Krüppel-like factor 8 (KLF8) plays important role in cell cycle and oncogenic transformation. Here we report the mechanisms by which KLF8 crosstalks with Wnt/β-catenin signaling pathway and regulates hepatocellular carcinoma (HCC) cells proliferation. We show that overexpression of KLF8 and nucleus accumulation of β-catenin in the human HCC samples are positively correlated. More importantly, KLF8 protein levels plus nucleus accumulation of β-catenin levels were significantly elevated in high-grade HCC compared to low-grade HCC. Using HCC HepG2 cells we find that, on the one hand both protein and mRNA of KLF8 are up-regulated under Wnt3a stimulation, on the other hand overexpression of KLF8 increases the cytoplasm and nucleus accumulation of β-catenin, recruits p300 to β-catenin/T-cell factor 4 (TCF4) transcription complex, enhances TOP flash report gene transcription, and induces Wnt/β-catenin signaling target genes c-Myc, cyclin D1 and Axin1 expression. Knockdown of KLF8 using shRNA inhibits Wnt3a induced transcription of TOP flash report gene and expression of c-Myc, cyclin D1 and Axin1. Knockdown of β-catenin by shRNA rescues the enhanced HepG2 and Hep3B cells proliferation ability induced by overexpression of KLF8. Canonical Wnt/β-catenin signaling pathway plays essential role in regulating developmental decision and adult tissue homeostasis. The key regulatory molecule is β-catenin, which is kept low level in cytoplasm by its binding to the cytoplasmic tail of E-cadherin and its degradation through a destruction complex consisting of Axin1, glycogen synthase kinase-3β (GSK-3β), adenomatous polyposis coli (APC) and casein kinase Iα (CKIα) in the absence of Wnt signaling. Activation of canonical Wnt signaling by Wnt binding to the 7-span transmembrane protein Frizzled (Fz) and the single-span low-density lipoprotein receptor-related protein (LRP) leads to inhibition of GSK-3β activity and translocation of β-catenin to the nucleus, where it interacts with the TCF/lymphoid enhancer factor (LEF) family of transcription factors for up-regulation of such target genes as Axin1, c-Myc and cyclin D1 KLF8 belongs to the Krüppel-like factor (KLF) family of transcription factors, which share homology in their three C2–H2 zinc finger DNA binding domains, play critical role in diverse processes, including regulation of the cell cycle progression, oncogenic transformation and cell invasion Here, we report that KLF8 is a novel participator in canonical Wnt/β-catenin signaling pathway. We show that activation of Wnt/β-catenin signaling increases the expression of KLF8, in the meanwhile, KLF8 stabilizes β-catenin, binds with β-catenin/TCF4 complex, enhances Wnt/β-catenin signaling transcription activation in HCC cells.5′-CTGATATTGATGGACAGTA-3′, and human KLF8 shRNA plasmids, including shRNA1, target sequence: 5′-CTGGTCGATATGGATAAACTCA-3′, shRNA2, target sequence: 5′-CAGCAGATCTTACATGTCA-3′, and shRNA control, target sequence: TGAGCAGGCGCATGTGCTG-3′, were obtained from Open Biosystems. The plasmid of KLF8 (MHS1010-98053352) was purchased from Open Biosystems and was subcloned into pCMV-tag5A vector with c-myc tag. Primary antibodies to β-catenin and cyclin D1 were purchased from BD Biosciences. Primary antibodies to α-tubulin and Sp1 were purchased from Sigma. Anti-c-Myc antibody was purchased from ATCC. Primary antibodies to KLF-8, Axin1, p300, and TCF4 were purchased from Abcam. Second antibodies anti-mouse IgG-HRP and anti-rabbit-HRP were purchased from Sigma. MTT assay reagents were purchased from DingGuo Biotech.HCC cell lines HepG2 and Hep3B, L Wnt-3A cells and control L cells, were purchased from American Type Culture Collection (ATCC), and were cultured according to the recommendations from ATCC. Wnt-3a conditioned medium and control L cell medium were harvested and treated HepG2 cells for 4 hours for western blot assay, 16 hours for qRT-PCR assay and report gene assay. Human β -catenin shRNA, target sequences: 2O2 in methanol for 30 min. After incubating with blocking buffer (10% BSA in TBS) at 37°C for 1 hour, tissues sections were incubated with primary antibodies in TBS containing 1% BSA at 4°C overnight, followed by incubation with a horseradish peroxidase anti-rabbit or –mouse IgG antibody. Color was then developed by incubation with an ImmunoPure Metal Enhanced Diaminobenzidine (DAB) Substrate kit (Pierce). After each incubation, tissue sections were washed three times in TBS over 10 min. Tissue sections were finally counterstained with hematoxylin.HCC samples were obtained from Eastern Hepatobiliary Surgery Hospital. 5-µm thick tissues sections were gotten and dewaxed, endogenous peroxidase was quenched by incubating with 3% Hg for 15 min at 4°C. For subcellular fractionation, cell-surface, cytoplasmic and nuclear extracts were prepared using Qproteome Cell Compartment Kit (Qiagen). Samples were subjected to SDS-PAGE, transferred to PVDF membranes (Millipore) and detected with appropriate primary antibodies followed by horseradish peroxidase-conjugated goat anti-mouse or rabbit IgG. The blotting signals were detected using SuperSignal West Dura Extended Duration Substrate (Pierce). Quantitative analyses of immunoblotting signals were obtained via densitometry analysis using LAS4000 Image Software (Fuji Film).Cells were washed with ice-cold phosphate buffer saline, pH 7.4 (PBS) and lysed with ice-cold radioimmunoprecipitation assay lysis buffer  at 4°C for 1 hrs. Samples were centrifuged at 12,000 For immunoprecipitation, 1 µg appropriate antibody was preincubated with 30 µl slurry of Protein A-agarose beads . Lysates (∼1 mg/sample) were incubated with the antibody-bound Protein A-agarose beads at 4°C overnight. After extensive washing with the radioimmunoprecipitation assay lysis buffer, samples were resuspended in the reducing SDS sample loading buffer, boiled for 5 min, and subjected to SDS-PAGE and immunoblotting.2 qPCR Primer pairs were purchased from SABiosciences. PCR and data collection were performed on the Mx3000 qPCR System (Stratagene). All quantitations were normalized to an endogenous β-actin control. The relative quantitation value for each target gene compared to the calibrator for that target is expressed as 2-(Ct-Cc) . The relative expression levels of samples are presented using a semi-log plot.Total RNA was extracted using the Absolutely RNA Miniprep Kit (Stratagene) and reverse transcribed using ThermoScript RT-PCR System (Invitrogen). The resulting cDNA was used for PCR using the SYBR-Green Master PCR Mix (Applied Biosystem) in triplicates. All RTTo evaluate β-catenin/TCF-dependent transcriptional activity, luciferase reporter assay was performed with a pair of luciferase reporter constructs TOP-FLASH and FOP-FLASH (Upstate Biotechnology). TOP-FLASH contains three copies of the TCF binding sites, and FOP-FLASH contains mutated TCF binding sites. Cells were transiently transfected in triplicate with one of these luciferase reporters and pCMV-β-galactosidase (Promega) using Lipofectamine 2000 (Invitrogen). 48 h after transfection, luciferase activity was determined using the Luciferase Assay System Kit (Promega). β-galactosidase activity was determined using the Luminescent β-gal Detection Kit II (BD Clontech) as an internal control.3 cells/ml) were plated into 96-well tissue culture plates (100 µl complete medium/well) and cultured at 37°C in 5% CO2 atmosphere. At different time points, tetrazolium salt was added (20 µl per well) and incubated at 37°C for 4 hr. The insoluble blue formazan product was solubilized by addition of 100 µl/well 10% SDS/5% isobutanol. The plates were read on a micro-titer plate reader using a test wavelength of 570 nm and a reference wavelength of 630 nm.Cells including all transfectants were grown in exponential phase and detached by trypsin treatment. Viable cells (5×10www.r-project.org).To compute correlation coefficients of immunostaining scores between two proteins, Spearman’s rank correlation coefficient was used. P values of less than 0.05 were considered statistically significant. All computations were made with R 2.9.0  of HCC samples. We then examined possible correlation of the positive KLF8 with β-catenin in these 65 cases of HCC by immunohistochemistry assay. Using continuous tissue sectioning, we found that 82.98% (39/47) of KLF8 positive HCC samples showed cytoplasmic and nuclear β-catenin accumulation, compared to only 38.89% (7/18) KLF8 negative HCC samples showed cytoplasmic and nuclear β-catenin accumulation p<0.05, . In HCC We then detected the clinicopathological correlation of KLF8 positive or/and cytoplasm and nuclear β-catenin accumulation in the 65 HCC samples. Expression of KLF8 was detected in 25% (2/8) of Stage I HCC samples, 61.9% (13/21) of Stage II HCC samples, 84.61% (22/26) Stage III HCC samples, and 100% (10/10) Stage IV HCC samples , which sWe next asked whether KLF8 was regulated by Wnt/β-catenin signaling pathway. Wnt3a conditioned medium was used to activate Wnt/β-catenin signaling pathway. Compared with control medium, the cytoplasmic β-catenin in HepG2 cells treated with Wnt3a conditioned medium was dramatically increased , which dWe next explored whether KLF8 affect Wnt/β-catenin signaling pathway. Luciferase reporter assay was employed here. HepG2 cells were co-transfected with a plasmid encoding the TCF4 binding sites luciferase reporter (TOP-Flash) or its control reportor (FOP-Flash) and the system control plasmid encoding the β-galactosidase reporter. The results showed that, ectopic expression of KLF8 increased TOP-Flash activity no matter with or without Wnt3a conditioned medium, but not FOP-Flash activity . To furtTo further investigate whether KLF8 regulated Wnt/β-catenin signaling transcription activity, we examined the expression of Axin1, c-Myc and cyclin D1, which are well characterized targeting genes of Wnt/β-catenin signaling. Indeed, ectopic expression of KLF8 triggered mRNA and protein expression of Axin1, c-Myc and cyclin D1 in HepG2 cells , whereasTo investigate how KLF8 regulated Wnt/β-catenin signaling pathway, we first explored whether KLF8 binded with β-catenin. Using co-immunoprecipitation (co-IP) assay, we found that β-catenin could bind with KLF8 in HepG2 cells treated with Wnt3a conditioned medium . ImportaOthers’ In this report, we showed that KLF8 is a novel targeted gene and regulator in canonical Wnt/β-catenin signaling pathway and this finding is possibly clinically relevant. We identified that KLF8 stabilizes β-catenin, binds with and recruits p300 to β-catenin/TCF4 transcription complex, and activates Wnt/β-catenin target genes transcription. In the meanwhile, activation of Wnt/β-catenin signaling pathway up-regulates both mRNA and protein level of KLF8. Knockdown of β-catenin rescued the phenotype of overexpressed KLF8 HepG2 and Hep3B cells showed that the crosstalk between KLF8 and Wnt/β-catenin signaling pathway plays essential roles in HCC cells proliferation. More importantly, we showed that the aberrant overexpression of KLF8 is strongly correlated to the aberrant nucleus accumulation of β-catenin in human HCC samples, which positively correlates with progressed HCC.The three major Wnt signaling pathways are involved in almost every aspect of embryonic development and in adult tissue homeostasis. The canonical Wnt/β-catenin signaling pathway governs cell proliferation, differentiation, migration, and invasion. It centers around β-catenin, which binds to TCF/LEF transcription factors and leads to the target genes transcription. In the inactive state, the cytoplasmic β-catenin is continuously degraded. When stimulated by Wnt ligand, β-catenin escapes the degradation, accumulates in the nucleus, binds with TCF transcription complex and induces Wnt target genes expression As a transcription factor, KLF8 can negatively regulate the globin and E-cadherin genes through contacting with the C-terminal Binding Protein (CtBP) corepressor, and positively regulate cyclinD1 genes by recruitment of the p300 or p300/CBP associated factor(PCAF) co-activator of the histone acetylase family HCC is the top 5 common malignant tumors and third cause of cancer-related death


Burkholderia mallei and Burkholderia pseudomallei are the causative organisms of Glanders and Melioidosis, respectively. Although now rare in Western countries, both organisms have recently gained much interest because of their unique potential as bioterrorism agents. These organisms are less familiar to medical and laboratory personnel than other select bioterrorism bacterial agents and thus heightened awareness of Glanders and Melioidosis is crucial in order to enable adequate emergency preparedness and response to deliberate release of B. mallei and B. pseudomallei. The microbiological diagnosis of both species in the clinical laboratory is complicated. This paper reviews the various challenges and pitfalls associated with the diagnosis of Melioidosis and Glanders in the clinical setting, with emphasis on the role of sentinel laboratories. Burkholderia mallei and Burkholderia pseudomallei are the causative organisms of Glanders and Melioidosis, respectively. Although now rare in Western countries, both organisms have recently gained much interest because of their unique potential as bioterrorism agents  acquire melioidosis through percutaneous inoculation, inhalation or ingestion, and more rarely sexual transmission. The percutaneous route is thought to be the predominant portal of entry, even for patients with pneumonic melioidosis, but pulmonary infection may occur directly via inhalation  and has B. pseudomallei infection and in endemic areas, melioidosis may be the most common cause of community-acquired pneumonia  with septic shock present in one-fifth, and appreciable mortality (up to 60%) , 12, 13.neumonia . Pneumonneumonia . Pulmonaneumonia .Apart from pulmonary infection, melioidosis may affect every organ system, resulting in genitourinary infection (including prostatic infection) , suppuraThe outcome of melioidosis is related to host risk factors, adequate therapy and disease severity; mortality is <10% in isolated bacteremia or localized disease without bacteremia, up to 40% with septicemic disease involving at least one focus (e.g. pneumonia) with bacteremia, and 40-90% when severe sepsis or septic shock occur .th century were occupational infections among laboratory workers, horse handlers, butchers and veterinarians  in the United States and involved a microbiology laboratory worker  Difficulty in diagnosing clinical B. mallei infection; and (c) Misidentification of the clinical isolate by routine laboratory methods.An occupational tly year 00 in theB. mallei shares many genetic elements with B. pseudomallei and both organisms have a similar allelic profile . Prostration, out of proportion to clinical signs is a classic finding . SepticeBurkholderia contains more than 20 valid species, most of which are environmental bacteria isolated from soil or water. Three main human pathogens recognized in this group are B. mallei, B. pseudomallei and B. cepacia. Uncommonly, infection may occur with rare taxons such as B. fungorum, B. gladioli (previously B. cocovenenans) and B. thailandensis. Burkholderia species are aerobic, non-spore-forming, gram-negative bacilli. All species except B. mallei are motile due to the presence of polar flagella. All grow on MacConkey agar and appear as non-fermenters  and arginine dihydrolase activity that is positive only with B. pseudomallei , Microbact 24E , VITEK-1 or VITEK-2 (bioMerieux) and MicroScan WalkAway . Good results for B. pseudomallei identification were reported in two studies with the API20NE system   has been carried out with different sets of human, animal and environmental B. pseudomallei and B. mallei isolates  . The APIisolates . B. pseuB. pseudomallei . However, sensitivity has been disappointing (66%)  to molecular detection of ng (66%) . Co-agglectively . Previouectively , 44. In pecimens .B. mallei and B. pseudomallei in clinical samples have gained much interest, due to the difficulties in identifying these organisms, especially in non-endemic areas and the biosafety level requirements for processing B. mallei and B. pseudomallei samples in classical methods  breakpoints for Pseudomonas species. Recently, CLSI recommendations for potential bioterrorism agents have included B. mallei and B. pseudomallei. In general, disk-diffusion is not recommended for Burkholderiaceae  for the susceptible, intermediate-resistance and resistant categories, are presented in Table Therapy of Melioidosis requires an intensive-phase of bactericidal agents (mainly ceftazidime-based or carbapenem-based regimens) and maintenance therapy with a combination of oral agents, usually including trimethoprim-sulfamethoxazole, doxycycline and chloramphenicol . No firmB. pseudomallei, and emergence of resistance during therapy is rare    and natudomallei . Fluoroqdomallei . A recendomallei . A post-domallei .B. mallei is generally similar to B. pseudomallei. A recent survey of 15 B. mallei isolates has shown 100% susceptibility of B. mallei to cefotaxime, ceftazidime, amoxicillin-clavulanate, piperacillin-tazobactam, imipenem, chloramphenicol, trimethoprim-sulfamethoxazole and tetracyclines   .B. mallei or B. pseudomallei should be referred immediately to a reference laboratory. Sentinel laboratory work-up should consist of the minimal phenotypic and biochemical tests that suggest this diagnosis. In cases where a high clinical index of suspicion exists, direct referral of clinical samples to a specialized setting may be most appropriate.Until new or improved diagnostic modalities become available at the level of the sentinel laboratory, isolates compatible with


Mid IR spectral analysis was used to calculate the content of the epoxy groups as a function of crosslinking time and the crosslinking degree of resin. The amount of all the epoxy species was estimated from IR spectra to changes during the crosslinking kinetics of epichlorhydrin.The use of fast FT-IR spectroscopy as a sensitive method to estimate a change of the crosslinking kinetics of epoxy resin with polyamine adducts is described in this study. A new epoxy formulation based on the use of polyamine adducts as the hardeners was analyzed. Crosslinking reactions of the different stoichiometric mixtures of the unmodified GY250 epoxy resin with the aliphatic EH606 and the cycloaliphatic EH637 polyamine adducts were studied using mid FT-IR spectroscopic techniques. As the crosslinking proceeded, the primary amine groups in polyamine adduct are converted to secondary and the tertiary amines. The decrease in the IR band intensity of epoxy groups at about 915 cm The dEpoxy resins crosslinked with aliphatic or cycloaliphatic amines are extensively used in protective coating applications . Since t1H-NMR, GPC, DSC, TGA spectral analyses [1H/13C-NMR spectroscopic methods [13C-NMR [3 etherate accelerator were measured from the dependences of degree of cure with time, using GPC, DSC and IR spectroscopy [Cure reactions of the stoichiometric mixtures of diglycidyl ether of bisphenol A (DGEBA) and two very low molecular weight aliphatic polyether diamines (PED) were studied by using mid and near infrared (IR) spectroscopic techniques . Both Raanalyses . The molanalyses . New epo methods . Physica[13C-NMR . The rattroscopy . A methotroscopy . The metA search for new improved components for coating systems requires a better understanding of the structure-property relationships of the materials . Althoug3N > R2NH > RNH2 [Aliphatic amines are well known quenchers of the aromatic hydrocarbons –35. It hH > RNH2 . Thus, dH > RNH2 ,37. The H > RNH2 .The reaction of the primary amine with the epoxide to form a secondary amine  and the N-alkylation or Hoffman elimination (termination).The crosslinking of epoxy resins can be achieved through two different reaction mechanisms, polymerization by addition and by steps . The mosIn this study, a newer analysis method to measure the distribution of small amounts epoxy functional groups presented in the crosslinked polymers using available equipment was searched. Consequently, the main objective of this research is to monitor the crosslinking reactions by fast IR spectroscopy from the epoxy GY250 resin based diglycidyl ether of bisphenol A, cured with different polyamine adducts .2.2.1.Unmodified medium viscous  epoxy resin GY250 based on diglycidyl ether of bisphenol A (DGEBA), supplied by Vantico AG (Switzerland), whose epoxy equivalent weight is 183–189 g/epoxy equivalent, and flash point is 200 °C, was used as received. The aliphatic EH606 and cycloaliphatic EH637 polyamine adducts, supplied by Solutia-Vianova (Austria), whose amine equivalent weight are 100 g/NH equivalent, were used as received. The chemical structure of DGEBA resin is shown in 2.2.3; Flash point (Pensky-Martens) (DIN EN ISO 2719) > 100 °C.Detailed characterization of the polyamine curing agents was done by manufacturer Company: Cytec-Liquid Coating Resins & Additives. Beckopox EH 606 is aliphatic polyamine adduct. It is a relatively low viscous hardener, which can be used with liquid solvent-free epoxy resins. At room temperature such systems have a long pot life of approx. 6–7 hours. Even at higher ambient temperatures, a sufficiently long pot life is exhibited. The hardener can also be used for preparing moldings. In spite of the long pot life, it will not influence the curing speed when preparing moldings, exothermy is very little. Beckopox EH 606 shows the very lowest reactivity of all Beckopox-hardener grades; therefore it is often used as a partner for highly reactive hardeners. Physical properties: H-equivalent weight (f.o.d.) is 100 g/mol. Dynamic viscosity (DIN EN ISO 3219) : 2,400–4,400 mPa·s; Amine value (reaction resins) (DIN 16945/5.6) (f.o.d.): 310–350 mg KOH/g; Iodine colour number (DIN 6162) ≤ 3; Density (liquids) (DIN EN ISO 2811-2) approx. (20 °C): 1,03 g/cm3; Flash point (Pensky-Martens) (DIN EN 22719) approx. 96 °C.Beckopox EH 637 is cycloaliphatic polyamine adduct. It is a modified polyamine with very low viscosity, for use together with liquid epoxy resins for flooring compounds with good chemical resistance and low yellowing tendency. For thin walled laminates a post cure of 2–3 hours at 40–50 °C is necessary prior to deforming. Physical properties: H-equivalent weight (f.o.d.) is 100 g/mol. Dynamic viscosity (DIN EN ISO 3219) : 80–120 mPa·s; Amine value (reaction resins) (DIN 16945 / 5.6) (f.o.d.): 300–350 mg KOH/g; Iodine colour number (DIN 6162) ≤ 2; Density (liquids) (DIN 53217-3) approx. (20 °C): 1,00 g/cm2.3.The first crosslinking process was carried out by addition of the amine hardener EH606 and the amine hardener EH637 in 2 g of the unmodified GY250 epoxy resin (stoichiometric ratio GY250/EH606/EH637 100:25:25). Other studied samples were prepared in different stoichiometric ratio: 100:30:20 and 100:20:30. Stoichiometric mixtures of GY250/EH606/EH637 were mixed using a glass rod and magnetic stirring, followed by crosslinking in a convection oven at 30 °C. At certain time intervals, samples were removed from the oven and IR spectra were taken at room temperature of 25 °C. Samples crosslinked at room temperature were stored in a desiccator.2.4.−1. For the epoxy grous characterization, the sample was molten between two plain KBr plates separated by a 0.2 mm thick spacer. To assure a fixed path length during crosslinking process, the edge of the samples were covered with high strength epoxy paste. The IR spectra of polyamine adducts films were performed between two quartz plates (5 × 2 × 0.4 cm) using a 0.2 mm thick spacer on the edges, with a Bomem MB-100 spectroscop. The measurement time for each spectrum was 20 s. The epoxy content of resins is frequently expressed as weight per epoxide or epoxide equivalent weight and epoxy functionalities were determined according published methods [A drop of provided mixture was placed between two KBr disks for the mid-IR characterization, using a BOMEM Hartmann&Braun MB-100 instrument, with a 40 scan averaging at a resolution of 2 cm methods .3.3.1.2 and –CH3 asymmetrical and symmetrical, –C–Ar–O–C stretching, and epoxy CH2–(O–CH–) ring stretching vibration (−1 (νC–H epoxy) and 915 cm−1 (γC–O epoxy). The 1,4-substitution of aromatic ring is seen at 830 cm−1 for GY250 epoxy resin.The structures of GY250 epoxy resin and used EH polyamine adducts were confirmed by IR spectral analyses. The three investigated components of the polymer system have very different IR spectra. The spectral differences of pure samples can be clearly seen in ibration . The pre−1 is due to the −NH2 vibration absorption of amine compounds. Another doublet peak observed between 1,350–1,380 cm−1 is due to the presence of isopropyl group vibration absorption. The doublet peak from 1-substitution of aromatic ring is seen at 735 and 698 cm−1 for EH637 polyamine adducts. The –C–N group absorption frequency is seen at 1,109 cm−1 for EH606 and 1,046 cm−1 for EH637. The ether C–O–R is seen at 1,025 cm−1. The aromatic –CH, and –C=C– vibrations are seen around 3,030 and 1,605 cm−1 region for both the polyamine adducts. The aliphatic –CH2 and –CH3 vibrations are seen between 3,000 and 2,850 cm−1 for both the polyamine adducts  is thus given by A1182,0 and A1182,t refer to the areas of the reference peak at time zero (0) and after certain time (t), respectively. A915,0 and A915,t are the areas of epoxy peak for uncrosslinked resin and partially crosslinked resin after a certain time, respectively.Epoxy conversion (αThe filled symbols (▪) in −1 was verified. The extent of reaction by IR is defined as follows:A0 is the peak area at time zero (0), and At is the peak area after certain time (t). By comparison, the exceptionally good agreement of these results with the error being <2% can be observed.The extent of crosslinking reaction of GY250/EH606/EH637 system by the IR peak area of the epoxy group absorbance at 3056 cm3.4.et al. [−1 in GY250/EH606/EH637 system during crosslinking process. The content of the epoxy group is determined by linear regression , using the following P is the peak area of absorbance at 3,056 cm−1 and Eg correspond to the epoxy groups content after a certain crosslinking time.The methods described by Zlatkovic et al. ,42,45,464.−1 was observed due to more effective quenching of the tertiary amine groups in EH polyamine adducts, in comparison to the IR band at about 1,182 cm−1. Moreover, a large decrease in the IR band intensity of GY250 resin at about 3,056 cm−1 was observed. Therefore, mid IR spectral analysis was used to calculate the concentration of the epoxy groups as a function of crosslinking time. The amount of all the epoxy species was estimated from mid IR spectra of GY250 in comparison with epichlorohydrin spectra. The GY250/EH606/EH637 system at 100:25:25 stoichiometric ratio betoken relatively good elasticity, adhesive resistance, and low water absorption compared with other aliphatic amine/epoxy resins.A new solvent free epoxy formulation based on the use of aliphatic and cycloaliphatic polyamine adducts as the hardeners was analyzed and characterized by fast FT-IR spectroscopy. IR spectroscopy proved to be a good method for monitoring and assignation of characteristic changes occuring during the crosslinking of epoxy resin. As the crosslinking proceeded, the primary amine groups in EH polyamine adducts are converted to secondary and tertiary amines. The decrease in the IR band intensity of GY250 resin at about 915 cm


WNT4 and CTNNB1 (encoding β catenin) is not significantly different between these tissues. Furthermore, reduced R-spondin1 function in the ovotestis of an individual  with a RSPO1 mutation leads to reduced β-catenin protein and WNT4 mRNA levels, consistent with down regulation of ovarian pathways. Transfection of wild-type RSPO1 cDNA resulted in weak dose-dependent activation of a β-catenin responsive TOPFLASH reporter (1.8 fold maximum), whereas co-transfection of CTNNB1 (encoding β-catenin) with RSPO1 resulted in dose-dependent synergistic augmentation of this reporter (approximately 10 fold). Furthermore, R-spondin1 showed strong nuclear localization in several different cell lines. Taken together, these data show that R-spondin1 is upregulated during critical stages of early human ovary development and may function as a tissue-specific amplifier of β-catenin signaling to oppose testis determination.Human testis development starts from around 42 days post conception with a transient wave of SRY expression followed by up-regulation of testis specific genes and a distinct set of morphological, paracrine and endocrine events. Although anatomical changes in the ovary are less marked, a distinct sub-set of ovary specific genes are also expressed during this time. The furin-domain containing peptide R-spondin1 (RSPO1) has recently emerged as an important regulator of ovary development through up-regulation of the WNT/β-catenin pathway to oppose testis formation. Here, we show that RSPO1 is upregulated in the ovary but not in the testis during critical early stages of gonad development in humans (between 6–9 weeks post conception), whereas the expression of the related genes  SRY in the developing testis and several other critical genes downstream of SRY have now been discovered from human and mouse studies Sex development is a complex process that requires the integrated interaction of a network of different endocrine and paracrine signaling pathways, transcription factors and steroid hormones, during a critical time period in embryogenesis. In humans, this process starts from around 6 weeks post-conception with the expression of NR0B1) may antagonize or oppose testis development For many years, testis development was believed to be the more active process. Although the morphological changes in the developing testis and in surrounding structures are more marked than in the developing ovary RSPO1 (encoding R-spondin1) in several patients with testicular DSD , palmoplantar hyperkeratosis and a predisposition to skin tumors (OMIM *609595) Rspo1 in mice results in testis development in chromosomal XX females RSPO1 predicted to result in an in-frame deletion of the first furin-like domain in R-spondin1 (p.Ile32_Ile95del) in a patient with 46,XX ovotesticular DSD (formerly “true hermaphroditism”) has provided further evidence for the critical role of this protein in humans One of the most significant advances in understanding ovary development came through the discovery of single-gene defects in Despite these advances, relatively little is known about the expression of R-spondin1 in early human ovary development or its downstream interactions with components of the Wnt signaling pathway. Here, we show upregulation of R-spondin1 in the human ovary at a critical time in development, augmentation of β-catenin signaling by RSPO1 in a human cell system, and nuclear localization of R-spondin1.CTNNB1 (ENSG00000168036), WNT4 (ENSG00000162552), and RSPO1 (ENSG00000169218) in human fetal gonads between 6–9 weeks post-conception showed significantly higher expression of RSPO1 in the developing ovary compared to testis, whereas no significant differences in the expression of CTNNB1 or WNT4 were seen  and from normal ovary (Ov) control. RT-PCR analysis showed no noticeable difference in β-catenin mRNA expression between these two tissues  and normal ovary (Ov) and testis (T) controls (WNT4 mRNA expression levels were markedly lower in the patient's ovotestis compared to ovary control. SOX9 mRNA (ENSG00000125398) expression was barely detectable both in wild-type ovary and in ovotestis, but strongly expressed in the testis control (Mutant (MT) R-spondin1 lacks the first furin-like domain and has been reported to be responsible for abnormal ovary development in a patient with 46,XX ovotesticular DSD  tissues . However tissues . Similarcontrols . WNT4 mR control .RSPO1 showed a modest but significant activation of the TOPFLASH reporter vector for the WT  and a complete lack of activity for the p.Ile32_Ile95del mutant plasmid (CTNNB1 (β-catenin) expression vector were studied  (The ability of wild-type (WT) and mutant  R-spondin1 to activate Wnt/β-catenin dependent targets was investigated using the TOPFLASH (TCF) reporter system. Transient gene expression assays of increasing concentrations of plasmids encoding WT or MT  plasmid . However.8 fold) .CTNNB1 vector expressing β-catenin and with increasing concentrations of WT or MT RSPO1  (ENSG00000107984), a potent inhibitor of the Wnt signaling pathway co-receptor LRP6 In order to investigate further the mechanism of interaction between RSPO1 and β-catenin, cells transfected with RSPO1/R-spondin1 and Plasmids encoding WT or MT RSPO1 (pRSPO1) and expression vectors for different tagged WT and MT proteins  were transiently transfected into several different cell lines . H295R cells expressing GFP-tagged WT RSPO1 showed that the protein was located mostly in the nucleus and partially in the cytoplasm . SimilarThe R-spondin family of proteins are a relatively recently identified group of furin-like domain containing factors that are thought to play an important role in tissue development and cellular differentiation. For example, alterations in RSPO4 activity have been reported to cause anonychia in humans Rspo1 gene has a sexually dimorphic expression pattern at specific developmental stages (e12.5–e15.5), with progressively higher expression in the ovary compared to the testis RSPO1 expression in the developing ovary plays an important role in opposing testis development pathways. In the present study, we provide evidence that a similar dimorphic pattern of RSPO1 gene expression occurs in the human during critical early stages of testis and ovary development (between 6–9 weeks post conception), confirming the likely role of this gene as a key component of the human gonad determination pathway.Studies in the developing mouse gonad have shown that the The mechanism through which R-spondins have their effects are not fully elucidated but it has been reported that R-spondin proteins act primarily through upregulation of β-catenin and canonical Wnt signaling pathways Rspo1 in XX mice results in the formation of testicular tissue in the ovary (ovotestis) and partial androgenization of female mice, with decreased β-catenin signaling and reduced expression of Wnt4Wnt4, but with no effect on Rspo1 expression. Taken together, these studies suggest that β-catenin is a component between R-spondin1 and Wnt4 WNT4 mRNA in the gonad tissue analyzed, showing in a biological system that these two factors are strikingly related to RSPO1. Further in vivo data from patients with RSPO1 mutations would be needed to confirm this finding, although to date reports of such cases are rare.Recent studies in the mouse have provided additional important insight into the specific actions of R-spondin1. For example, deletion of SOX9 expression in the ovotestis from our patient did not show any increase in SOX9 mRNA levels. This finding also supports mouse studies described above, as Sox9 mRNA was not increased following deletion of either Rspo1 or Ctnnb1/β-catenin in the developing ovary SOX9 directly, but influences it at the protein level. Unfortunately, it was not possible to undertake measurements of SOX9 protein in our patient sample. Nevertheless, it seems likely that increased SOX9 stability in the ovary following reduction in RSPO1 or β-catenin is a central mechanism leading to testis or ovotestis development within these tissues.Analysis of CTNNB1 and WNT4 mRNA are similar in testes and ovaries during these critical stages of early ovary development. Thus, R-spondin1 may act as a tissue (ovary) and time specific amplifier of β-catenin activity. By our functional experiments we have also shown that the mutant RSPO1 protein in our patient was not able to activate the TCF promoter, confirming in vitro an essential role of the furin domain for RSPO1 activity or stability.As β-catenin-mediated activation is fairly ubiquitous throughout cellular development and function, the R-spondin family of proteins may play an important role in mediating tissue-specific events. By using human cell-based assay systems, we have shown that R-spondin1 can act to augment β-catenin signaling in a dose-dependent manner. This effect could have a significant biological effect at times when relative dosage effects of sex-determining genes and proteins are important. It is emerging that gene dosage effects are crucial during early gonad development in humans and other species. Furthermore, we have provided the first evidence in humans to shown that expression levels of R-spondin proteins may also interact with members of the Wnt pathway in a synergistic manner. For example, it has been reported that human Rspo1 synergizes with Wnt3a to stabilize β-catenin and that this synergy occurs in the extracellular environment RSPO1, are actively upregulated at critical stages of development to oppose pathways that would otherwise lead to testis development, by several different mechanisms of action.Taken together, these findings confirm that R-spondin1 is a key regulator of ovary development in humans and that this factor may represent a tissue- and time-specific modulator of β-catenin signaling. Our results support the model that ovary-specific genes, such as Anonymized human embryonic/fetal material was obtained from the Medical Research Council-Wellcome Trust Human Developmental Biology Resource with written consent and ethical approval from the local Research Ethics Committee of University College London Hospitals and the Royal Free Hospital Research Ethics Committee (08/H0712/34). We obtained tissue samples for genetic and molecular studies after receiving informed consent from the patient, following approval from the Research Ethics Committee of the San Camillo-Forlanini Hospital.Human testes and ovaries (n = 3 each) from Carnegie stage (CS) 18–21 , Fetal stage 1 (F1) (8wpc) and F2 (9wpc) were obtained on ice and preserved in RNA later (Ambion). Fetal heart tissue (8wpc) was used as a control. RNA was extracted using the Trizol method and the concentration and ratio of absorbance at 260 nm to 280 nm (A260/A280 ratio) were measured using a NanoDrop ND-1000 Spectrophotometer . First-strand cDNA was synthesized using the SuperScript II Reverse Transcriptase (Invitrogen) and random primers according to the manufacturer's instructions. The amount of input RNA in each reaction was calculated to be 200 ng.CTNNB1, WNT4, and RSPO1 (Applied Biosystems). Amplification was performed in a total volume of 20 µl per reaction using TaqMan® Gene Expression Master Mix and a StepOne Real-Time PCR system (Applied Biosystems). Thermocycling conditions consisted of an initial step of 2 min at 50°C, denaturation of 10 min at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization and relative quantification of gene expression was performed according to the 2-ΔΔCt method Primers and TaqMan® probes were obtained for CTNNB1 (encoding β-catenin), SOX9 and WNT4 were amplified using gene-specific primers (sequences and conditions available on request). RT-PCR of GAPDH was used as a positive control.Total RNA was isolated from patient's gonadal tissue and from normal human ovary and testis using the Chomczynski and Sacchi extraction protocol Western blots to assess total β-catenin expression were performed using total proteins extracted from patient's gonadal tissue and normal human ovary as described previously, using a mouse monoclonal anti-β-catenin antibody  CTNNB1 (IMAGE Consortium CloneID 6151332).A DNA fragment encoding full-length wild-type (WT) R-spondin1 was amplified from normal ovary tissue. A corresponding DNA fragment encoding mutant  human R-spondin1 was amplified from the ovotestis of a patient with ovotesticular DSD 2 at 37°C (10% CO2 for NT2/D1). NCI-H295R human adrenal carcinoma cells (ATCC) were grown in DMEM/F12 1∶1 containing 2.5% Nu-Serum and ITS+ Premix (1X) (BD Biosciences).Human embryonic kidney 293T (HEK293T) cells , human embryonic kidney tsa201 cells Renilla luciferase (Promega) was used as an internal control of transfection efficiency. Luciferase assays were performed 24 hours after transfection using a FLUOstar Optima fluorescence microplate reader (BMG Labtech) and luciferase activity was normalized to Renilla co-expression. Results are shown as the mean ± SEM for triplicate samples of at least three independent experiments.Transient gene expression assays were performed using Lipofectamine2000 (Invitrogen) and a Dual-Luciferase Reporter Assay System (Promega) in accordance with the manufacturer's instructions. Cells (tsa201) were seeded in 96-well plates (TPP) 24 h prior to transfection. Varying amounts (0–100 ng/well) of either WT or MT pRSPO1 plasmids were transfected with or without varying amounts (0–5 ng/well) of β-catenin vector. The TOPFLASH reporter plasmid (100 ng/well), containing TCF-binding sites linked to luciferase, was used to assess target gene activation Recombinant mouse R-spondin1  (R&D Systems) was added to culture media 24 hours prior to luciferase assay and 48 hours prior to immunocytochemistry studies.Human recombinant DKK1 protein  (R&D Systems) was used to block LRP6 activity. This peptide was added to cultured media 2 hours prior to transfection or stimulation.Empty vector, WT and MT pRSPO1, p-RSPO1-GFP, or pRSPO1-HA expression vectors (0.8 µg/chamber) were transfected into several different cell lines  seeded in 8-chamber slides using Lipofectamine2000 (Invitrogen). After 24–48 hours, cells were fixed and permeabilized with TritonX-100 (0.3%) prior to immunohistochemistry using standard protocols. The following primary antibodies were used: rabbit polyclonal anti-HA (1∶25) (Sigma), human/mouse monoclonal anti-RSPO1 (1∶150-1∶200) (R&D Systems) and rabbit polyclonal α-C23 anti-nucleolin (1∶100) (Santa Cruz Biotechnology). Nuclear counter staining was performed with Vectashield containing DAPI (Vector Laboratories). Cells were visualized on a Zeiss Axioskop microscope and images captured using a ZeissAxiocam camera.HEK293T cells were transiently transfected with either WT or MT pRSPO1-HA expression vectors (0.8 µg/well) using Lipofectamine 2000 (Invitrogen). After 48–72 hours, cytoplasmic and nuclear protein extracts were prepared using a modified version of the Dignam protocol P values were calculated using GraphPad and the statistical analysis function in Microsoft Excel® (2007 version). Significance is expressed at *p≤0.05; **p≤0.01; ***p≤0.001.All values are expressed as mean ± SEM unless indicated otherwise. Comparison of means between multiple groups was performed using one-way analysis of variance (ANOVA) with Bonferroni corrections for multiple comparisons where significant differences were found. Comparison of means between two groups was undertaken using unpaired two-tailed Student's t-tests.


Lineage allocation of the marrow mesenchymal stem cells (MSCs) to osteoblasts and adipocytes is dependent on both Wnt signaling and PPARγ2 activity. Activation of PPARγ2, an essential regulator of energy metabolism and insulin sensitivity, stimulates adipocyte and suppresses osteoblast differentiation and bone formation, and correlates with decreased bone mass and increased fracture rate. In contrast, activation of Wnt signaling promotes osteoblast differentiation, augments bone accrual and reduces total body fat. This study examined the cross-talk between PPARγ2 and β-catenin, a key mediator of canonical Wnt signaling, on MSC lineage determination. Rosiglitazone-activated PPARγ2 induced rapid proteolytic degradation of β-catenin, which was prevented by either inhibiting glycogen synthase kinase 3 beta (GSK3β) activity, or blocking pro-adipocytic activity of PPARγ2 using selective antagonist GW9662 or mutation within PPARγ2 protein. Stabilization of β-catenin suppressed PPARγ2 pro-adipocytic but not anti-osteoblastic activity. Moreover, β-catenin stabilization decreased PPARγ2-mediated insulin signaling as measured by insulin receptor and FoxO1 gene expression, and protein levels of phosphorylated Akt (pAkt). Cellular knockdown of β-catenin with siRNA increased expression of adipocyte but did not affect osteoblast gene markers. Interestingly, the expression of Wnt10b was suppressed by anti-osteoblastic, but not by pro-adipocytic activity of PPARγ2. Moreover, β-catenin stabilization in the presence of activated PPARγ2 did not restore Wnt10b expression indicating a dominant role of PPARγ2 in negative regulation of pro-osteoblastic activity of Wnt signaling. In conclusion, β-catenin and PPARγ2 are in cross-talk which results in sequestration of pro-adipocytic and insulin sensitizing activity. The anti-osteoblastic activity of PPARγ2 is independent of this interaction. Regulation of marrow MSC fate toward adipocyte or osteoblast lineage involves multiple mechanisms including modulation of lineage-specific transcription factors PPARγ nuclear receptor is an essential regulator of energy metabolism and a key transcription factor for adipocyte differentiation In vitro studies suggest a role for PPARγ2 isoform in commitment of marrow MSCs to adipocytic lineage at the expense of osteoblastic lineage PPARγ2 expression and activity increases in marrow MSCs with aging and upon treatment with TZDs, and it correlates with decreased number of osteoblasts and decreased bone formation, and increased number of adipocytes and accumulation of fat in the bone marrow Osteoblast differentiation is regulated by a number of osteogenic pathways, including Wnt signaling β-Catenin-mediated Wnt signaling requires interaction with other transcriptional regulators. Besides TCF/LEF complex, β-catenin may interact with a number of transcription factors and nuclear receptors including PPARγ Differentiation of marrow MSCs towards osteoblasts relies on functional Wnt10b/β-catenin canonical signaling In this study, we demonstrate that PPARγ2 pro-adipocytic and pro-insulin signaling activities require degradation of β-catenin protein and that the degradation of β-catenin does not directly affect anti-osteoblastic activity of PPARγ2. This activity rather depends on Wnt10b, which is under negative control of PPARγ2 and under positive, PPARγ2-independent, control of β-catenin.ueous Non-Radioactive Cell Proliferation Assay (Promega). All other chemicals and reagents were purchased from Sigma-Aldrich.Specific reagents for this study were obtained from the following sources: MEM-α medium , DMEM medium and fetal bovine serum , G418 , protease inhibitor , phosphatase inhibitor , Opti-MEM, Lipofectamine, and Plus Reagent (Invitrogen), rosiglitazone , GW9662 . The following primary antibodies were used: anti-β-catenin , anti-β-actin , anti-Akt and anti-phospho-Akt  anti-PPARγ . The following secondary antibodies were used: goat anti-mouse IRDye 800CW and donkey anti-goat IRDye 600 , chicken anti-goat Alexa Fluor 594 and goat anti-mouse Alexa Fluor 488 , goat serum . β-Catenin-specific siRNA containing a mixture of four different 20–25 nt oligonucleotides was purchased from Santa Cruz Biotechnology. The following kits were used; BCA Protein Assay (Thermo Scientific), RNeasy Mini , DNase I (Invitrogen), iScript cDNA synthesis , QuikChange Site-Directed Mutagenesis , Power SYBR Green PCR Master Mix , Dual Luciferase Reporter Assay System , Cell Titer 96 AQLuciferase gene reporter constructs, TOP-Flash and FOP-Flash , were used to measure β-catenin transcriptional activity. The TOP-Flash construct contains three copies of the TCF/LEF sites, whereas FOP-Flash has mutated copies of TCF/LEF sites and is used as a control for measuring nonspecific activation of the reporter. β-Catenin expression plasmid constructed on a pSPORT6 vector backbone was purchased from Invitrogen. pEF-PPARγ2 expression construct, a kind gift from Dr. J. Gimble , was generated by cloning PPARγ2 cDNA into pEF-BOS expression vector downstream of the translation elongation factor 1α (EF-1α) promoter sequence, which permits the levels of ectopically expressed transcript to be within the physiological range  [39]. PP2.Murine marrow-derived U-33 cells represent a clonal cell line spontaneously immortalized in the long term bone marrow culture Cell lysate fractionation was performed as described previously U-33/γ2 cells were briefly washed with PBS buffer and permeabilized by incubating with ice cold methanol for 10 min. After washing, cells were blocked using 5% goat serum in 0.2% Triton-X for 1 h and incubated with mouse anti-β-catenin (1∶50) and/or goat anti-PPARγ2 (1∶50) diluted in 5% goat serum containing 0.1% Triton-X for 1 h at room temperature. To visualize β-catenin and PPARγ2 cells were incubated with either goat anti-mouse Alexa Fluor 488 or chicken anti-goat Alexa Fluor 594 respectively for 1 h at room temperature. As a negative control, cells were incubated with Alexa Fluor antibodies without prior incubation with primary antibodies. Finally, the cells were mounted using Prolong Gold anti-fade reagent with DAPI (Invitrogen). Images were taken within 24–48 h after immunostaining.Total RNA was extracted using RNeasy Mini kit. Its purity and concentration were determined using Agilent 2100 Bioanalyzer . After DNase treatment, 0.75µg of RNA was converted to cDNA using the iScript cDNA synthesis kit. The amount of cDNA corresponding to 7.5 ng of RNA was used for each reaction containing Power SYBR Green mix and was processed using StepOne Plus System . Relative gene expression was determined by the ΔΔ-Ct method using 18S RNA levels for normalization. Primers were designed using Primer Express 3.0 software (Applied Biosystems). All primers used in this study are listed in 5 cells) were transfected with 200 ng of β-catenin siRNA or nonspecific random siRNA as a negative control using Lipofectamine 2000. Seventy two hours after transfection, total RNA and protein were extracted and analyzed for β-catenin knockdown and expression of target genes.U-33/γ2 cells  activity was measured as previously described ueous Non-Radioactive Cell Proliferation Assay kit.For adipogenesis assay, U-33/γ2 cells were seeded in 6-well plate at the 1×10All experiments were performed in triplicates. Statistical analysis of results was conducted using one-way ANOVA and t-test, as applicable. All data showed represent means and standard deviation of the means (SD). Statistical significance was set to p<0.05.The U-33/γ2 cells, and their negative control U-33/c cells, represent a model of marrow MSC differentiation under control of PPARγ2 transcription factor Despite the late response of β-catenin gene expression, its protein levels were decreased much earlier after Rosi treatment . In cytoPresented results indicate that the PPARγ2 negative regulation of β-catenin protein levels involves two mechanisms; a rapid proteolytic degradation and a long-term suppression of β-catenin gene expression.Phosphorylation by glycogen synthase kinase 3β (GSK3β) targets β-catenin for proteosomal degradation. LiCl prevents β-catenin phosphorylation which includes inactivating autophosphorylation of GSK3β Consistent with β-catenin translocation to the nucleus, a protective effect of LiCl was also seen at the level of β-catenin transcriptional activity tested in luciferase gene reporter assay using TOP-Flash construct carrying β-catenin responsive TCF/LEF elements . As expeIn U-33/γ2 cells, activation of PPARγ2 with Rosi increases expression of adipocyte-specific genes, induces adipocyte formation, and simultaneously decreases the expression of osteoblast-specific gene markers and suppresses osteoblast phenotype Since PPARγ activation with anti-diabetic Rosi increases insulin signaling in adipocytes, we examined the effect of β-catenin stabilization on the expression of gene markers of this pathway. Upon Rosi treatment, both insulin receptor and FoxO1 gene expression increased respectively by 14- and 5-fold, however stabilization of β-catenin in the presence of activated PPARγ2 decreased this effect by 2-fold . FurtherIn contrast, β-catenin stabilization did not protect against the PPARγ2-mediated suppression of osteoblast phenotype. As shown in In order to assess a contribution of PPARγ2 pro-adipocytic activity to β-catenin stability, we inhibited Rosi-induced PPARγ2 activity with GW9662 selective antagonist previously shown to block adipogenesis induced by TZD treatment To test whether PPARγ2 anti-osteoblastic activity is dependent on the protein domain conferring the pro-adipocytic activity and β-catenin degradation we introduced previously reported mutation of PPARγ1 into PPARγ2 protein sequence 2-fold decrease in β-catenin protein levels .(TIF)Click here for additional data file.Figure S2GSK3 β inhibitor 6-bromoindirubin-3′-oxime (BIO) protects β-catenin from PPARγ2 mediated degradation and suppresses adipogenesis. U-33/γ2 cells were pre-treated with 5 µM BIO for 2 h followed by treatment with either 5 µM BIO or in combination of BIO with 1 µM Rosi for 24 h. Non-treated cells or treated with 1 µM Rosi alone were used as controls. A. Western blot analysis of cytoplasmic levels of β -catenin protein. β -actin was used as a loading control. Each lane was loaded with 30 µg of protein lysate. B. Oil Red O staining of lipids. Red: lipid droplets; Purple: cell cytoplasm. V – vehicle; R – Rosi; B – BIO; BR – BIO+Rosi.(TIF)Click here for additional data file.Figure S3Representative images of U-33/γ2 cells used for counting populations of β-catenin-positive cells and β-catenin-positive nuclei. Letter symbols indicate cell treatment; V-vehicle, R-rosiglitazone, LiCl-lithium chloride, LiCl+R-lithium chloride and rosiglitazone, GW-GW9662, GW+R-GW9662 and rosiglitazone. Images are shown in green channels of the RGB images, solid arrows indicate positive nuclei, open arrows indicate negative nuclei. Total number of cells was determined by counting DAPI-stained nuclei on RGB image. Results are presented in graphs on (TIF)Click here for additional data file.Table S1DNA primers used for a determination of gene expression in real time q-PCR.(DOC)Click here for additional data file.


Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive diseases of banana. Toxins produced by Foc have been proposed to play an important role during the pathogenic process. The objectives of this study were to investigate the contamination of banana with toxins produced by Foc, and to elucidate their role in pathogenesis.Fusarium wilt, caused by the fungal pathogen Foc representing races 1 and 4 were isolated from diseased bananas in five Chinese provinces. Two toxins were consistently associated with Foc, fusaric acid (FA) and beauvericin (BEA). Cytotoxicity of the two toxins on banana protoplast was determined using the Alamar Blue assay. The virulence of 20 Foc isolates was further tested by inoculating tissue culture banana plantlets, and the contents of toxins determined in banana roots, pseudostems and leaves. Virulence of Foc isolates correlated well with toxin deposition in the host plant. To determine the natural occurrence of the two toxins in banana plants with Fusarium wilt symptoms, samples were collected before harvest from the pseudostems, fruit and leaves from 10 Pisang Awak ‘Guangfen #1’ and 10 Cavendish ‘Brazilian’ plants. Fusaric acid and BEA were detected in all the tissues, including the fruits.Twenty isolates of Foc in banana. The toxins produced by Foc, and their levels of contamination of banana fruits, however, were too low to be of concern to human and animal health. Rather, these toxins appear to contribute to the pathogenicity of the fungus during infection of banana plants.The current study provides the first investigation of toxins produced by  Fusarium comprises a large number of plant-associated fungal species with the potential to cause economic damage and reduce the quality of agricultural crops. These include wilt pathogens such as F. oxysporum and F. solani, both which cause the eventual demise of their plant hosts, and mycotoxin-producers such as F. graminearum and F. verticillioides, which contaminate food and feed with secondary metabolites (mycotoxins) harmful to humans and animals. Some toxins produced by Fusarium species are important for the establishment of the fungus in host tissue, and are known as phytotoxins. Numerous mycotoxins and phytotoxins produced by Fusarium species have now been described, along with their potential to cause disease to plants and animals The genus Fusarium species is F. oxysporum, a soil-borne saprophyte with specificity to more than 150 different plant hosts formae speciales. Pathogenic F. oxysporum isolates infect their hosts through the roots, invade the xylem vessels and eventually cause a lethal wilting of the infected plant. Wilting is the result of the restrictive movement of water in the vascular bundles F. oxysporum can be contributed to toxic metabolites produced by the fungus. Toxins produced by F. oxysporum include beauvericin (BEA), the enniatines, fusaric acid (FA), moniliformin, naphthazarins and sambutoxin Fusarium species with health consequences to humans and animals include fumonisins, the trichothecenes  and zearalenone.One of the most economically important + pumping and causes K+ leaking. It also suppresses mitochondrial oxygen absorption and causes malic acid oxidation Phytotoxins such as FA cause pathological changes in the plant, including browning of vascular cells and plant necrosis Beauveria bassiana, which attacks insects Fusarium fujikuroi species complex F. oxysporum from corn formae speciales of F. oxysporumet al. F. oxysporum f. sp. melonis demonstrated that BEA is neither related to its pathogenicity, nor to the differential specificity of its races Beauvericin is produced by the entomopathogenic fungus Fusarium oxysporum f. sp. cubense (Foc), the causal agent of banana Fusarium wilt, is considered one of the most important pathogens of banana (Musa spp.) plants Foc may result in loss of food and income to millions of people. Furthermore, chlamydospores produced by the fungus will survive in infested soils for decades, making the continued production of bananas difficult Foc race present in banana fields, resistant varieties can be planted to continue production Foc race 1, but Cavendish bananas are resistant to this race and Foc race 2. Cavendish bananas, however, are susceptible to Foc race 4 isolates, which also attack all other banana varieties susceptible to Foc races 1 and 2 Foc can be differentiated from susceptible ones by treating the apical meristem with a fungal crude filtrate. Xu et al. Foc which caused Fusarium wilt of banana. However, Morpurgo et al. in vivo and in vitro behavior of banana shoot tip cultures treated with FA and fungal crude filtrates, and those inoculated with Foc. Their results, therefore, suggested that the use of crude filtrate and the non-host specific toxin FA in a screening program for selecting resistant banana genotypes is not necessarily accurate. More recently, however, BEA was associated with Foc isolates collected from apple banana in the Americas Matsumoto et al. Foc to banana production, most studies on the pathogen have focused on its diversity, geographical distribution, symptomology, epidemiology and management Foc-infected banana. The first objective of this study, thus, was to confirm the range of toxins produced by Foc. A second objective was to correlate toxin production in banana plants to fungal virulence. Such information may provide new information to elucidate pathogenicity of Foc to banana plants. The final objective was to determine the concentration of Foc toxins in banana plants. This may signify whether infection of banana plants with Foc presents a food safety risk.Due to the devastating effect of Foc isolates obtained from diseased banana pseudostems were used for studies on secondary metabolite production and no specific permits were required for the described field studies. These isolates were all collected from Cavendish cv ‘Brazilian’ (AAA) and Pisang Awak cv ‘Guangfen #1’ (ABB) banana plants with Fusarium wilt symptoms in Guangdong, Hainan, Fujian, Yunnan and Guangxi Provinces in China. The fungal cultures were all identified as F. oxysporum based on cultural appearance, the morphology of macroconidia, the production of microconidia in false head on monophialides, and the presence of single or pairs of chlamydospores. They were confirmed as the banana Fusarium wilt pathogen by completing pathogenicity tests, and divided into vegetative compatibility groups (VCGs) according to heterokaryon formation with known Foc VCG testers . The Foc isolates included seven Foc race 1 isolates and 33 Foc race 4 isolates  (Forty ed data)  and 2. AFusarium oxysporum f. sp. cubense isolates were inoculated onto potato dextrose agar (PDA) plates and cultured in the dark at 28°C. After 15 days, the mycelia were harvested, sonicated and the metabolites extracted with methanol. The extracts were analyzed by high performance liquid chromatography - tandem mass spectrometry  F. oxysporum and other phytopathogenic Fusarium species Foc to banana was first determined on cell protoplast. Banana protoplasts were obtained from embryogenic cell suspensions (ECS) 7 protoplasts/ml packed cell volume (PCV), according to the method of Xiao et al. 2.The effect of FA and BEA produced by 4 cells in 100 µl of test solution. The results were calculated with a multifunctional microplate reader after 6, 12, 24, 36, 48 and 72 hours. Damage to cells was also observed under a laser scanning confocal microscope  with a wavelength of 488 nm. Each reaction was repeated three times, and the statistical software used was SAS 8.0 (P<0.05).Cytotoxicity of FA and BEA to banana protoplasts was tested using the Alamar Blue assay according to manufacturers’ recommendations . The FA and BEA were first dissolved in 0.5% methanol to prepare a stock solution of 1 mM, which was then serially diluted to concentrations of 200, 50, 10 and 1 µM using pre-plasmolysis buffer. The control treatment included equal amounts of methanol and pre-plasmolysis buffer. The reactions were performed in 96-well plates, each well containing 2×10The effect of FA and BEA was further tested on banana pseudostems and tissue culture plantlets. The toxins were dissolved in 0.5% methanol to prepare a stock solution of 1 mM, which was then serially diluted to obtain concentrations of 500, 300, 200 and 100 µM. For treatment, the lower pseudostems of 6-week-old Cavendish cv ‘Brazilian’ (AAA) banana tissue culture plantlets were surface-disinfected with ethanol and dried. The base of each pseudostem was treated with 200 µl of FA and BEA solutions at different concentrations. For controls, the pseudostems were treated with the same amount of distilled water and 0.5% methanol, respectively. Treated pseudostems were placed on a layer of Whatman filter paper which was soaked in sterilized water in a Petri dish (90-cm diameter). The lid of the Petri dish was wrapped with parafilm to maintain the moisture. In addition, ‘Brazilian’ banana tissue-culture plantlets were transplanted into pre-plasmolysis buffer in test tubes containing 1, 5, 10, 20, 50, 100, 200, 300 and 500 µM FA and BEA. Both the Petri dishes and the treated pseudostems or tissue culture plantlets were incubated in a growth chamber with a photoperiod of 12 hours of light per day for 10 days at 3000 lux at 25°C. Each treatment was repeated three times, and statistically compared using SAS 8.0 software.Foc race 1 isolates were inoculated onto the ‘GuangFen #1’ plantlets, while Foc race 4 isolates were inoculated onto ‘Brazilian’ plantlets. The planting medium  was first sterilized in an autoclave, and the plants was inoculated with a spore suspension at a concentration of 105 conidia/g soil, as described by Sun and Su Healthy tissue-culture plantlets of ‘Brazilian’ (AAA) and Pisang Awak cv ‘Guangfen #1’ (ABB) bananas at the 5- to 6-leaf stage were used for pathogenicity studies. in planta mycotoxin contamination. The fruit, pseudostem and leaves of each plant were sampled at the fruit ripening stage, FA and BEA extracted with methanol Ten ‘Guangfen #1’ and ten ‘Brazilian’ banana plants that developed Fusarium wilt symptoms under field conditions were selected for analyzing oC, atomization gas (N2) pressure 0.6 Mpa, auxiliary gas (N2) pressure 0.6 MPa; curtain gas (N2) pressure 0.4 MPa, focusing voltage (FP) 375 ev; collision gas (N2) pressure 0.4 MPa; NEB 8, CUR 10, and CAD 8. In order to quantify FA in the samples, the standard curve was obtained with solutions at 0.5, 1, 2, 5, 10, 20, 50, 100 µg/ml. If the concentration of FA in the sample was higher or the estimated curve was larger than the linearity range of the standard curve, the concentration of the sample was diluted with methanol.The content of FA and BEA was determined using LC/MS/MS analysis. For FA analysis, mobile phase A consisted of acetonitrile:water:formic acid = 5∶95:0.1 (v:v:v), and mobile phase B of acetonitrile:water:formic acid = 95∶5:0.1 (v:v:v), with a flow rate of 0.2 ml/min using a gradient elution method. The column temperature was set at room temperature. Mass spectrometry employed an electron spray source in the positive ion mode 5.5 kv, and the scan mode was set up as selected reaction monitoring (SRM) method. During quantitative analysis, the ion reaction for FA was 180.1/134.2, for phenacetin it was 180.1/138.3 with the following parameters: scan time 0.1 s, heating temperature 350−1 dry weight.For BEA analysis, LC/MS/MS was performed using a Finnigan LCQ Deca XP system  comprising a Surveyor liquid chromatograph, a Surveyor autosampler and an LCQ Deca XP quadrupole ion trap mass spectrometer equipped with an ESI source. The HPLC separation was performed using an ODS HYPERSIL column . Elution was carried out using a mobile phase comprising water (15%) and methanol (85%) at a flow rate of 0.15 ml/min. LC/MS/MS conditions were as follows: spray voltage 3 kv, sheath gas flow 15 (arbitrary units). Relative collision energy was set at 60%. The BEA standard curve was obtained with solutions at 6, 15, 30, 60, 150, 300, 600 ng/ml. All analyses were performed in triplicates, and the mean values are reported here. Results were expressed at µg gFoc isolates selected  F. oxysporum f.sp. tuberosi (potato) F. oxysporum f. sp. lycopersici (tomato) Foc isolates. The damage of banana protoplasts, root and pseudostems caused by Foc toxins increased cell membrane permeability and induced apoptosis F. oxysporumformae speciales of F. oxysporumFoc race 1 and race 4 isolates attacking Pisang Awak and Cavendish bananas, respectively.A positive relationship has previously been reported between FA content and Fusarium oxysporum f. sp. cubense can be isolated from all banana tissue, but is difficult to find in the upper stalk. It has also never been isolated from the fruit. In fact, both Foc race 1 and 4 isolates died when they were inoculated onto a medium made of banana pulp . This situation appears to be somewhat different for melon, where BEA was detected inside the fruit of two cultivars after F. oxysporum f. sp. melonis was applied to the fruit surface Foc. The translocation of mycotoxins in plants is not an unusual event, and has been reported in corn and wheat Foc in banana plants. According to toxicological test in mice, the lethal concentration LC50 of FA is 80 mg/kg 5 kg bananas contaminated with FA at a concentration of 12.13–52.34 ng/g to be killed. Although the toxicity of FA may increase when combined with other mycotoxins Fusaric acid and BEA is not considered as mycotoxins with significant health consequences to humans and animals, and food and feed contaminated with these metabolites are therefore not regulated in any country. Still, both FA Foc isolates proved to be more pronounced in vitro than in planta. This could be the result of sample collection, as toxins might be concentrated in some affected tissue and very low in other plant parts, which may result in their concentration being profoundly diluted in collected material. It is, however, more likely that the differences is attributed to very different environments; one in which the fungus is artificially cultured, and the other where it has to overcome plant structural and chemical barriers. More interesting is the significantly higher production of FA than BEA in plants. One can speculate the reason for this to be that (1) BEA is not stable, especially inside the banana plant where enzymes may catalyze its conversion to other compound, or that (2) the environment inside the plants is not favorable for BEA synthesis, and suppressed the expression of related genes. Yet, FA and BEA are simultaneously produced by Foc in banana, and it is important that their additive and synergistic effects in the pathogenic process be further investigated in future.Mycotoxin production by


This suggests that cadherins play a crucial role in β-catenin-dependent transcription.The Wnt/β-catenin signalling pathway shares a key component, β-catenin, with the cadherin-based adhesion system. The signalling function of β-catenin is conferred by a soluble cytoplasmic pool that is unstable in the absence of a Wnt signal, whilst the adhesion function is based on a cadherin-bound, stable pool at the membrane. The cadherin complex is dynamic, allowing for cell-cell rearrangements such as epithelial-mesenchymal transition (EMT), where the complex turns over through internalisation. Potential interplay between the two pools remains poorly understood, but cadherins are generally considered negative regulators of Wnt signalling because they sequester cytoplasmic β-catenin. Here we explore how cellular changes at EMT affect the signalling capacity of β-catenin using two models of EMT: hepatocyte growth factor (HGF) treatment of MDCK cells, and gastrulation in embryonic development. We show that EMT not only provides a pool of signalling-competent β-catenin following internalisation of cadherin, but also significantly facilitates activation of the Wnt pathway in response to both Wnt signals and exogenous β-catenin. We further demonstrate that availability of β-catenin in the cytoplasm does not necessarily correlate with Wnt/β-catenin pathway activity, since blocking endocytosis or depleting endogenous cadherin abolishes pathway activation despite the presence of β-catenin in the cytoplasm. Lastly we present data suggesting that cadherins are required for augmented activation of the Wnt/β-catenin pathway  During embryogenesis and in adulthood, cells undergo dynamic morphological rearrangements, including changes in cell-cell adhesion, which are essential for driving both morphogenetic processes, such as gastrulation, and differentiation into distinct tissues. These cellular changes critically depend upon precise interactions between neighbouring cells and tight regulation of gene expression; dysregulation of either may lead to perturbed cell proliferation, adhesion, migration or differentiation, all of which are hallmarks of diseases such as cancer.de novo synthesised, cadherin-free form of β-catenin to enter the nucleus and, together with LEF/TCF, activate Wnt target genes The cadherin/catenin-based adhesion system is the major means by which cells adhere to one another. β-catenin, a central structural component of this adhesion complex, also acts as a transcriptional co-activator in the Wnt signalling pathway, a pathway used reiteratively during development to control cell fate decisions and one implicated in cancer in many tissues. The current, and somewhat uncontested, understanding of this pathway is that Wnt receptor activation inhibits the degradation of newly synthesised β-catenin, allowing for a Wnt3a or the Wnt-regulated mesodermal transcription factor Tbx6, these delaminated cells form additional neural tubes instead of somites The concept of ligand-independent Wnt signalling is further suggested by the fact that in some developmental situations there appears to be no obligate correlation between Wnt ligand expression and Wnt pathway activation. This is the case for migrating neural crest cells; despite not expressing any known Wnt ligand, the Wnt pathway is active in these cells in vivo events could, therefore, be explained by an alternative phenomenon: differential sensitivity to Wnt signals between epithelial and mesenchymal cells. Compared to epithelial cells, mesenchymal cells generally exhibit a much less regimented structure and weaker intercellular adhesion. Thus, it is possible that the cytoarchitectural changes that occur at EMT render the mesenchymally transformed cells more sensitive to Wnt signals originating from neighbouring tissue, the Wnt ligand-expressing ectoderm in the cases of migrating neural crest and mesodermal cells.Importantly, both of these events see cells undergoing epithelial-mesenchymal transition (EMT), a process involving dissociation of adherens junctions and changes in cell morphology. The above To gain more insight into the relationship between the two functions of β-catenin, and to explore how cellular changes at EMT affect the signalling capacity of β-catenin we used two models of EMT: hepatocyte growth factor (HGF) treatment of MDCK cells, and gastrulation in embryonic development. We find that cadherins do not merely provide a pool of β-catenin capable of feeding into the Wnt pathway, but rather play a positive role in rendering β-catenin competent in signalling.For studying the Wnt signalling pathway, HEK293 cells are widely used as they are very responsive to exogenous Wnt ligands and transfected β-catenin, as judged by activation of the established β-catenin/LEF/TCF reporter TOPflash . In contde novo β-catenin synthesis, but rather to a possible movement of pre-existing β-catenin from the membrane to the cytosol. We did not detect a reduction in β-catenin in membrane fraction after HGF treatment, presumably due to the relatively large amount in the membrane fraction.In MDCK cells, β-catenin is predominantly at the membrane, where it colocalises with E-cadherin . TreatinTo test whether the HGF-induced EMT causes activation of Wnt target genes, we looked at the expression levels of both the potential endogenous target MMP-13 To verify the shift of β-catenin from the membrane to the cytoplasm, we examined the role of endocytosis. E-cadherin undergoes a dynamic turnover; it is continuously internalised in a Dynamin- and Rab5-dependent manner, then is either recycled to the cell surface or degraded β-catenin DNA, exogenous β-catenin is found in the cytoplasm in cells treated with HGF, regardless of absence or presence of dynasore  cadherins maintain a pool of β-catenin that can be made available for signal transduction upon EMT; (2) mesenchymally transformed cells are highly responsive to Wnt ligands and overexpressed β-catenin in the activation of the Wnt pathway; (3) the internalisation process, presumably that of the cadherin complex, is a prerequisite for the augmented activation upon EMT; and (4) cadherins are required for and play an instructive role in providing β-catenin for Wnt signalling, such that binding of β-catenin to cadherins renders β-catenin competent in the Wnt pathway.in vitro data to explain the potential in vivo phenomenon of ligand-independent Wnt pathway activation, we have found that conversion of epithelial cells to a mesenchymal morphology activates the Wnt pathway in two ways. Firstly, as an immediate response, the conversion is associated with an increase of β-catenin in the cytosol, where it then becomes available to activate target genes, which we show to be dependent on endocytosis and independent of protein synthesis. Secondly, we show that the cellular changes that occur at EMT augment Wnt pathway readout by exogenous Wnt ligand and transfected β-catenin, and that this augmentation also requires intact endocytic processes. Moreover, we also find that cadherins play a positive role in β-catenin-dependent transcription: it is crucial for β-catenin to bind cadherin in order to activate downstream genes; β-catenin with reduced affinity for E-cadherin (the Y654E mutant) shows impaired activation of transcription, while loss of E-cadherin reduces Wnt transcriptional readout by both Wnt3a ligand and transfected β-catenin. Requirement of cadherins is further verified in an in vivo context, where knockout of N-cadherin leads to reduced Wnt pathway activity in ingressing cells during gastrulation.In an effort to obtain in vitro causes an increase of cytosolic β-catenin in an ‘active’ form We have shown that cells with distinct epithelial features are refractory to exogenous Wnt signals, in comparison to mesenchymal-like cells, but that their conversion to mesenchyme by HGF greatly facilitates the utilisation of β-catenin for signalling. This stimulation of canonical β-catenin signals by HGF, and the subsequent increase in transcriptional readout at the immediate-early stage, appears to be a result of movement of pre-existing β-catenin from the membrane to the cytoplasm, since it occurs independently of protein synthesis. This supports previous studies that suggest that β-catenin from the membrane can enter the nucleus and be used for transcription In addition to describing the mobilisation and transcriptional utilisation of β-catenin from the membrane, we demonstrate another fundamental cell biological phenomenon: that the cellular alterations occurring at EMT augment Wnt pathway readout in response to exogenous Wnt ligand and transfected β-catenin. The result that transfected β-catenin shows high transcriptional activity in cells that have undergone EMT, together with the results that both inhibition of internalisation and depletion of endogenous cadherin abolish the augmentation despite the presence of β-catenin in the cytoplasm, implies control at the subcellular, rather than extracellular, level. The increase in E-cadherin internalisation at EMT It might be tempting to suggest that HGF potentiates transcriptional activity of β-catenin by increasing its stability. HGF does indeed increase the amount of stable β-catenin in the cytosol, as detected by ABC immunoblotting, and this is reflected in the augmented activity of TOPflash . HoweverXenopus, Drosophila and cell culture models The result that cadherins are required for activation of the Wnt pathway, both in MDCK  and HEK2Importantly, however, it has been shown more recently that whilst knockdown of E-cadherin augments β-catenin-dependent transcription in colon cancer cells in which the Wnt pathway is active, it has no effect in nontransformed keratinocytes that do not display Wnt signalling in vitro and in vivo contexts  by outcompeting β-catenin's alternative binding partner, the Axin/APC degradation complex It has been shown that newly synthesised β-catenin is bound to E-cadherin in the ER and that this coupling facilitates efficient exit of the complex from the ER and its subsequent trafficking to the basolateral membrane in vitro, with the lineage decision depending on the surface area of the substrate on which the cells are allowed to grow Drosophila embryos, mechanical deformation during morphogenetic movement induces specific gene expression There are a number of examples where cell shape affects pathway activation and, in turn, gene regulation. For example, mesenchymal stem cells differentiate into either adipocytes or osteoblasts HEK293T (ATCC) and MDCK cells TOPflash reporter (Upstate) and Renilla luciferase (Promega), together with control or β-catenin DNA where necessary, were transfected using Polyfect (Qiagen) following the manufacturer's protocol. Cells were lysed 2 days after transfection and processed for the Dual Luciferase Assay System (Promega). All data were triplicate and analysed using student's t-test. For N-cadherin siRNA, lipofectamine 2000 (Invitrogen) was used following the manufacturer's protocol. Control and N-cadherin siRNA were obtained from Santa Cruz .2, with proteinase inhibitor cocktail (Roche) and 1 mM AEBSF) with a cell scraper. The extract was dounce homogenised and spun at 15,000 g for 45 minutes at 4°C, and the supernatant was collected as the cytosolic fraction. The pellet was resuspended in 100 µl lysis buffer  and 1 mM AEBSF). After keeping on ice for 10 minutes, it was spun at 10,000 g for 10 minutes at 4°C, and the supernatant were collected as the membrane fraction. Both fractions were incubated with Laemmli sample buffer containing 5% β-mercaptoethanol at 100°C for 5 minutes before loading onto the gel. Antibodies used for Westerns are; anti-β-catenin (Sigma), anti-active-β-catenin , anti-E-cadherin (BD Bioscience), anti-β-tubulin (Sigma), anti-transferrin receptor (Santa Cruz), anti-flag (Sigma) and anti-myc (Sigma). A431 cell lysate as a positive control for anti-active-β-catenin antibody was provided by Millipore.To collect cytosolic and membrane fractions, cells were grown in 35 mm dishes and were collected with 100 µl cold hypotonic buffer , anti-E-cadherin (BD Biosciences), anti-transferrin receptor (Invitrogen), anti-Flag (Santa Cruz).Cells grown on coverslips were fixed with 3% paraformaldehyde in PBS for 30 minutes at 4°C, followed by incubation in 50 mM NHT against GAPDH using student's t-test.MDCK cells were transfected with TOPflash reporter DNA. On the next day, cells were split into small dishes and treated with HGF and/or cycloheximide for 6 hours. RNA was collected using Trizol (Invitrogen) following manufacturer's protocol. Additional DNase treatment was performed to eliminate any contamination of DNA. After cDNA synthesis, PCR reaction was performed using Express SYBR GreenER (Invitrogen) on ABI 7500 (Applied Biosystems). Data of triplicate average Ct were normalised with that of GAPDH and then further converted to fold difference relative to no-HGF, following manufacture's protocol (Applied Biosystems). P-values were calculated using ΔCN-cadherin flox mice with LoxP sites at the regions flanking the exon 1 of N-cadherin


The development of traumatic stress studies during the past decades has much profited from professionals from the United States and from Europe. However, these professional societies, the International Society for Traumatic Stress Studies and the European Society for Traumatic Stress Studies (ESTSS) still struggle to find an equal common pathway. This is a personal retrospective view of Berthold Gersons, past president of ESTSS on behalf of the 20th anniversary of ESTSS. The celebration of 20 years of European Society for Traumatic Stress Studies (ESTSS) brings us back to 1993 when ESTSS was formally launched as the European Society for Traumatic Stress Studies in Bergen, Norway during the Third European Conference on Traumatic Stress. However, the First European Conference was already held in 1988 in Lincoln, UK. The roots of ESTSS started 25 years ago! (I)STSS was “born” in 1985 with Charles Figley as its first president Bloom, . On bothWybrand Op den Velde, psychiatrist in Amsterdam who held monthly lectures at his hospital in Amsterdam and who has been a pioneer on World War II studies in The Netherlands; Wim Wolters, child psychologist and my colleague professor at Utrecht University; and Bessel van der Kolk who was born in The Netherlands but became a psychiatrist in the United States, lives in Boston and is a pioneer on traumatic stress studies. I myself was trained as a psychiatrist and psycho-analyst in Amsterdam, but until 1990 was mainly active in the field of community psychiatry and prevention. Through my encounter with police officers in Amsterdam who developed PTSD after shooting incidents, I became interested in traumatic stress, in treatment of PTSD and in prevention STSS was in 1989 at the Fifth Annual Meeting of the Society for Traumatic Stress Studies (STSS) in San Francisco. I became familiar with STSS through three Dutch colleagues: Gersons, . I will Gersons, . The meeIn 1989, I was also asked by Charles Figley accompanied by Shad Mashed to organize the First World Conference on Traumatic Stress, to be held in Amsterdam in 1992 for STSS. Unaware of the competition between STSS and the pre-ESTSS group headed by Walter de Loos, I helped with people like Charles Figley and Yael Danieli who wanted to make their American organization an international one. Together with Jos Weerts and Carl Steinmetz, we organized a five-day conference with 700 participants from all over the world and with highlights like a pioneer panel with Leon Eitinger, Jan Bastiaans, Henry Krystal, and Lawrence Kolb, with the Dutch Queen at the opening ceremony and a reception with the famous painting, ‘Night Watch’ of Rembrandt in the background. I proposed to the STSS board to put an “I” in their name, to make it clear they wanted to be a real international society. However, because of the still hybrid character of ISTSS, the wish to be a serious international organization has been hampered always because in reality 80% of its members are from the United States. Also, after this first world conference in June 1992, their annual meeting was held on American soil in November of that year in Los Angeles. I was a member of the ISTSS board from 1996 to 1999 and I have been member of many program committees of ISTSS meetings and a two-times member of the editorial board of JTS. But ISTSS never became really an international organization. Board meetings were always dominated by topics important for the United States. Up until 1993, ISTSS followed a strategy to become recognized as an international umbrella organization by affiliating only Traumatic Stress Societies overlooking continents, such as the Australasian STSS and the ESTSS. After the Gulf War in 1993, ISTSS dropped this strategy by affiliating with the Kuwaiti SSTS. When the new German Speaking STSS (DEGPT) in 1998 was founded on their request, they were affiliated directly without asking for approval of the ESTSS.I was not involved with the 1988 Lincoln conference but in all other biannual ESTSS conferencesESTSS meetings differ so much in these aspects. Mostly, there is one conference venue and many hotels in the neighborhood. The meetings often start with live music from the country, a welcome from the minister or a local representative of the city council, the patronage of someone from the royalty or the president. Receptions are often in beautiful old town halls with much history, far away from anonymous hotels, with speeches from the city's government.Of course, Europe is called the “old” continent where history is everywhere and where local culture, food, language, and pride but also much suffering are felt present. Roderick Orner also started a series of very successful local ESTSS meetings on early interventions. At the same time, the ESTSS Board could convene in those places where the ESTSS workshops were held.However, this was more the “romantic” side of ESTSS. When I joined the board in 1999, it was Bas Schreuder who went to the notary to get official bylaws for ESTSS and to also start a small professional secretariat at his mental health organization Centrum 45 in the Netherlands. When Dean Ajdukovic took over the presidency from Bas, I became treasurer of a society that often did not receive any money from successful conferences and with a decreasing membership. There were no elections and the board was only renewed by cooptation. Under my presidency, we devised a strategic plan to change ESTSS into a large umbrella organization with local STSS in the different European countries. The first elections were held and candidates needed to present themselves with a bio and an idea on which contribution they wanted to make to ESTSS. Learning from ISTSS that a one-year presidency term is too short to consolidate developments, we chose a 4-year period, 1 year as president-elect, 2 years as president and 1 year as past-president. The presidents after me like Jon Bisson and Miranda Olff have implemented this strategy very successfully (see Bisson, ESTSS and ISTSS were born in the same decade but with a different background and geopolitical orientation. They are also culturally different, i.e., ISTSS wants to be as scientific as possible while ESTSS acknowledge the value of narratives, oral histories, and not yet evidence-based practices. ESTSS biannual meetings are more visited by people from all over the world as the ISTSS meetings. This rivalry between ESTSS and ISTSS will continue and is to some extent productive. However, I am longing for a better balance in which ISTSS acknowledges their American membership and perhaps a new international umbrella can come into existence.


Chronic activation of Wnt/ß-catenin signaling is found in a variety of human malignancies including melanoma, colorectal and hepatocellular carcinomas. Interestingly, expression of the HCMV-encoded chemokine receptor US28 in intestinal epithelial cells promotes intestinal neoplasia in transgenic mice, which is associated with increased nuclear accumulation of ß-catenin. In this study we show that this viral receptor constitutively activates ß-catenin and enhances ß-catenin-dependent transcription. Our data illustrate that this viral receptor does not activate ß-catenin via the classical Wnt/Frizzled signaling pathway. Analysis of US28 mediated signaling indicates the involvement of the Rho-Rho kinase (ROCK) pathway in the activation of ß-catenin. Moreover, cells infected with HCMV show significant increases in ß-catenin stabilization and signaling, which is mediated to a large extent by expression of US28. The modulation of the ß-catenin signal transduction pathway by a viral chemokine receptor provides alternative regulation of this pathway, with potential relevance for the development of colon cancer and virus-associated diseases. US28 is one of four GPCRs encoded by the HCMV In this study we show that the viral chemokine receptor US28 positively modulates the ß-catenin pathway via a non-conventional novel pathway, involving Rho kinase.129A mutant) were kept under the selective pressure of neomycin (400 µg/ml) (Sigma) to ensure homogenous receptor expression. Transient transfections of HEK293T cells were performed with the polyelthyleneimine (PEI) method Human HEK293T, human glioblastoma U373-MG, human foreskin fibroblasts (HFF) and NIH-3T3 mouse fibroblast cells were all obtained from ATCC, and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (PAA Laboratories), supplemented with penicillin (50 IU/ml), streptomycin (50 µg/ml) (PAA Laboratories) and 10% fetal bovine serum (FBS) (PAA Laboratories), heat-inactivated FBS and bovine serum (Gibco) respectively. NIH-3T3-stable cell lines  and 25 ng of US28 receptor DNA (wild type or G-protein-uncoupled mutant R129A) unless indicated differently and 25 ng of DNA encoding G-proteins (RGS2 and Lsc-RGS G protein scavengers were kindly provided by Dr. B. Moepps). Comparable TOPflash reportergene transfection protocols were used for U373-MG and NIH-3T3 cells, respectively. Total DNA amounts were kept constant by addition of empty vector. Inhibitors Y27632  were incubated overnight and added directly after transfection. 200 ng/ml human recombinant Wnt3a  was incubated overnight to activate the canonical Wnt signaling pathway. Luciferase activity was measured 24 h post transfection  with a Victor2 multilabel plate reader from (PerkinElmer Life Sciences). Statistical analyses, * or ** indicating p<0.05 or p<0.001, using Anova and Bonferroni post test 95% confidence interval.10Human Foreskin Fibroblasts (HFF) infected at a multiplicity of infection (MOI) of 1 on IBIDI slides with the TB40wt and TB40-ΔUS28 strains, respectively. Anti-IEA (Milipore) and anti-non-phospho-ß-catenin antibodies  were used to visualize IEA and activated ß-catenin. After transfection (24 h) of the TOPflash reporter gene in U373-MG cells different HCMV Titan strains (WT or -ΔUS28) were used to infect U373-MG at an MOI of 2. Multiple viral stocks  were assayed in triplicate. The rate of infectivity was controlled by back titration and IEA staining on parallel clear plates. 48 h post-infection luciferase activity was measured.Stably transfected NIH-3T3 cells  were analyzed for radiolabelled chemokine binding and inositol phosphate accumulation as previously described 129A) were lysed in radioimmunoprecipitation assay buffer supplemented with α-Complete Protease Inhibitor Cocktail (Hoffmann-la Roche), 1 mM PMSF, 1 mM NaVO4 and 1 mM NaF. Samples were normalized using the BCA total protein determination kit . Antibodies were used for detection of active ß-catenin , total ß-catenin (BD Transduction Laboratories), mouse monoclonal ß-actin expression (Sigma), Gαq (Santa-Cruz) and P-LRP6 (Ser1490) .Biorad minigel and electroblot systems (Biorad) were used to perform SDS-PAGE and subsequent protein transfer onto 0.45 µm nitrocellulose or PVDF membranes. After an overnight serum starvation in medium containing 0.5% bovine serum, NIH-3T3 stable cell lines  in these studies. US28-WT expressing NIH-3T3 cells displayed [125I]-CCL5 binding and increased inositol phosphate production compared to mock-transfected cells (129A) receptor displayed [125I]-CCL5 binding but no constitutive signaling (129A expressing cells (129A) did not display ß-catenin activation. Also in HEK293T cells, increasing expression of US28 resulted in dose-dependent activation of the ß-catenin signaling pathway as demonstrated using the TOPflash reporter gene . In line with this notion, activation of TOPflash activity through Wnt3a and US28 were additive  (data not shown). Co-expression of Gαq enhanced US28-mediated TOPflash reporter gene activation, while knocking down Gαq with shGαq resulted in over 50% inhibition of US28-mediated TOPflash activation , but not wild type Gα13, resulted in enhanced TOPflash activation in mock cells . These data clearly indicate a role for US28 in regulation of ß-catenin signaling during HMCV-infection.The HCMV Titan 2B strain (referred to as WT) We have demonstrated that HCMV partly through expression of the constitutively active chemokine receptor US28 induces ß-catenin signaling upon infection. Indeed, mounting evidence links viral infection to ß-catenin hyperactivation. For instance, the Epstein-Barr virus (EBV) activates ß-catenin in latently infected B lymphocytes Interestingly, expression of the HCMV-encoded chemokine receptor US28 in intestinal epithelial cells promotes intestinal neoplasia in transgenic mice q and Gα12/13 proteins is essential for the observed activation of ß-catenin signaling. Overexpression, scavenging and/or downmodulation of either G protein greatly affect US28 mediated ß-catenin signaling. The reported ligand-independent, constitutive, activity displayed by US28 is primarily exerted through activation of Gαq proteins q proteins and RhoA, the small G protein that is activated by Gαq proteins through RhoGEF 12/13 and RhoA q and Gα12/13 in US28 mediated ß-catenin signaling and reported coupling of US28 to RhoA we postulated a role for its downstream target Rho kinase (ROCK) in US28 mediated activation of ß-catenin. Inhibition of ROCK, with the specific inhibitor Y27632, substantiates a role for the Rho-ROCK axis in the US28 induced activation of the ß-catenin pathway. Exposure of US28 expressing cells to the chemokine CCL5 result in further increases in ß-catenin signaling, infering involvement of Gα12/13 proteins which is in line with previous studies indicating the involvement of these G proteins in US28-mediated responses 12/13 and Gαq proteins converging at RhoA/ROCK . Increases in ß-catenin nuclear localization were also reported upon infection of murine CMV Of importance is that significant increases of ß-catenin stabilization and signaling are observed in HCMV-infected HFFs and glioblastoma cells. This increase in ß-catenin signaling upon HCMV infection is mediated to a large extent by expression of US28, as shown using the deletion mutant HCMV-?US28. Besides US28, other CMV encoded proteins, including another viral GPCR UL33, also contribute to the observed increase in β-catenin signalling upon HCMV infection Click here for additional data file.Figure S2Back titration of wild-type HCMV and HCMV ΔUS28. The levels of infection were assessed by staining for Immediate Early (IEA). The amount of IEA+ cells is not significantly different between the different viral strains. This backtitration was performed on the samples that were used for the analysis shown in (EPS)Click here for additional data file.Figure S3Activation of Tcf/Lef by UL33. UL33 and TOPflash were co-transfected, and luciferase activity was analysed 24 hours post-transfection. UL33 induces Tcf/Lef activation strongly.(EPS)Click here for additional data file.