular killing of say Fig. . Media iin vitro antibiotic susceptibilities to strains of B. mallei has been published. The recommendations for treatments of glanders are largely based on knowledge of pathogenesis of melioidosis, a human disease caused by a closely related species B. pseudomallei. Currently, ceftazidime is the first antibiotic of choice for treatment of acute melioidosis [B. mallei has a susceptibility profile similar to B. pseudomallei, the MICs are usually lower in case of B. mallei [in vitro susceptibility and clinical outcome of the treatments for melioidosis, the development of effective treatments has been difficult [B. mallei and B. pseudomallei, share morphological, biochemical and antigenic characteristics, and could be expected that similar problems will occur in the case of B. mallei. There is a need for an extensive evaluation of susceptibility of antibiotics to these pathogens beyond in vitro studies.Limited data of ioidosis . The preioidosis . Althoug. mallei . Due to ifficult ,16,17. BB. mallei. Only intraperitoneal pathogenesis of glanders has been well described in the mouse model [Animal models to study equine glanders have been established  while thse model  with morse model .in vitro of B. mallei to ceftazidime and levofloxacin, and their efficacy in vivo using intranasal infection in BALB/c mice, as inhalation would be the most likely route of infection in the event of bioterrorism threat. In previous in vitro studies, ceftazidime proved to be effective against B. mallei among others including imipenem, doxycycline, piperacillin, ciprofloxacin [B. mallei but not B. pseudomallei [B. mallei strain ATCC 23344 is susceptible to a concentration as low as 2.5 μg/ml of levofloxacin and 5 μg/ml of ceftazidime. These results confirmed prior studies evaluating susceptibility of 15 isolates of B. mallei to 35 antimicrobial agents [B. mallei. However, the high percentage of resistant strains of B. pseudomallei to levofloxacin and the emergence of ceftazidime-resistant clinical isolates of B. pseudomallei would affect the recommendations of these drugs as useful treatment for both glanders and melioidosis, underlining the need for supplementary monitoring of the effectiveness of the recommended antimicrobials.Here, we evaluated the susceptibilities floxacin ,9. Levofdomallei . Levoflodomallei . Our resin vitro were substantiated in our in vivo experiments with all treated mice surviving at least 34 days post infection. The intranasal infection of mice with 5 × 105 CFUs of B. mallei resulted in 90% death in untreated control mice. Treatment with antibiotics used in this study prevented the development of an acute lethal form of disease but lacked the ability to provide complete clearance of the bacterial infection. By 34 days post-infection, bacteria were largely cleared from the lungs with no significant differences between treatments. Interestingly, in our intranasal infection model, the spleen appears to be the major target tissue for glanders infection and a site of multifocal abscesses. Similar findings were documented in studies with intraperitoneal glanders in mice and hamsters [4 CFU/g of spleen. The antibiotics administrated 24 hours post-infection for 10 days led to the development of a chronic, non-lethal abscess infection suggesting that B. mallei may have the propensity for latency, as does the very closely related organism B. pseudomallei [B. mallei [B. pseudomallei infected mice [in vitro MIC and the ability of the bacteria to reside in privileged intracellular sites  should be considered as an important factor in effective treatment. For that reason, we tested levofloxacin in our study since fluoroquinolones are known to penetrate renal, lung and bronchial track tissues achieving a high intracellular concentration exceeding levels of the drug in serum [The effectiveness of levofloxacin and ceftazidime hamsters ,24. The domallei . Efficacted mice . Combinein serum . Both anin vitro against B. mallei, failed to eradicate bacterium and resulted in development of a chronic, non-lethal form of glanders. Both antibiotics demonstrated some utility for treatment of glanders, including the ability for intracellular penetration and clearance of organisms in vitro, despite bacterial burdens recovered in vivo following i.p. antibiotic treatment.The current study showed that both ceftazidime and levofloxacin, despite good activity B. mallei strain ATCC 23344 (China 7) was cultured on Luria-Bertani supplemented with 4% glycerol (LBG) agar plates for 48 h at 37°C. Isolated colonies were sub-cultured to LBG broth, and cultures were incubated at 37°C until optical density readings at 600 nm (OD600) reached an exponential phase of growth. Bacteria were pelleted by centrifugation, washed and re-suspended in sterile 1× phosphate-buffered saline  to obtain the desired CFU/ml. All procedures were performed in a biosafety level 3 laboratory.B. mallei ATCC 23344 and disks containing the antibiotic dilutions placed on top of the inoculated agar. The plates were incubated at 37°C for 24–48 h. Zones were measured and the mean diameter was calculated. The interpretation of results was based on the NCCLS zone diameters used for non-Enterobacteriaceae. For the broth dilution method, an inoculum of 105 CFU of washed B. mallei per ml was used, and the test was conducted in LBG for 24 h at 37°C. The interpretation of results was based on the NCCLS MIC breakpoints for non-Enterobacteriaceae and MIC for B. pseudomallei [The minimal inhibitory concentrations of the antibiotics were tested by the Kirby-Bauer disk diffusion method and the dilution method as previously described . Briefly, fresh antibiotic-containing disks  were used for susceptibility testing. LBG plates were inoculated with domallei . The inhAnimal studies were carried out in accordance with the Animal Care and Use Committee's guidelines as recommended by the National Institutes of Health. Female, 6- to 8-week-old, BALB/c mice were obtained from Harlan Sprague Dawley, Inc. . Animals were provided with rodent feed and water ad libitum and maintained on 12 h light cycle.5 CFUs of B. mallei ATCC 23344, in a total volume of 50 μl in PBS solution given to both nares. Treatment with antibiotic via intraperitoneal route (i.p.) started 24 hours after infection, once a day, for 10 days. Doses of antibiotics used in this study were in the range of high doses used in humans: ceftazidime 100 mg/kg/day and levofloxacin 20 mg/kg/day. The animals were weighed prior to challenge and doses of antibiotics were adjusted accordingly. Levofloxacin  and ceftazidime  were purchased through local UTMB Pharmacy and doses for injection were prepared and stored according to manufacturer's instructions.Groups of 10 animals were inoculated via intranasal (i.n.) route with 5 × 10Five animals from each group of antibiotic treated animals and survivors from non-treated control animals, were sacrificed and lungs and spleen were harvested for CFU determination. Organs were weighed, homogenized in 5 ml sterile PBS, plated in duplicates on LBG and incubated at 37°C for 2 days prior to CFU determinations. For comparison, spleen weights from healthy non infected but antibiotic treated animals were also evaluated. CFU were expressed as the mean ± SEM. Organs  from additional remaining survivors were used for histological examinations.5) onto Corning Costar 24 well plates  with DMEM and incubated overnight at 37°C with 5% CO2. Bacteria were added at an MOI of 25:1 to J774A.1 cells in duplicate. The high MOI was used to guaranty that every macrophage was able to take up a large number of bacteria that survived the phagocytic activity of the cell but were killed by our experimental antibiotic treatment. Inoculated wells were centrifuged at 800 × g for 2 minutes and incubated for 2 hours at 37°C with 5% CO2 followed by a PBS wash (×3) and 2, 4 and 8 hours incubation with antibiotics. Media in control wells contained 250 μg/ml kanamycin for first 2 h postinfection and 100 μg/ml kanamycin for the rest of the assay to prevent the growth of extracellular bacteria [Murine J774A.1 cells were seeded . Comparisons of spleen weights were performed using ANOVA and LOG transformed values of bacterial load was analyzed by Student's t-test. P value ≤ 0.05 was considered significant.MICs: Minimal inhibitory concentrations; i.p.: intraperitoneal; i.n.: intranasal.BMJ designed and conducted experiments and drafted the manuscript. GCW contributed to design and conduct of experiments and drafting manuscript, AGT conducted and provided analysis of the bacterial work, DME conceived the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


The cytoplasmic domain of cadherins is known to associate with the intracellular proteins, catenins, which link cadherins to the actin-based cytoskeleton. In this study, we immunohistochemically investigated the expression of beta-catenin as well as E-cadherin and alpha-catenin in 86 human colorectal cancers, and we analysed their coexpression pattern and relationship to clinicopathological factors. In cancerous tissues, the frequency of reduced expression of beta-catenin  was similar to that of E-cadherin , but less than that of alpha-catenin . All three molecules were expressed strongly, as was the normal epithelium, in 36 cases (42%), whereas the rest  showed reduction in one of the molecules. The reduction of beta-catenin expression was significantly correlated with dedifferentiation, Duke's stage, lymph node metastasis and liver metastasis. Next, we examined tyrosine phosphorylation in the protein complex immunoprecipitated with E-cadherin, as E-cadherin function is down-regulated by receptor-type tyrosine kinase in vitro. It was of interest that up-regulation of tyrosine phosphorylation of beta-catenin was more frequently observed in cancerous tissues than in the matching normal mucosa. These results suggest that beta-catenin may have important regulatory roles within an E-cadherin-mediated adhesion system in human colorectal cancers.


Primary cell cultures demonstrate constitutive tcf activation in aggressive fibromatosis. Expression and co-immunoprecipitation studies suggest that β-catenin binds and activates tcf-3 in this tumour. This is the first demonstration of tcf-3 activation by β-catenin stabilization in a human neoplastic process. © 2001 Cancer Research Campaign


Infants' sensitivity to social or behavioral contingency has been examined in the field of developmental psychology and behavioral sciences, mainly using a double video paradigm or a still face paradigm. These studies have shown that infants distinguish other individuals' contingent behaviors from non-contingent ones. The present experiment systematically examined if this ability extends to the detection of non-humanoids' contingent actions in a communicative context. We examined two- to three-year-olds' understanding of contingent actions produced by a non-humanoid robot. The robot either responded contingently to the actions of the participants (contingent condition) or programmatically reproduced the same sequence of actions to another participant (non-contingent condition). The results revealed that the participants exhibited different patterns of response depending on whether or not the robot responded contingently. It was also found that the participants did not respond positively to the contingent actions of the robot in the earlier periods of the experimental sessions. This might reflect the conflict between the non-humanlike appearance of the robot and its humanlike contingent actions, which presumably led the children to experience the uncanny valley effect. The detection of behavioral contingency is one of the abilities that play an important role in human interaction from early stages of development. Previous studies of sensitivity to behavioral contingency have shown that even infants can distinguish whether or not others' reactions are contingent on their own actions. One of the experimental procedures used to examine this sensitivity is the still-face paradigm, in which the experimenter facing the infant participant stops contingent interaction, along with facial and vocal signals e.g., . AnotherAlthough the double-video paradigm is a powerful and useful tool for assessing infants' reactions to behavioral contingency, it has limitations. First, only humans can serve as stimuli producing contingent actions, since they can react flexibly to the infant's actions. This limitation makes it difficult to separate the effect of contingency from humans' physical appearance or species-specific actions. Meltzoff and colleagues argued that “like-me” detection, which can be regarded as a kind of conspecific recognition, might function as a foundation of social cognition A second limitation is that interaction through TV monitors, rather than face-to-face interaction, is crucial to equate stimuli between the contingent and non-contingent conditions. Despite that infants can discriminate between two- and three-dimensional displays, and they respond more readily to a live adult One way to overcome these limitations is to use a non-humanoid robot as the interacting partner. This might allow us to test children's detection of contingency in a non-human agent in relatively natural settings. Further, by programmed recording the actions performed by the robot for a participant in the contingent condition can be reproduced exactly for another participant in the non-contingent condition. Previous studies have suggested that infants attribute mental states to non-human objects including robots when the latter appear to interact with a person In the present experiment, we used a creature-like robot that interacts with people through actions and sounds As the target stimulus for the experiment, we used a robot named Keepon, which was built to study the development of communication M = 30.7 months, SD = 6.5 months; 8 boys and 8 girls). Their mothers also participated in the experiment. The data for an additional 6 children were excluded from the analysis because of their fussiness during the experiment. All participants were registered on the voluntary panel for infant study at Kyushu University. Written informed consent was obtained from the caretakers of the children before the experiment was conducted. The procedure of the present study complied with the “Ethical Principles of Psychologists and Code of Conduct” .The participants were 16 children aged 2 to 3 years  and tested in one session.In the contingent condition (C-condition), an experimenter (K.Y.) remotely controlled Keepon through the PC to respond contingently to the actions of the participants and attempted to generate natural interactions as far as possible see . The seqIn the NC-condition, Keepon replayed the same sequence of actions as it performed in the C-condition, but now facing a new participant of the same sex and almost the same age (within three months) as in the C-condition. Thus, actions expressed by Keepon were not contingent on the actions of the participant in the NC-condition, but the sequence of actions presented to the 2 participants was exactly the same between the conditions.The experiment was conducted in an experimental booth (190×312 cm) built in a room of the university. During the experiment, the participant was allowed to move and play freely in the experimental booth. On the other hand, the mother was instructed to sit still on the floor midway between Keepon and the toys and to not speak or influence the child, although she was allowed to reply briefly or nod her head in response to questions or comments. The experiment continued until it became apparent that the participant had lost interest in the situation, up to a maximum of 10 minutes.In addition to Keepon's eye-camera, we installed 2 cameras in the experimental booth in order to complement Keepon's recording of the participant's responses as well as to record the mother's behavior. This was because it became difficult to record the participants' responses solely through Keepon's eye-camera, especially in the NC-condition. Thus, the participants' responses were video recorded from different perspectives through the multiple cameras.We analyzed the data from the participant's first look at Keepon. Since the minimum duration of a session was 400 sec, the first 400 sec of each video record was set as the target of our analysis. The first and second authors coded each participant's looking behavior and position in the booth on a second-by-second basis. For looking behavior, targets coded were Keepon, mother, or toys. For three randomly selected sessions, 18.8% of the collected data, a new coder blind to the aim of the research independently coded the data. The average inter-coder agreement score was κ = .84, indicating high inter-observer reliability.2 in area. The locations of all the participants in the booth were coded in the same manner as described above. The concordance rate was .93 on average, indicating high reliability of the coding.To define the participants' position in the booth, we operationally divided the floor of the booth into six spaces and defined the spaces around Keepon, the mother, and the other toys as the K-, M-, and T-areas, respectively. All the spaces were 0.99 mAs the response measures, we took the participants' looking time at Keepon (LTk), the mother (LTm), and the toys (LTt) and the time they spent in the three areas . Specifically, looking time was considered to reflect the participants' interest and the time spent in the three areas indicated the physical proximity of the participants to Keepon, the mother, or the toys.The statistical analyses consisted of analysis of variance (ANOVA) followed by post-hoc analysis using Ryan's method F  = 4.89, p = .044) and target  = 12.58, p<.001). The interaction between condition and target was not significant  = 0.30, p = .74). A post-hoc analysis demonstrated that LTk and LTt was longer than LTm (LTk > LTm: t (28) = 4.99, p<.001, nominal level = .017; LTt > LTm: t (28) = 2.97, p = .006, nominal level = .033).We compared total LTk, LTm, and LTt in the C- and NC-conditions . A 2  three-way mixed ANOVA. As shown in F  = 6.82, p<.001) and three-way interaction among the three factors  = 2.60, p = .023), along with the significant main effect of target and condition as already described above. Analysis of the three-way interaction (target × condition × block) revealed that LTt in the C-condition was longer than in the NC-condition during the 2nd block  = 7.57, p = .007). The simple main effect of block of LTk was significant in the NC-condition  = 11.51, p<.001) and the simple main effect of block of LTt was significant in both conditions  = 3.07, p = .030; NC: F  = 9.34, p<.001). .To investigate how LTk, LTm, and LTt changed with time, we divided the test session (duration of 400 s) into four blocks (100 s per block) and conducted a 2 (condition: C and NC) ×3  ×4 (block: 1st block was significantly shorter than during the 2nd block (t (126) = 2.80, p = .006, nominal level = .008) and marginally shorter than during the 3rd block (t (126) = 2.01, p = .046, nominal level = .025) and the 4th block (t (126) = 2.37, p = .019, nominal level = .013). On the other hand, in NC-condition, LTt during the 4th block was significantly longer than 1st, 2nd, and 3rd blocks (4th>1st: t (126) = 4.53, p<.001, nominal level = .013; 4th>2nd: t (126) = 4.55, p<.001, nominal level = .008; 4th>3rd: t (126) = 2.46, p = .015, nominal level = .025), and marginally longer during the 3rd block than during the 1st block (t (126) = 2.08, p = .040, nominal level = .025). Moreover, in the NC-condition, LTk during the 1st and 2nd blocks were significantly longer than during the 3rd and 4th blocks (1st>3rd: t (126) = 2.90, p = .004, nominal level = .013; 1st>4th: t (126) = 5.11, p<.001, nominal level = .008; 2nd>3rd: t (126) = 2.59, p = .011, nominal level = .025; 2nd>4th: t (126) = 4.79, p<.001, nominal level = .013), and marginally longer during the 3rd block than during the 4th block (t (126) = 2.20, p = .029, nominal level = .025).A post-hoc analysis of the simple main effects showed that in the C-condition, LTt during the 1st, 2nd, 3rd, and 4th) three-way mixed ANOVA yielded the following: (a) a marginal main effect of area  = 2.82, p = .077), (b) a significant interaction between condition and block  = 4.42, p = .009), and (c) a three-way interaction among the three factors  = 3.25, p = .006). Analysis of the condition × block interaction showed that the time spent in the three areas was longer in the C-condition than in the NC-condition in the 1st block  = 5.76, p = .020) and 2nd block  = 5.66, p = .021). The simple main effect of block in the C-condition approached significance  = 2.40, p = .081).The time spent by each participant in each of the three areas  was used for the analysis. As shown in st block, the time spent in the M-area was marginally longer in the C-condition than in the NC-condition  = 3.59, p = .060), while in the 4th block, it was marginally longer in the NC-condition than in the C-condition  = 3.38, p = .068). The simple main effect of area was significant in the 1st block in the C-condition  = 3.14, p = .047), and significant in the 4th block in the NC-condition  = 3.20, p = .044). In addition, the simple main effect of block in the M-area was significant in both conditions  = 6.73, p<.001; NC: F  = 2.82, p = .042).Analysis of the three-way interaction (condition × area × block) revealed that in the 1st block (M > T: t (112) = 2.22, p = .029, nominal level = .017; M > K: t (112) = 2.12, p = .036, nominal level = .033), while in the NC-condition, they spent more time in the M-area than in the K-area during the 4th block (t (112) = 2.53, p = .013, nominal level = .017). Moreover, in the C-condition, the time spent in the M-area was longer during the 1st and 2nd blocks than during the 4th block (1st>4th: t (126) = 4.10, p<.001, nominal level = .008; 2nd>4th: t (126) = 3.34, p = .001, nominal level = .013), and marginally longer during the 1st block than during the 3rd block (t (126) = 2.51, p = .013, nominal level = .013). In contrast, in the NC-condition, they tended to spend more time in the M-area during the 3rd and 4th blocks than during the 1st block (4th>1st: t (126) = 2.47, p = .015, nominal level = .008; 3rd>1st: t (126) = 2.29, p = .024, nominal level = .013).A post-hoc analysis of the simple main effects demonstrated that in the C-condition, children tended to spend more time in the M-area than in the T- and K-areas during the 1The present study provides evidence that behavioral contingency between the participant and the non-humanoid robot can have a marked effect on the children's reaction. The 2–3 year old children changed their behaviors depending on whether or not the robot's actions were contingent on their own actions.This might empirically support the view that the children attribute mental states to non-human objects that appear to interact with people, as suggested in younger children or infants st block, there was no significant difference between the C- and NC-conditions in LTk, LTm, or LTt. However, the mean time spent in the M-area in the 1st block was marginally higher in the C-condition than in the NC-condition. In addition, in the C-condition, the time spent in the M-area tended to be higher than in the other two areas in the 1st block, whereas no such tendency was found in the NC-condition. These differences indicate that the area used by the participants was limited to the space around their mothers during the 1st block in the C-condition; however, in the NC-condition, they moved around more freely. In other words, the participant stayed by the mother and restricted the chance to approach to Keepon with keeping attention to it at the earlier stages of the C-condition.The significant interaction between the blocks and conditions highlights another interesting aspect of the present results. In the 1Such a response can be reasonably interpreted as reflecting the participants' hesitation to interact with Keepon when they experienced it reacting contingently. We consider the possibility that this response pattern could be related to the uncanny valley hypothesis Animals including humans use multiple signals to accurately detect conspecifics Though the present study tested 2- to 3-year-old children, previous studies have demonstrated that even 2-month-old infants can detect social contingency in adult-infant interactions and tend to show positive responses to it st block was lower than other 3 blocks), the time spent near to the mother decreased as the session proceeded. A previous study suggested that 60 sec of observation altered 10-month-old infants' perception of agency of a humanoid robot The results also suggest that children who interacted with Keepon in the C-condition overcame the uncanny valley with experience: though the looking time at Keepon, mother, and toys remained almost stable over the session (except that LTt in the 1However, we cannot conclude on the basis of our results that the participants began to interact communicatively with Keepon during the 400 sec experimental session. Conceivably, the discrepancy between the non-humanlike appearance of Keepon and its humanlike contingent actions was too large for the children to integrate within the limited time span. Another possibility should be the gap between the detection of agency and engagement through overtly communicative interaction with the agent in face-to-face situations. These possibilities remain to be addressed in future studies aimed at clarifying the basis for human-robot interaction in more detail. In addition, relevant experience with nonhuman agents before the age of 2 years, such as toys, might have influenced the present results. Since strict control of the developmental history of children is not practical, a cross-cultural study would be one way to reveal the effect of experience on the perception of human-likeness.Presenting various robots that differ in appearance and performance would open a new direction of research to address empirically what forms the basis of human interaction, as Keepon functioned as a platform to shed light on a new aspect of the uncanny valley effect and as a valuable mirror to reflect humans' perception of themselves.


Nevertheless, cells displaying nuclear β-catenin accumulation are nonrandomly distributed throughout the tumour mass and preferentially localise along the invasive front where parenchymal cells are in direct contact with the stromal microenvironment. Here, we discuss the putative role played by stromal cell types in regulating β-catenin intracellular accumulation in a paracrine fashion. As such, the tumour microenvironment is likely to maintain the cancer stem cell phenotype in a subset of cells, thus mediating invasion and metastasis.Sporadic cases of colorectal cancer are primarily initiated by gene mutations in members of the canonical Wnt pathway, ultimately resulting in  The complex binds and phosphorylates β-catenin at serine and threonine residues, thus targeting it for ubiquitination and proteolytic degradation. In contrast, oncogenic mutations in β-catenin render it resistant to Ser/Thr phosphorylation and proteolytic degradation. Upon its cytoplasmic stabilisation and subsequent nuclear translocation, β-catenin binds to members of the TCF/LEF family of transcription factors, thus modulating expression of a broad range of target genes (http://www.stanford.edu/~rnusse/pathways/targets.html). Although the presence of these initiating mutations predicts nuclear β-catenin accumulation throughout the tumour mass, heterogeneous intracellular distributions are observed within primary colorectal tumours and their metastases. In particular, tumour cells located at the invasive front and those migrating into the adjacent stromal tissue are earmarked by nuclear β-catenin accumulation , thus actively contributing to malignancy.Several intrinsic (cell autonomous and/or autocrine) and extrinsic  factors may explain the observed heterogeneity of Wnt/our mass . Here, wα-smooth muscle actin (α-SMA) characterises these myofibroblasts and underlies contractile force tension that facilitates healing. Myofibroblasts produce a variety of growth factors, prostaglandins, cytokines, chemokines, and extracellular matrix components that facilitate tissue repair and survival. Myofibroblasts arise through a multitude of processes, including transdifferentiation of resident fibroblasts, epithelial-to-mesenchymal transition (EMT) of parenchymal cells, recruitment, and differentiation of pericytes , and from bone marrow-derived circulating immature fibrocytes  induces β-catenin stabilisation in colorectal cancer cells via c-MET-dependent inhibition of GSKβ activity and its Tyr phosphorylation  reflects its activation by TGFβ stimulation and directly correlates with prognosis and survival in human colorectal cancer , and migrate towards the CCR1 ligand CCL9, highly increased in the tumour epithelium. κB (nuclear factor-κB), a key mediator of inflammation, has a critical role in the development of tumours resulting from chronic inflammation or exogenous mutagens, induced by exposure to dextran sulphate sodium salt and Azoxymethane (AOM). Moreover, genetic ablation of MyD88, a signalling adaptor of Toll-like receptors in the innate immune system, was shown to reduce mortality due to intestinal tumorigenesis in ApcMin+/ mice  transcription factor  intestinal tumorigenesis , et genes . Transfoβ-catenin is likely to represent a central node where different signals converge and are subsequently coordinated to regulate tissue homeostasis under physiological conditions and cancer stemness in the context of tumour–stroma interactions. Because the putative β-catenin interaction partners are themselves regulated by extracellular stimuli, it is plausible that the subsequent effects on β-catenin activation and possibly cancer stemness are modulated in a context-dependent manner. In fact, β-catenin has been reported to interact directly with several growth factor receptors, including EGFR , Met (the receptor for HGF), TGFRII (the receptor that is activated upon TGF stimulation), and KIT  therapeutic strategies, and, thus, prognosis and survival for colorectal cancer patients. Stromal cells have even been suggested as possible targets for tailor-made therapeutic interventions for intestinal tumorigenesis, rather than parenchymal cells. Here, we propose that the functional characterisation of additional β-catenin-binding partners in these alleged CSCs will improve our understanding of malignancy and invasion and open future perspectives for a metastasis-free survival to colorectal cancer patients.Despite the clear genetic prerequisite for mutations in downstream components of the Wnt/


Cancer progression is linked to a partially dedifferentiated epithelial cell phenotype. The signaling pathways Wnt, Hedgehog, TGF-β and Notch have been implicated in experimental and developmental epithelial mesenchymal transition (EMT). Recent findings from our laboratory confirm that active Wnt/β-catenin signaling is critically involved in invasive ductal carcinomas (IDCs) of breast.In the current study, we analyzed the expression patterns and relationships between the key Wnt/β-catenin signaling components- E-cadherin, Slug and GSK3β in IDCs of breast.Of the 98 IDCs analyzed, 53 (54%) showed loss/or reduced membranous staining of E-cadherin in tumor cells. Nuclear accumulation of Slug was observed in 33 (34%) IDCs examined. Loss or reduced level of cytoplasmic GSK3β expression was observed in 52/98 (53%) cases; while 34/98 (35%) tumors showed nuclear accumulation of GSK3β. Statistical analysis revealed associations of nuclear Slug expression with loss of membranous E-cadherin (p = 0.001); nuclear β-catenin (p = 0.001), and cytoplasmic β-catenin (p = 0.005), suggesting Slug mediated E-cadherin suppression via the activation of Wnt/β-catenin signaling pathway in IDCs. Our study also demonstrated significant correlation between GSK3β nuclear localization and tumor grade (p = 0.02), suggesting its association with tumor progression.The present study for the first time provided the clinical evidence in support of Wnt/β-catenin signaling upregulation in IDCs and key components of this pathway - E-cadherin, Slug and GSK3β with β-catenin in implementing EMT in these cells. Breast cancer is a major cause of female mortality in the Western world. In India, it is the second most common cancer among females, while in the metropolitan cities; it ranks as the most common cancer . The incCDH1 gene in about 50% of lobular carcinomas of the breast . Figure The expression and sub-cellular distribution of β-catenin in these IDCs has been reported by us earlier in the same cohort . Here inOf the 98 IDCs analyzed, 33 tumors showed nuclear accumulation of Slug. Of these 33 tumors, 17 (51%) IDCs showed nuclear localization of β-catenin protein (p ὄ 0.01); while 23 (70%) cases showed loss of membranous E-cadherin expression (p ὄ 0.01). Twelve of 33 (36%) IDCs showed both nuclear localization of β-catenin protein and loss of membranous E-cadherin.Recent studies demonstrate that canonical Wnt signaling pathway is associated with both stem cell and tumor cell development -29. In mCDH1 in IDCs; CDH1 hypermethylation frequency was found to be 50% in IDCs [During EMT, the epithelial cells acquire fibroblast-like properties and show reduced intercellular adhesion and increased motility; this process is associated with functional loss of E-cadherin ,33. Furt in IDCs . Taking  in IDCs -12.CDH1 [In the present study, we observed loss/or reduced expression of E-cadherin in 54% (53/98) of IDCs. Similar E-cadherin loss was reported in 85% cases in a series of 71 ductal carcinomas and correlated with promoter methylation of CDH1 . In our CDH1 . In the in vivo repressor of E-cadherin as compared to snail in breast carcinomas [Slug mediated loss of E-cadherin expression in IDCs is another finding of our study. Although several transcription factors including Snail and Slug have been implicated in E-cadherin repression, herein we have analyzed the expression of Slug only, because it has been proposed to be a likely rcinomas ,37. Nuclrcinomas , and corrcinomas ,12. HoweThe other important component of Wnt pathway investigated in our study is GSK3β, a multikinase involved in Wnt, Akt and Hedgehog pathways, all of which are involved in determination of cell fate and morphology; inhibition of GSK3β activity or expression results in bonafide EMT ,38,39. WIn a recent study, we demonstrated the relationship of loss of E-cadherin and APC proteins with activation of Wnt/β-catenin signaling driving EMT . We demo+/CD24- population harbors stem cell properties. These CD44+/CD24- cells, express low or undetectable levels of epithelial markers (E-cadherin and β-catenin) and high levels of mesenchymal markers (vimentin and fibronectin), suggesting that these cells have undergone EMT [Our current findings also support the concept that generation of cancer stem cells (CSCs) - the acquisition of the stemness and tumorigenic characters is driven by induction of EMT . In breagone EMT . Expressgone EMT ,43,44. Igone EMT . In the In the present study, we provide clinical evidence in support of up-regulation of Wnt/β-catenin signaling in invasive ductal carcinoma of breast and key components of this signaling pathway such as E-cadherin, Slug, GSK3β and β-catenin to be associated with Epithelial Mesenchymal Transition (EMT) process and pathogenesis of IDCs. Moreover, a better understanding of the pathways  that trigger EMT and cancer cell self renewal (cancer stem cells) might lead to new therapeutic approaches for cancer patients by developing molecular tools that interfere with them.IDC: Invasive Ductal Carcinoma; EMT: Epithelial Mesenchymal Transition; PI3K: Phosphoinositide 3-kinase; GSK3β: Glycogen Synthase Kinase3β; Dvl: Disheveled.The authors declare that they have no competing interests.CPP planned, set up the experiments, collected the data, analyzed and interpreted the results and drafted the manuscript. GR participated in the study design, interpretation of the results and in editorial support. SM conducted histopathological evaluation of all clinical specimens and assessment of immunohistochemical staining data. DB enrolled the patients in the study, provided the clinical specimens, patient data, follow up and clinical knowledge. RP enrolled the patients in the study, provided the clinical specimens, patient data and clinical knowledge. RR planned, supervised, provided financial and technical support for the study and writing of the manuscript. All authors have read and approved the final manuscript.The authors declare that they have no competing interests.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/325/prepub


A functional or physical connection between these β-catenin pools has been suggested in previous studies, but not conclusively demonstrated to date. To further examine this intersection, we treated A431 cell colonies with lysophosphatidic acid (LPA), which forces rapid and synchronized dissociation of AJ. A combination of immunostaining, time-lapse microscopy using photoactivatable-GFP-tagged β-catenin, and image analyses indicate that the cadherin-bound pool of β-catenin, internalized together with E-cadherin, accumulates at the perinuclear endocytic recycling compartment (ERC) upon AJ dissociation, and can be translocated into the cell nucleus upon Wnt pathway activation. These results suggest that the ERC may be a site of residence for β-catenin destined to enter the nucleus, and that dissociation of AJ may influence β-catenin levels in the ERC, effectively affecting β-catenin substrate levels available downstream for the Wnt pathway. This intersection provides a mechanism for integrating cell-cell adhesion with Wnt signaling and could be critical in developmental and cancer processes that rely on β-catenin-dependent gene expression. Morphogenesis and integrity of tissues requires interactions between neighboring cells and properly coordinated regulation of gene expression In cell-cell adhesion, the recruitment of cytosolic β-catenin to the plasma membrane and its tight association to E-cadherin is required for formation and stabilization of adherens junctions (AJ) APC are found in 61% of patients APC mutations generally have direct alterations in β-catenin, TCF, or other molecules in the same pathway APC is also the responsible initiating genetic event in the colorectal cancer syndrome familial adenomatous polyposis Another pool of β-catenin, thus far thought to be separate and functionally distinct from AJ associated β-catenin either AJ formation or Wnt signaling. However, relatively little consideration has been given to the possibility that the presence of β-catenin in both pathways may point to a mechanistic link between two core cell processes of fundamental importance in their own right The functions of β-catenin are intensely studied separately in Colonies of the A431 squamous carcinoma cell line are rapidly dissociated by exposure to lysophosphatidic acid (LPA) In order to positively identify the specific perinuclear compartment(s) where β-catenin and E-cadherin accumulated, we also compared localization of β-catenin with transferrin. Transferrin bound to its cell surface receptor is internalized by endocytosis, and has been well characterized as a classic marker for the perinuclear endocytic recycling compartment (ERC) APC mutations are known to inactivate GSK-3β and result in β-catenin translocation from the cytoplasm to the nucleus. To test whether ERC-targeted β-catenin from disrupted AJ could be translocated to the nucleus upon Wnt activation, we exposed LPA-treated cells to lithium chloride (LiCl), a GSK-3β inhibitor often used for stimulating downstream activation of the Wnt pathway In addition to its role as an AJ component, β-catenin also plays an important role in the nucleus as an activator of the transcription factor TCF/LEF It has been reported that extended exposure  of cells to LiCl  may have toxic effects To confirm that, in these experiments, ERC-resident β-catenin was translocated inside the nucleus, we reconstructed its physical location by confocal microscopy generated z-stack analyses. To further confirm the microscopy results with an independent approach, we lysed cells and performed cytosol/nuclear fractionation followed by western blotting with antibodies specific for β-catenin or PCNA, a nuclear antigen. As shown in It has been suggested that GSK-3β inhibition by LiCl releases β-catenin from its fate of proteasome-mediated destruction Our data to this point clearly indicate that rapid disruption of AJ results in increased nuclear β-catenin when GSK-3β function is impaired. However, microscopic images including immunofluorescence staining or western blot data present only a stationary snapshot of β-catenin localization and do not provide information about dynamic translocation of β-catenin. In order to obtain direct evidence that the β-catenin that is subsequently localized in the nucleus truly originated from the AJ pool, we monitored β-catenin movement in living cells by pulse-chasing β-catenin fused with photoactivatable (PA)-GFP In order to see the effect of LPA-enhanced nuclear translocation of β-catenin on gene expression, we measured TCF/LEF-dependent transcriptional activity using a TOPflash reporter plasmid. After overnight starvation, cells were stimulated by PBS or LPA, in the presence or absence of LiCl for 18 h, and assessed for transcriptional activity by measuring luciferase activity. A431 cells showed significantly elevated LEF/TCF dependent transcription with LiCl alone  In contrast, our present study points to an additional, more direct mechanism of coordination that links adhesion and transcription function of β-catenin in a more straight-forward manner, which suggests functional integration between adhesion and Wnt-dependent differentiation. Of course, since multiple mechanisms may control pool distribution of β-catenin, studies are warranted to determine how these mechanisms may interface with each other. Thus, the AJ-Wnt β-catenin transport pathway we describe here does not conflict with, but rather complements, biochemical regulation of β-catenin allotment to distinct pools.et al.++ removal. We repeated those experiments and obtained results comparable to LPA treatment, although ERC localization and nuclear translocation of β-catenin were partially confounded by side effects on cell morphology, possibly induced by the Ca++ deprivation , which is consistent with previous reports APC mutations uncouple AJ stability from Wnt-mediated regulation and, whether or not the Wnt pathway is activated, cadherin-bound β-catenin traveling from dissociated AJ may end up in the nucleus and presumably alters transcriptional regulation.On a speculative note, the AJ-to-Wnt β-catenin connection provides an intuitive mechanism to integrate cell-cell adhesion with gene expression . More explicitly, the implications of an intersection between cell-cell adhesion and Wnt pathways are manifold. First, in this scenario, AJ integrity becomes a key factor to determine susceptibility to Wnt signaling. For instance, at constant Wnt activity, the amount of β-catenin that reaches the nucleus may increase if AJ are disrupted. That is, the intensity of Wnt signaling response could be affected by AJ stability. Thus, cells would modulate gene expression by integrating AJ status with Wnt-like external signaling. Second, the effects of the AJ/Wnt axis may be compounded by other transcriptional changes induced by AJ disruption. For example, a recent report While our results clearly demonstrate that the cadherin-bound pool of β-catenin can be made available to Wnt signaling The β-catenin-ΔGSK-GFP mutant used in photo-activation experiments also revealed that LPA suppresses the gene expression that is elevated by mutating β-catenin . ResultsThere are multiple possible explanations for these mixed results. The first possibility is that the cadherin-bound/Wnt-bound β-catenin intersection is devoid of physiological significance. The second possibility is that regulation of Wnt-dependent gene expression is more complex than we expected, and increased levels of AJ-bound β-catenin are compensated for by other mechanisms  was purchased from Avanti Polar Lipids , and stored at −80°C at a stock concentration of 100 µM. The monoclonal antibodies against β-catenin, E-cadherin, PCNA, and EEA1 were obtained from BD Biosciences . The polyclonal rabbit IgG antibody against β-catenin, the mAb rabbit IgG against Rab11, mouse IgG mAb against Golign97, Alexa-Fluor™488-anti-rabbit IgG, Alexa-Fluor™568-anti-mouse IgG, and Alexa-Fluor™647-transferrin were purchased from Invitrogen . GSK inhibitor VIII and XIII, and proteasome inhibitor MG132 were purchased from EMD Biosciences . KCl and LiCl were purchased from Sigma . The construct expressing β-catenin-ΔGSK-GFP was a generous gift from A. I. Barth and W. J. Nelson, (Stanford University) and the vector containing photo-activatable-(PA)-GFP-C1 cDNA was a gift from A. Kenworthy (Vanderbilt University). The expressing plasmid of mRFP-actin was a gift from A. Weaver (Vanderbilt University) and TOPflash reporter plasmid was from E. Lee (Vanderbilt University).2 at 37°C.A431 (human epidermoid squamous carcinoma cell line), HEK293 (a human embryonic kidney cell line), HCT-8, HCT-15, HCT-116, and HT-29 (human colon carcinoma cell lines) were obtained from American Type Culture Collection  and maintained in DMEM  supplemented with 10% fetal bovine serum  and 2 mM L-glutamine, and kept in constant culture in a humidified incubator with 5% COFor the live cell imaging, A431 cells were co-transfected with β-catenin-ΔGSK-PA-GFP and mRFP-actin (3∶1) by using FuGENE® HD . The cDNA of β-catenin-ΔGSK was subcloned into PA-GFP expressing vector by using SacII and BamHI restriction enzyme sites.Immunofluorescence labeling for confocal microscopy was performed as described previously with minor modifications Cells expressing β-catenin-ΔGSK-PA-GFP and mRFP-actin were starved for 18 h and placed on a Zeiss LSM-510 stage equipped with a humidified and heated chamber (37°C). The AJ and nuclear areas of transfected cells were identified based on the mRFP-actin image , and the junctional area alone was activated by 405-nm excitation light. The green fluorescence (488 nm excitation) of each area was monitored at various time points over the course of 25 min. The fluorescence intensity was measured and compared using a Zeiss LSM-510 microscope and image analysis software. Raw nuclear and AJ associated fluorescence values were converted to nuclear/AJ ratios to remove bias from variability in cell size/morphology and initial fluorescence intensities captured at 0 h. Ratios (300–1500 sec) were then normalized against corresponding measurements at 0 h, resulting in values presented as the fold-change of nuclear/AJ ratios over time.After LPA stimulation, cells were washed in ice-cold PBS and collected by centrifuge at 700×g. The nuclear fraction and cytoplasmic fractions were extracted by using NE-PER® Nuclear and Cytoplasmic Extraction Reagents  according to the manufacturer's manual. Isolated fractions were separated on 8% SDS-polyacrylamide gel and analyzed by immunoblot using Odyssey Infrared Imaging System .TCF/LEF-dependent transcriptional activity was assessed by using TOPflash reporter plasmid. Briefly, the reporter gene was transfected into cells grown to semi-confluence in 12-well plates, and cultured for a further 6–8 h. After overnight starvation, cells were stimulated by PBS or LPA (5 µM) in the presence or absence of LiCl (20 mM) or Wnt3a-L-cell conditioned media (collected by centrifugation at 1500 g for 10 min to remove cell debris) for 18 h. Luciferase activity was measured by using Steady-Glo Luciferase Assay System  according to the manufacturer's instructions.In order to see the effect of β-catenin-GFP or β-catenin-ΔGSK-GFP, they were co-transfected into cells with TOPflash reporter gene and the luciferase activity was measured with or without LPA stimulation, as described above.Data analysis was performed using SPSS, version 16 . Differences between cell treatments were examined using two-sided Student's t-tests, and were considered significant when P<0.05.Figure S1LPA induces redistribution of E-cadherin, α-catenin and β-catenin. A431 cells were stimulated with 1 µM LPA for 3 h and stained with Alexa-FluorTM 568-phalloidin and Alexa-FluorTM 488 anti-mouse IgG to detect F-actin (red) and E-cadherin (green), respectively. α- and β-catenin were visualized the same way. Images  were captured with a Zeiss Axioplan-2 fluorescence microscope. LPA-treated cells displayed disorganized β-catenin, α-catenin, and E-cadherin expression, as expected in disintegrating AJ.(3.16 MB EPS)Click here for additional data file.Figure S2β-catenin is targeted to the perinuclear endocytic recycling compartment. A431 cells were serum-starved, treated with 1 µM LPA or PBS for 1 h, immunostained for indicated antibodies, and imaged with a Zeiss LSM510 confocal microscope . In LPA-treated cells, β-catenin (green) was not colocalized with lysosome  or the Golgi , but partially colocalized with early endosomes .(6.50 MB EPS)Click here for additional data file.Figure S3Ca++-deprivation mimics LPA effects. A431 cells were serum-starved for 18 h, treated with 1 µM LPA or PBS for 1 h in the presence or absence of 1.8 mM Ca++ in serum-free media, immunostained for β-catenin, and imaged with a Zeiss LSM510 confocal microscope . In normal, higher Ca++ levels (1.8 mM), no nuclear β-catenin was observed with or without the Wnt activator LiCl; in contrast, in low Ca++ concentrations, upon dissociation of AJ, β-catenin is transported to a perinuclear region (presumably the ERC) and translocated to the nucleus in the presence of LiCl. Compared with LPA stimulation, side-by-side, results with low Ca++ were not as clearly interpretable, possibly due to disturbances of cell shape.(1.97 MB EPS)Click here for additional data file.Figure S4Proteasome inhibitor, MG132, promotes β-catenin translocation to nucleus. A431 cells were serum-starved, treated with 1 µM LPA or PBS for 1 h, subsequently treated with 40 mM LiCl or 10 µM MG132 (a proteasome inhibitor) for 1 h, immunostained for β-catenin for 45 min, and examined with a Zeiss LSM510 confocal microscope . LPA increased the nuclear accumulation of β-catenin in cells co-stimulated by MG132.(3.81 MB EPS)Click here for additional data file.Figure S5TCF/LEF-dependent transcriptional activity TOPflash analysis of Wnt3a treated A431. In order to measure TCF/LEF-dependent transcriptional activity of the A431 cell line, cells were transfected with a TOPflash reporter plasmid. After overnight starvation, cells were stimulated by either PBS or LPA, in the presence or absence of Wnt3a-L-cell conditional medium (Wnt3a) for 18 h, and were assessed for luciferase activity using the Steady-Glo Luciferase assay system. Both PBS and LPA alone showed negligible levels of transcriptional activity (N = 3). In contrast, cells treated with Wnt3a showed significant increase in signal , which was significantly reduced by co-treatment with LPA .(0.48 MB EPS)Click here for additional data file.Figure S62-catenin mutated A431 cells. In order to see the effect of Î2-catenin-GFP or Î2-catenin-GSK-GFP, each of these constructs was co-transfected into A431 cells with TOPflash reporter gene. After overnight starvation, cells were stimulated by PBS or LPA, in the presence or absence of LiCl or Wnt3a for 18 h, and assessed for transcriptional activity using the Steady-Glo Luciferase assay system. Each PBS, Î2-catenin-GFP, and Î2-catenin-GFP+LPA alone showed negligible levels of transcriptional activity (N = 3). In contrast, Î2-catenin-GFP cells treated with LiCl showed a significant increase in signal . Further, Î2-catenin-GSK mutants exhibited significantly decreased signal upon LPA treatment . Oppositely, cells treated with Wnt3a significantly increased gene expression .TCF/LEF-dependent transcriptional activity TOPflash analysis of Î(0.68 MB EPS)Click here for additional data file.Figure S7Colon cancer cell lines display no discernable pattern of β-catenin transcriptional expression. In order to measure TCF/LEF-dependent transcriptional activity of colon cancer cells, HCT-8, HCT-15, HCT-116, and HT-29 cell lines were transfected with a TOPflash reporter plasmid. After overnight starvation, cells were stimulated by either PBS or LPA, in the presence or absence of LiCl for 18 h, and were assessed for luciferase activity using the Steady-Glo Luciferase assay system. Colon cancer cells exhibited no discernable pattern of expression.(0.72 MB EPS)Click here for additional data file.


Intermediate progenitor cells constitute a second proliferative cell type in the developing mammalian cerebral cortex. Little is known about the factors that govern the production of intermediate progenitors. Although persistent expression of stabilized β-catenin was found to delay the maturation of radial glial progenitors into intermediate progenitors, the relationship between β-catenin signaling and intermediate progenitors remains poorly understood. Using a transgenic reporter mouse for Axin2, a direct target of Wnt/β-catenin signaling, we observed that β-catenin signaling is decreased in intermediate progenitor cells relative to radial glial progenitors. Conditional deletion of β-catenin from mouse cortical neural progenitors increased intermediate progenitor numbers, while conditional expression of stabilized β-catenin reduced the intermediate progenitor population. Together, these findings provide evidence that β-catenin signaling in radial progenitors negatively regulates intermediate progenitor cell number during cortical development. During mammalian cortical neurogenesis, neural progenitors proliferate to produce neurons that form the six-layered cerebral cortex. Two distinct populations of progenitor cells produce the excitatory projection neurons that populate the cortical plate. Radial progenitors, also known as radial glia, have a characteristic polarized epithelial morphology with an apical process that abuts the lateral ventricle and a basal process that extends toward the pial surface of the developing cortex Intermediate progenitors arise from radial glia Although basal mitotic figures in the developing cortex were first described over three decades ago Tbr2) transcription factor is both necessary and sufficient for the development of intermediate progenitors in the developing cortex Tbr2 in the developing cortex led to the loss of intermediate progenitor cells Tbr2 is sufficient to induce intermediate progenitor cell fate in radial progenitor cells Ngn2 also regulates the progression of progenitor to postmitotic neuron, but its role in intermediate progenitor development changes during cortical development. Loss of Ngn2 or misexpression of Mash1 (repressed by Ngn2) early in cortical development leads to an expansion of the basal intermediate progenitor pool at the expense of early neuronal production Ngn2 promotes increased basal divisions Insm1 reduces the number of intermediate progenitors, while overexpression increases basal divisions and Tbr2 expression The production of intermediate progenitors from radial progenitors appears to require transcription factors important in the stepwise maturation of neurons from dividing progenitors. Recent studies suggest that the T-brain gene-2 , a GFP variant designed to be rapidly degraded  RP23-235G22 at the ATG transcriptional initiation site for the Eomes (Tbr2) gene so that EGFP expression reflects the endogenous expression of Tbr2. GFP-expressing (representing Tbr2-expressing) cells and GFP-negative cells were flow sorted from the dorsal neocortex of E13.5 embryos lox(ex2–6) mice, in which exons 2  to 6 of β-catenin are flanked by LoxP sites fl/fl/Nes-Cre+ (β-catfl/fl/Cre+) and β-cateninfl/fl/Nes-Cre- (β-catfl/fl/Cre-) cortices . To assess the numbers of basal-dividing intermediate progenitors, we stained β-catfl/fl/Cre+ and β-catfl/fl/Cre- cortical sections for the phosphorylated form of histone H3 (PH3) , whereas mitotic apical progenitors showed no change in density  vs. 104.3±5.3 cells/mm, in β-catfl/fl/Cre- ; P = 0.9627, data not shown).To investigate whether downregulation of β-catenin is sufficient to induce the production of intermediate progenitors, we crossed β-catenincortices . Deletiocortices  led to aH3 (PH3) . Loss ofTo examine whether downregulating β-catenin signaling specifically may regulate the transition from radial glial cells to intermediate progenitors, we electroporated E13.5 WT brains with a DNA construct driving expression of an N-terminal truncated TCF4. This form of TCF4 lacks the binding site for β-catenin and functions as a dominant negative by competing with endogenous TCF/LEF members for promoter fl/fl/Cre+) significantly increased the fraction of total cells expressing Tbr2 in all three regions  between dorsal and lateral cortex Tbr2 expression ) Because loss of β-catenin appears to increase the occurrence of basal intermediate progenitors, we investigated whether increasing β-catenin could reduce intermediate progenitor numbers. The N-terminal portion of β-catenin containing GSK3β phosphorylation sites required for its degradation are encoded by exon 3 of the β-catenin locus, and removal of this region results in the production of a stabilized protein lox(Ex3)/+; NesCre (hereafter referred to as ΔEx3 transgenic) exhibited large expansion in ventricular surface area compared with control mice . While expression of Wnt/β-catenin target genes can reveal reductions in signal, using Tbr2Insm1Foxg1 and Pax6 has resulted in a corresponding decreased number of intermediate progenitors and reduced expression of Tbr2Prior studies have shown that radial progenitors can generate cortical neurons directly or indirectly via production of intermediate progenitors As the fraction of intermediate progenitor divisions that generate additional intermediate progenitors (proliferative) appears to decline steadily during cortical development Moreover, our recent observations that the degree of β-catenin signaling declines in the VZ from mid to late cortical development provide further support for the notion that higher β-catenin signaling inhibits intermediate progenitor production increased Tbr2 cell numbers. The differences in timing between our two studies may provide additional insights. Kuwahara et al. utilized retroviral infection of stabilized β-catenin at E12.5, while our approach used Nestin Cre mice to produce stabilized β-catenin beginning at E10.5. In fact, the differences in outcomes support findings from their group suggesting that β-catenin signaling has distinct functions early vs. late in cortical development, with β-catenin promoting self-renewal at E10.5 vs. differentiation at later stages Although our study suggests that β-catenin reduces the number of intermediate progenitors, a recent study suggests that β-catenin promotes intermediate progenitor production Foxg1 and COUP-TFI are expressed in low medial to high lateral gradients that could potentially oppose β-catenin signaling Foxg1 haploinsufficiency has been shown to decrease the population of intermediate progenitor cells COUP-TFI overexpression increased intermediate progenitors, while the loss of COUP-TFI decreased the intermediate progenitor population Inquiry into the interactions between β-catenin and other signaling pathways will likely yield further insight into the regulation of intermediate progenitor cells. Several transcription factors such as Recent studies support the importance of other β-catenin interactions in the developing cortex. N-cadherin associates with β-catenin at the apical end feet of radial glial cells, mediating cell-cell adhesion and polarity. The observation that N-cadherin plays an important role in regulating β-catenin signaling in the developing cortex It is also important to note that β-catenin has been shown to interact with non-TCF transcription factors such as the SOX family Tbr2 or Insm1.While the genetic approaches used to conditionally activate or delete β-catenin provide insights onto the role of β-catenin in intermediate progenitor development, further studies are required to determine whether β-catenin controls intermediate progenitor development through direct regulation of genes such as lox(ex2–6) mice (B6.129-Ctnnb1<tm2Kem>/KnwJ) http://jaxmice.jax.org/pub-cgi/protocols/protocols.sh?objtype=protocol&protocol_id=471 (β-cateninlox(ex2–6)) and http://jaxmice.jax.org/pub-cgi/protocols/protocols.sh?objtype=protocol&protocol_id=288 (Cre). Axin2-d2EGFP mice lox(ex3) mice were a gift from M. Taketo . The construction and genotyping protocols for these mice have been described All animals were treated according to protocols reviewed and approved by the animal care and use committee at Northwestern University, Animal Study Protocol # 2006-0131, approved by the Northwestern University Office for the Protection of Research Subjects Institutional Animal Care and Use Committee. Beta-cateninE13.5 Eomes::GFP embryos  were mated to C57Bl6  to generate embryos for flow cytometric sorting of intermediate progenitors (see below).lox(ex3) mice, all coronal images here represent sections taken through the midsection of the eye as a standard landmark.Embryonic brain samples were fixed overnight in 4% paraformaldehyde (PFA). Samples were then prepared for sectioning by either vibratome or cryostat. Samples that were to be sectioned by vibratome (Axin2-d2EGFP), were removed from fix and embedded in a 1.5% solution of agarose is phosphate buffered saline (PBS). Once the agarose polymerized, samples were post-fixed in 4% PFA for an additional 30 minutes, mounted, and vibratome sectioned in the coronal plane at 100 µm. Samples to be cryosectioned were transferred to a 30% sucrose solution after fixation for ∼24 hours and then embedded and flash frozen in O.T.C. compound . Samples were sectioned in the coronal plane at a thickness of 12 µm and stored at −80°C until use. Because of extreme disruption of the neocortical architecture in the Cre+ Beta-cateninPrimary antibodies used were GFP , Tbr2  and Phospho-Histone H3 (Ser10) . Secondary antibodies  and DAPI (1∶1000) nucleic acid dye were purchased from Invitrogen.For immunofluorescence, vibratome sections were incubated for 12 h in blocking buffer (5% goat serum and 0.3% Triton X in PBS) at 4°C, followed by incubation for 24 hours in primary antibody diluted with blocking buffer at 4°C. After washing in PBS, sections were incubated with secondary antibody and DAPI diluted in PBS for 12 hours at 4°C.For cryosections, slides were incubated for 30 minutes in blocking buffer (same as above) at room temperature, and then incubated with primary antibody solution for 16 hours at 4°C, followed by incubation with secondary antibody and DAPI for 2 hours at room temperature. To maintain consistent rostral-caudal positioning, coronal sections through the plane of the embryonic eye structures were used for all analysis.http://rsbweb.nih.gov/ij/). Linear ventricular measurements were calculated by measuring the distance along the ventricular surface of the cortex from the point at which the dorsal and medial walls of the lateral ventricle intersect to the lateral ganglionic eminence. One section per brain was counted/measured. Distances were measured using Metatmorph version 6.3r1 . Lower power images used in some figures were collected with a Nikon  TE2000-U inverted fluorescence microscope and Metamorph acquisition software.Images used for analysis were acquired with a Zeiss  LSM510 confocal microscope in 1 µm optical sections. Identical settings were used to acquire control and experimental images for all experiments. Composites of overlapping high power images were assembled using Photoshop CS4 . Cell counts were compiled using ImageJ (Embryonic Day (E)14.5 Axin2-d2EGFP mice were sacrificed and vibratome sections were prepared and co-immunostained with anti-GFP, anti-Tbr2 and DAPI as described above. One micron optical section images were obtained on a Zeiss confocal microscope, with each of the 3 above markers in a separate channel. All sections used were stained together and imaged in the same session with the same settings to minimize intersample variance. Using Metamorph image analysis software, each individual cell in the VZ and SVZ was identified by nuclear DAPI expression. Using the software, a region was created over each cell's nucleus. These regions were saved and transferred to the images containing the Tbr2 image and the d2EGFP image. Again using Metamorph, the mean pixel intensity for both the Tbr2 fluorescence and the corresponding d2EGFP fluorescence of each cell was calculated. This data was then used to compute the difference in fluorescence intensity between Tbr2+ and Tbr2- cells.Dorsal neocortices from E13.5 Eomes::GFP embryos were rapidly dissected, meninges removed, then dissociated in 1% trypsin/EDTA (Gibco) for subsequent fluorescence activated cell sorting. Cortices from two GFP+ embryos were used per sorted sample and 3 samples were generated per developmental time point. Cells were sorted into GFP+ and GFP- populations on a Becton Dickinson FACS Vantage Flow Cytometer Cell Sorter . Cells were sorted directly into collection tubes containing RNALater™ (Qiagen) to minimize post-sort RNA degradation.High quality total RNA was isolated using the RNAeasy™ (Qiagen) kit and flash frozen. Oneµl of RNA was run on an Agilent 2100 Bioanalyzer using the RNA PicoChip to assess RNA structural quality and quantity. Amplified and terminal-labeled cDNAs were then generated by the WT Sense Target Labeling Protocol (Affymetrix).T analysis was run in singleplex using a StepOne Plus Real-time PCR system (Applied Biosystems). FAM-conjugated Taqman primers for mouse Axin2 or mouse Actin (Applied Biosystems) were mixed with Taqman Master mix, H2O, and cDNA made from the FACS-sorted cells isolated from the E13.5 Eomes::GFP brains. Identical volumes of cDNA were loaded for all samples, and samples were run in duplicate. The CT (cycle number at which fluorescence is detected above threshold) was determined by the StepOne software algorithm. ΔCT was obtained by subtracting the mean CT value of the endogenous target (Actin) from the mean CT of the experimental target (Axin2). ΔΔCT was calculated by setting a choosing a baseline sample (ΔΔCT set to 0) and comparing all other samples to this baseline. Results are reported in the form of relative quantification, or RQ = 2∧(- ΔΔCT). RQ values were averaged for cDNA from three different Tbr2-egfp brains, with one RQ average for GFP positive-sorted (Tbr2+) cells and one RQ average for GFP negative-sorted (Tbr2-) cells . Error bars represent SEM, and student's t-test was used to calculate significance.Comparative CData was compiled using Microsoft Excel 2008 . All statistics were computed using Prism 3.0 software .Figure S1Beta-catenin signaling regulates VZ precursors E13.5 embryos were electroporated with (A) Δ 90β-catenin-GFP (N = 3), (B) DNTCF4-GFP (N = 3), or (C) GFP control (N = 3). After 30 hours embryos were sacrificed and stained with antibodies raised against GFP and PAX6, a marker for VZ precursors. The fraction of GFP expressing cells that were also PAX6+ for each group was graphed (D). The fraction of PAX6+ electroporated cells in the three experimental groups was significantly different . Increased β-catenin signaling by Δ 90β-catenin-GFP increased the fraction of cells that retained PAX6 positivity (0.534±0.015 ) while blocking β-catenin signaling decreased the fraction (0.173±0.006), when compared to control (0.339±0.031). Scale bars are 50 µm. n = 3.(0.84 MB TIF)Click here for additional data file.Table S1Summary of embryos and cell counts. cKO refers to beta-catenin fl/fl Nes-cre+ mice; GOF refers to beta-catenin ex3fl/+ Nes-Cre+ mice.(0.03 MB DOC)Click here for additional data file.