Wnt/β-catenin signalling regulates cell fate, survival, proliferation and differentiation at many stages of mammalian development and pathology. Mutations of two key proteins in the pathway, APC and β-catenin, have been implicated in a range of cancers, including colorectal cancer. Activation of Wnt signalling has been associated with the stabilization and nuclear accumulation of β-catenin and consequential up-regulation of β-catenin/TCF gene transcription. In 2003, Lee et al. constructed a computational model of Wnt signalling supported by experimental data from analysis of time-dependent concentration of Wnt signalling proteins in Xenopus egg extracts. Subsequent studies have used the Xenopus quantitative data to infer Wnt pathway dynamics in other systems. As a basis for understanding Wnt signalling in mammalian cells, a confocal live cell imaging measurement technique is developed to measure the cell and nuclear volumes of MDCK, HEK293T cells and 3 human colorectal cancer cell lines and the concentrations of Wnt signalling proteins β-catenin, Axin, APC, GSK3β and E-cadherin. These parameters provide the basis for formulating Wnt signalling models for kidney/intestinal epithelial mammalian cells. There are significant differences in concentrations of key proteins between Xenopus extracts and mammalian whole cell lysates. Higher concentrations of Axin and lower concentrations of APC are present in mammalian cells. Axin concentrations are greater than APC in kidney epithelial cells, whereas in intestinal epithelial cells the APC concentration is higher than Axin. Computational simulations based on Lee's model, with this new data, suggest a need for a recalibration of the model.A quantitative understanding of Wnt signalling in mammalian cells, in particular human colorectal cancers requires a detailed understanding of the concentrations of key protein complexes over time. Simulations of Wnt signalling in mammalian cells can be initiated with the parameters measured in this report. Wnt signalling regulates survival, proliferation and differentiation at various stages of development Interpreting the roles of particular signalling proteins is complex. For example, β-catenin is not only involved in gene transcription, but is also a key member of a cell-cell adhesion complex with E-cadherin The multifunctional nature of these proteins means that the Wnt signalling pathway also interacts with other major signalling pathways. Critically, the cellular adhesion pathway is tightly linked to the Wnt pathway - APC et al. et al. model; but the interpretation of predictions for mammalian systems is limited by the lack of corresponding mammalian data for the concentrations of key Wnt signalling proteins. In order to establish a quantitative basis for understanding of Wnt signalling in mammalian cells and human cancer, knowledge of the concentrations of key Wnt signalling proteins in mammalian cells is essential.Understanding the dynamics of Wnt signalling and other pathways requires a systems-level computational modelling approach. Only then is it likely that the behaviour of these cellular pathways, in response to specific stimuli or mutations, can be predicted et al.'s Xenopus study In this study we report initial estimates of the concentration of β-catenin, Axin, APC, GSK3β and E-cadherin in five mammalian cell lines  et al. et al.'s Xenopus extract, despite having a higher Axin concentration. These mammalian experimental results provide a basis for formulating computational models of the mammalian Wnt pathway. The differences observed between the two systems support the need for caution when translating the Xenopus Wnt signalling model The results presented here demonstrate significant differences in the concentrations of Wnt signalling proteins between the Xenopus egg extracts 2 linear fits (0.78 to 0.999)  volumes of mammalian cells in the resting (or ‘steady state’) and the dividing state. The whole cell and cell compartment volumes of five mammalian cell lines were determined: at least 50 cells were analysed for each cell line. Live cells stained with fluorescent compartment markers, namely Hoechst 33342 (nuclei), Calcein AM (cytosol) and Vybrant DiI (membrane) were prepared and imaged using confocal microscopy. The acquired image stacks show that the trypsinised cells are generally spheroidal or ellipsoidal in appearance . After io 0.999) , and S152 linear correlation for the non-dividing population is still reasonably correlated (see 2 0.58 to 0.99).Using this relationship, the resting  and dividing (D) populations of cells can be identified  of the two populations (N and ND) are tabulated in The measured compartmental volumes for non-dividing cells are tabulated in i.e. a larger cytosolic compartment, and 1∶1∶0.5 i.e. a larger nuclear compartment, respectively.Approximately half of the cellular volume of non-dividing cells is associated with the cytoplasm , while nThe confocal quantification technique not only provided consistent whole cell volume quantification, it also provides the cell compartment volume data. Cell compartment data are crucial for modelling of the intracellular pathways as protein concentrations are rarely evenly distributed throughout cells. Other advantages of this technique include the ease of application with relatively simple staining, imaging and cell selection steps involved. Furthermore, the analytical quantification has been partially automated in MATLAB. This procedure can be further developed for extensive and bulk quantification of large datasets. The availability of such tools to biochemist and/or computational biologists will provide a fast and reproducible alternative to acquiring crucial quantitative data needed for systems and computational modelling. In addition to the absence of whole cell volume data for the mammalian cells, there is also an absence of compartment volume data in the current literature. In recent years, it has become clearer that many of the mechanisms in the cellular pathway involve translocation between compartments within the cell. To understand and model the relative changes and translocation of proteins between compartments, an understanding of the levels of specific proteins in different regions or compartment of the cell is needed. These proteins concentrations are crucial for estimating the reaction rates in the computational models of signal transduction pathways.The total protein amounts per cell, as shown in Quantification results show that MDCK cells have the highest amounts of total protein per cell among the cell types investigated. Subsequently, the concentrations of the four key Wnt proteins and E-cadherin were calculated and are summarised in Measurements of total β-catenin concentrations in the functional pools or compartments  of the cell lines are as shown in It should be noted that for SW480 and SW480APC cells the culture conditions (i.e. confluency levels) have a significant impact on the consequent spatial localisation of β-catenin. This effect has been investigated briefly using both sub-cellular fractionation approach and a confocal imaging quantitation see . This deFurther to that, “active” β-catenin (ABC) concentrations Analysis of intestinal epithelial cells  and kidney epithelial mammalian cell lines (HEK293T and MDCK) reveals a pattern between the key scaffold proteins Axin and APC levels, whereas no trend is seen in the β-catenin concentrations between these two cell groups. The initial observation is that Axin and APC are present at comparatively low concentrations in intestinal mammalian cells. The absence of a pattern in β-catenin levels is unexpected as the β-catenin levels are hypothesized to be modulated by the degradation complex (which includes APC and Axin). As such, higher wild-type Axin or APC levels should imply a corresponding lower β-catenin levels and vice versa, but this is not observed here. It is also noted that the concentration of Axin is higher than that of APC in the kidney cells; however, in the intestinal cells APC concentrations are higher than Axin concentrations. The significantly higher level of Axin than APC, for kidney mammalian cell lines, as compared to intestinal mammalian cell lines, is shown by the small APC∶Axin ratio (<0.1) for kidney mammalian cell lines in contrast to an APC∶Axin ratio greater than 0.5 for intestinal mammalian cell lines see . It is nCaco-2 has a single APC with a nonsense mutation at codon 1367, a C to T transition changing Gln (CAG) to a stop codon (TAG) These observed differences in Axin and APC concentrations between kidney and intestinal mammalian cells has not been reported previously and will be critical information in the development of mammalian Wnt pathway models.et al. (2003), the levels of APC and GSK3β were assumed to be maintained at a comparatively high level as biochemical degradation experiments indicated reasonably slow turnover of APC and GSK3β with no clear degradation observed within 3 hours. Therefore no synthesis or degradation for the two proteins is explicitly parameterized in their model.The total protein concentrations for β-catenin, Axin, APC and GSK3β for the Xenopus egg extract and the WCL from the kidney epithelial mammalian cell lines, (HEK293T and MDCK) and three intestinal epithelial mammalian cells lines  are shown in et al.'s estimations and the results acquired in this study. Several differences were noted. First, comparing kidney epithelial mammalian cells with Xenopus, the β-catenin concentrations are 10 to 40 times higher in the kidney epithelial mammalian cells than that reported in the Xenopus egg extracts. Levels of GSK3β are similar in both systems. There is a marked difference in the concentrations of Axin. Lee and colleague (2003) proposed that Axin is the rate limiting factor in the Xenopus Wnt signalling pathway, as the measured total concentration of 0.02 nM are two to three orders magnitude lower than the other measured protein concentrations in the Wnt signalling pathway. Certainly Lee et al.'s Axin concentrations were much lower than the 110–150 nM range detected in mammalian cells, and yet we see that β-catenin concentrations are higher in mammalian cells despite Axin presumably no longer being rate-limiting. Furthermore, no substantial differences in β-catenin between kidney epithelial mammalian and intestinal epithelial mammalian cells are observed in spite of the differing levels of Axin. These observations are therefore inconsistent with the dogma that β-catenin degradation, facilitated by the degradation complex, is limited by the level of Axin. The situation is further complicated by an unexpectedly low APC level in kidney epithelial mammalian cells. One possible explanation for this discrepancy between the two cellular systems may be due to the presence of self-associated aggregations or vesicles of Axin in the cytoplasm in HEK293T and MDCK cells known as puncta In view of establishing a better understanding of the differences in protein levels between the two cellular systems, comparison were made between Lee higher (10 to 20 times) in the intestinal epithelial mammalian cells than the Xenopus egg extracts. Similarly to kidney epithelial mammalian cells, Axin levels are even more elevated (1000–1600 times higher) in intestinal epithelial mammalian cells than in the Xenopus extracts. In contrast, APC levels are 3 to 6 times lower in intestinal epithelial mammalian cells. It is surprising that the difference in β-catenin levels between kidney epithelial mammalian and intestinal epithelial mammalian cells are not as substantial as one would have expected. This implies that the pool of β-catenin regulated by the Wnt pathway might be small in comparison to the total amount of β-catenin protein. The GSK3β in Caco-2 cells are similar to the Xenopus extract, whereas in SW480 the GSK3β levels are 2 to 5 times lower than the Xenopus extracts.Comparing intestinal epithelial mammalian cells with Xenopus, again the β-catenin concentrations are et al. (2003). This is surprising considering APC is widely considered to be a tumour suppressor protein and therefore is expected to be at a significant level in order to serve that particular function. Again despite differing levels of APC between kidney epithelial mammalian cells and intestinal epithelial mammalian cells, there are not substantial differences in β-catenin levels, adding to the complication of the roles of Axin and APC in β-catenin modulation or the subset of β-catenin affected by this modulation.Overall, APC concentrations in these cell lines are significantly lower than that reported in the Xenopus egg extract by Lee In general, mammalian cells  have lower APC and higher Axin and β-catenin levels than the Xenopus extract. One might expect these differences in these key protein concentration to substantially change the dynamics of signalling through the Wnt pathway. All of these observed differences might be due to the different cellular systems involved  or the different functional needs  of the tissues, and so for the cells of different tissue origin. Xenopus extract was based on the cytoplasm of Xenopus egg while the mammalian cells are epithelial cells. Nonetheless, these results highlight the care needed in using data from a range of species or even cell types within a species and the requirement of more species specific quantitative data. These steady state protein quantifications will be crucial data for progressive computationally modelling of the mammalian Wnt pathway.et al. proposed a computational model  based on that described by Lee et al. 2003 The Lee et al. model using mammalian concentrations indicate significant differences. et al. model is not representative of mammalian cells. At a minimum, additional calibration of rates of production or degradation, reaction rates, or new reactions would be required to obtain agreement between observed total concentrations and model simulations.Comparison of open-system, steady-state results . Furthermore, concentrations of proteins for the majority of the complexes in the degradation cycle are low and primarily limited by the low level of steady state free Axin. total Axin in all the cell types tends toward a very low level (<0.02 nM). The steady state level of β-catenin is lower than the initial starting total concentration  ratios. For HEK293T and SW480APC, more than 75% of the initial total β-catenin concentration is retained at steady state, while Lee et al.'s (2003) model retained 97% of the initial total protein. However for MDCK, Caco-2 and SW480, less than 40% of the initial protein concentration is maintained. The I∶SS ratio for MDCK is particularly small at 1∶0.09 with only 9% of the initial β-catenin protein retained. It is noted that the low I∶SS ratio appears to correspond with smaller SS Axin level (<0.004 nM) and higher GSK3β concentration (both components of the degradation complex). As no input and output fluxes are present in the Lee et al. (2003) model Using the Lee et al. (2003) model. This low Axin level means that there would be a very low and perhaps limiting concentration of degradation complex available to degrade β-catenin. This Axin limitation can be further substantiated by the low levels of protein complexes formed (<0.0005 nM) for the different cell lines , the total steady state concentration of Axin is predicted to be extremely low (<0.02 nM) in the Lee ll lines To test the uniqueness of the steady state solution for particular initial concentrations, simulations were also repeated for a different pathway to the open-system steady-state, namely, from the initial concentrations directly to the open- system steady-state . In otheet al.'s model has to be re-calibrated using the mammalian experimental data as a prerequisite for further analysis. Therefore, optimisations of the reactions constants, turnover rates or reaction topology will be required. This is the subject of investigation of our subsequent work which includes experimental analysis and computational predictions of the temporal behaviours of the Wnt signalling pathway components in several mammalian cells. Further, temporal data required to validate β-catenin dynamics in the mammalian whole cells were acquired to calibrate and validate the computational models (manuscript in preparation).In the case of mammalian cells, Lee et al.'s model was the first comprehensive computational model of the Wnt pathway and is notable for its successful integration of computational-and experimental approaches. It has triggered several subsequent computational studies that extended the Lee et al. model et al. model (0.02 nM) requires careful interpretations of Axin-mediated degradation kinetics. Preferably relevant data should be acquired to enable model validation.Lee et al.Two interesting studies on Wnt modelling were published in recent years by van Leeuwen Mammalian cell lines were used in this study: HEK293T, a human kidney epithelial The following primary monoclonal antibodies were used in this study: anti-GSK3β , anti-β-catenin , anti-Active-β-catenin , anti-E-cadherin , anti-β-tubulin . The following primary polyclonal antibodies were used: anti-APC  and anti-Axin  antibodies. The following secondary antibodies were used: goat anti-mouse IRDye 800CW  and anti-rabbit IRDye 800CW .The following cell labelling solutions were used in this study: BD™ Calcein AM Fluorescent Dye  used as a green fluorescent live cell marker 6-m Axin-HA, full-length H6-APC-EE and the intracellular domain of E-cadherin. The expression and purification of all recombinant proteins are as described in Recombinant proteins used in this study include β-cateninEE, full-length FLAG-GSK3β, HTrypsinised cells from the 5 cell cultures was stained with Calcein AM fluorescent dye, Hoechst 33342 nucleic acid stain and Vybrant DiI cell labelling solution to mark the cytoplasm, nuclei and membrane respectively. The stained cells were embedded evenly in agarose mixture within a Sykes Moore Chamber. 3D image stacks of the embedded cells were acquired using the Olympus FV1000 confocal microscope and processed using Metamorph Premier image processing software. Image analysis and cell compartment calculations were performed in Matlab Utilizing the measured volume and quantitative western blot analysis technique, the whole cell protein concentrations of key Wnt proteins β-catenin, GSK3β, Axin and APC as well as the cellular adhesion protein E-cadherin for the five mammalian cell lines HEK293T, MDCK, Caco-2, SW480 and SW480APC were systematically quantified and analysed. The compartment concentrations of β-catenin  were further quantified for the cell lines. Details of the measurement procedures can be found in The dependency of the sub-cellular distribution of β-catenin  on the cell culture conditions for SW480 and SW480APC cells was investigated using confocal imaging. The relative 3D compartment quantification of β-catenin in SW480 and SW480APC was conducted as per described in the et al. 2003 et al. 2003 et al.'s model in the context of mammalian whole cell system was investigated by integrating the newly acquired quantitative data .The response of Lee et al.'s model et al.'s assumptions for model reduction would hold true for the mammalian systems. Therefore a non-presumptive approach with all the reactions was used. In this integration analysis, Lee et al. model was solved in MATLAB et al. study using the Xenopus data.The computational model utilised in the study is a reconstruction of Lee et al. model is appropriate for mammalian systems, it is expected the total protein level are to be maintained after redistribution in an open system.The experimental WCL concentrations measured are total concentration for each individual protein, without information of the distribution of these proteins among the various complexes. The first step in the computational analysis then involves steady state redistribution of the proteins among its complexes see . This anet al. model is steady state representative of Wnt signalling in mammalian cells, steady state simulation (Phase A and B) are conducted with et al. Specifically, Lee et al. model is simulated without Wnt stimulation (Wnt = 0) and starting from the initial concentrations . The system ODEs were solved to obtain the new steady states with proteins redistributing among their various complexes initially in a closed system (Phase A), followed by an open system (Phase B) To determine if Lee Figure S1Steps for image stack analysis and selection of cells using metamorph. Fluorescence labelled cytosol , membrane (Vybrant DiI in red) and nuclei (Hoechst 33342 in blue).(TIF)Click here for additional data file.Figure S2Procedures for image processing and quantification (Steps 1 to 3). Import, separate channels and segment image. Step 1, IMPORT: Import image stack TIFF for each individual selected cell. Step 2, CHANNEL SEPARATION: Separate independent channel information. Step 3, IMAGE SEGMENTATION: Threshold, filter and fill holes to generate binary masks.(TIF)Click here for additional data file.Figure S3Procedures for image processing and quantification (Steps 4 and 5). 3D object filtering, processing and quantification.(TIF)Click here for additional data file.Figure S4Procedures for image processing and quantification (Steps 6 to 8). Data consolidation, cell population analysis and volume distribution analysis.(TIF)Click here for additional data file.Figure S5Western blots for quantitative analysis of whole cell lysates (WCL) for Wnt signalling proteins. (A) β-catenin (B) APC (C) Axin (D) GSK3β (E) E-cadherin in the different cell lines with recombinant proteins as standards.(TIF)Click here for additional data file.Figure S6Procedures for quantifying levels of proteins in WCL. This figure uses β-catenin as the specific protein investigated. Mass of total protein, TP per lane (A) calculated based on known amount of protein loaded. The total mass of specific protein (P) per lane (B) was calculated using the standard curve of known amounts of the P in the same western blot. Mass of P per ng TP loaded (C) calculated by dividing B with A. P per ng TP (D) calculated by averaging n independent sets of C. Average TP per cell (E) acquired from cell count and BCA assay experiments and used to calculate average P per cell (F) by multiplying D with E. Relative molecular weight of P, (Mr) used to calculate nanomole of P per cell, dividing F with Mr. Whole cell volume of a resting cell (H) was measured in this study and used to calculate the Molar concentration of P per cell (I), dividing G with H. Final concentration of P per cell calculated in nM per cell (J).(TIF)Click here for additional data file.Figure S7Western blots for quantitative compartment analysis of β-catenin. β-catenin levels in (A) HEK293T, (B) Caco-2, (C) MDCK, (D) SW480 and (E) SW480APC with recombinant β-catenin as protein standards.(TIF)Click here for additional data file.Figure S8Western blots for quantitative compartment analysis of “active” β-catenin. (A) Western blot of two identical sets of recombinant β-catenin probed for total or “active” β-catenin. The standard curves generated by these two sets were used for correlating protein levels detected by the two antibodies. “Active” β-catenin levels in (B) HEK293T, (C) Caco-2, (D) MDCK, (E) SW480 and (F) SW480APC with recombinant β-catenin as protein standards.(TIF)Click here for additional data file.Figure S9Procedures for quantifying proteins levels for sub-cellular fractions. The mass of β-catenin per lane (E) from the western blot was calculated using known amounts of recombinant β-catenin. Mass of β-catenin in the loading sample (F) and subsequently total cell volume (G) was calculated by scaling to the original cell volume (B). Dividing F by total cell count (A) gives the mass of β-catenin per cell (H). Relative molecular weight of β-catenin (Mr) was then used to calculate nanomole of β-catenin per cell (I = H÷Mr). Whole cell volume of a resting cell (J) was acquired in this study and used to calculate the molar concentration of β-catenin per cell (K = I÷J). Final concentration of β-catenin per cell calculated in nM (L).(TIF)Click here for additional data file.Figure S10Dependency of β-catenin distribution in SW480 and SW480APC on confluency. 3D confocal imaging and compartment analysis at different confluency: (A) SW480 and (B) SW480APC at low confluency; (C) SW480 and (D) SW480APC at high confluency. Panels show the (i) overlay (DAPI in blue and β-catenin in green), (ii) DAPI, (iii) β-catenin, (iv) nuclear β-catenin intensity and (v) non-nuclear β-catenin intensity images. (E) Results of compartmental analysis in intensity per voxel shows higher nuclear∶non-nuclear β-catenin ratio (>1) for low confluency samples. Lower nuclear∶non-nuclear β-catenin ratio (<1) is observed for highly confluent samples. (F) Sub-cellular fractionation results for both cell-lines at difference confluency in β-catenin concentrations (nM). Increasing cytosolic and membrane β-catenin concentrations are observed with increasing confluency with the nuclear β-catenin levels remaining constant. (G and H) β-catenin compartment distribution (in % of compartment summation), nuclear β-catenin distribution decreases and the cytosolic β-catenin level increases with increasing confluency. The membrane levels remained constant. Note: panels ii–v are grayscaled for visual clarity.(TIF)Click here for additional data file.Figure S11Sub-cellular distribution of “active” β-catenin in mammalian cells. (A) Compartment concentrations of “active” β-catenin in HEK293T, Caco-2, MDCK, SW480 and SW480APC cells. (B) Percentage distribution of “active” β-catenin for the five cell lines.(TIF)Click here for additional data file.Figure S12Cell population sizing and selection (HEK293T). Population statistics of HEK293T, showing the (A) histograms of normalised volume ; (B) Scatter plot of fluorescent marked volumes  showing a good linear relationship; (C) Scatter plot of fluorescent marked volumes  showing population isolation and the cut-off volumes applied.(TIF)Click here for additional data file.Figure S13Cell population sizing and selection (MDCK). Population statistics of MDCK, showing the (A) histograms of normalised volume ; (B) Scatter plot of fluorescent marked volumes  showing a good linear relationship; (C) Scatter plot of fluorescent marked volumes  showing population isolation and the cut-off volumes applied.(TIF)Click here for additional data file.Figure S14Cell population sizing and selection (Caco-2). Population statistics of Caco-2, showing the (A) histograms of normalised volume ; (B) Scatter plot of fluorescent marked volumes  showing a good linear relationship; (C) Scatter plot of fluorescent marked volumes  showing population isolation and the cut-off volumes applied.(TIF)Click here for additional data file.Figure S15Cell population sizing and selection (SW480). Population statistics of SW480, showing the (A) histograms of normalised volume ; (B) Scatter plot of fluorescent marked volumes  showing a good linear relationship; (C) Scatter plot of fluorescent marked volumes  showing population isolation and the cut-off volumes applied.(TIF)Click here for additional data file.Text S1Preparation and Purification of Recombinant Proteins.(DOC)Click here for additional data file.Text S2Protocol for confocal cell compartment volume measurements and analysis.(DOC)Click here for additional data file.Text S3Measurement Protocol for Protein Concentrations in Mammalian Cells.(DOC)Click here for additional data file.


We hypothesised that prolonging the metabolic half-life of 68Ga-DOTAVAP-P1 could further improve its imaging characteristics. In this study, we evaluated a new analogue of 68Ga-DOTAVAP-P1 modified with a mini-polyethylene glycol (PEG) spacer (68Ga-DOTAVAP-PEG-P1) for in vivo imaging of inflammation.Vascular adhesion protein-1 (VAP-1) is an adhesion molecule that plays a key role in recruiting leucocytes into sites of inflammation. We have previously shown that 68Ga-DOTAVAP-P1 and 68Ga-DOTAVAP-PEG-P1 were evaluated in vivo by dynamic PET imaging and ex vivo by measuring the radioactivity of excised tissues. In addition, plasma samples were analysed by radio-HPLC for the in vivo stability of the peptides.Whole-body distribution kinetics and visualisation of inflammation in a rat model by the peptides 68Ga-DOTAVAP-P1 and 0.53 ± 0.01 for 68Ga-DOTAVAP-PEG-P1 by PET. In addition, inflammation-to-muscle ratios were 6.7 ± 1.3 and 7.3 ± 2.1 for 68Ga-DOTAVAP-P1 and 68Ga-DOTAVAP-PEG-P1, respectively. The proportion of unchanged peptide in circulation at 60 min after injection was significantly higher for 68Ga-DOTAVAP-PEG-P1 (76%) than for 68Ga-DOTAVAP-P1 (19%).The peptide with the mini-PEG spacer showed slower renal excretion but similar liver uptake as the original peptide. At 60 min after injection, the standardised uptake value of the inflammation site was 0.33 ± 0.07 for 68Ga-DOTAVAP-P1 peptide, leading to higher target-to-background ratios and improved in vivo PET imaging of inflammation.The eight-carbon mini-PEG spacer prolonged the metabolic half-life of the  In vivo imaging of inflammation is a demanding task, and novel molecular imaging targets are called for. The gold standard in nuclear medicine is the radiolabelling of white blood cells, which is both time consuming and potentially hazardous for the technical personnel.in vivo imaging of inflammation. This approach may open new opportunities for diagnosing, therapy planning and monitoring of the treatment efficacy, as well as for the drug discovery and development processes [Vascular adhesion protein-1 (VAP-1) is an inflammation-inducible endothelial adhesion protein involved in the leucocyte trafficking from the blood stream into the tissues. VAP-1 is stored in intracellular granules within endothelial cells. However, upon inflammation, it is rapidly translocated to the endothelial cell surface, for example, in the synovial tissue in rheumatoid arthritis and at the site of ischemic reperfusion injury . Thereforocesses -6.in vivo imaging of leucocyte trafficking. The linear peptide, VAP-P1, has been characterised by Yegutkin et al. and proven to bind the enzymatic groove of VAP-1 and dose-dependently inhibit VAP-1-dependent lymphocyte rolling and firm adhesion to primary endothelial cells [68Ga-labelled DOTA-conjugated VAP-P1 peptide (68Ga-DOTAVAP-P1) is able to delineate inflammation in rats by a VAP-1-specific way using positron emission tomography (PET) [68Ga-DOTAVAP-P1 peptide has relatively short plasma half-life and very rapid clearance by the kidneys to the urine.Peptide-based imaging agents are small molecules that possess favourable properties such as rapid diffusion in target tissue, rapid clearance from the blood circulation and non-target tissues, easy and low-cost synthesis, and low toxicity and immunogenicity. We are particularly interested in developing radiolabelled peptides for VAP-1 targeting for the purposes of al cells . We havehy (PET) . DisadvaPEGylation, the process by which polyethylene glycol (PEG) chains or its derivatives, e.g., mini-PEGs are attached to a peptide, has been used for modifying the properties of radiolabelled compounds, such as antibodies and peptides, in order to improve their imaging characteristics. The goal of PEGylation is mainly to improve the tracer's kinetics and distribution pattern by increasing its metabolic half-life and by lowering its non-specific binding. By increasing the molecular mass of the peptide and by shielding it from proteolytic enzymes, PEGylation may modify its biodistribution and pharmacokinetics . Thus, t68Ga-DOTAVAP-P1 would further improve its potential for in vivo imaging of inflammation. In this study, we evaluated a new mini-PEG spacered analogue of 68Ga-DOTAVAP-P1 (68Ga-DOTAVAP-PEG-P1) for in vivo PET imaging of inflammation.We hypothesised that prolonging the metabolic half-life of The DOTA-conjugated peptides were purchased from Almac Sciences , ABX advanced biochemical compounds GmbH  and NeoMPS .68Ga as previously described [68Ga-DOTAVAP-P1 and 68Ga-DOTAVAP-PEG-P1. Briefly, 68Ga was obtained in the form of 68GaCl3 from a 68Ge/68Ga generator  by elution with 0.1 M HCl. The 68GaCl3 eluate (500 μl) was mixed with sodium acetate  to give a pH of approximately 5.5. Then, DOTA-peptide (35 nmol) was added and the mixture was incubated at 100°C for 20 min. No further purification was needed.Linear 9-amino acid DOTA-chelated peptide (GGGGKGGGG) with and without a PEG linker  between the DOTA and the N terminal amino acid was labelled with 2, 125 Å, 10 μm; Waters Corporation, Milford, MA, USA). The HPLC conditions for 68Ga-DOTAVAP-P1 have been described previously [68Ga-DOTAVAP-PEG-P1 were slightly different and as follows: flow rate = 4 ml/min, λ = 215 nm, A = 2.5 mM trifluoroacetic acid, B = acetonitrile and C = 50 mM phosphoric acid. Linear A/B/C gradient was 100/0/0 for 0 to 3 min, 40/60/0 for 3 to 9 min, and 0/0/100 for 9 to 16 min. The radio-HPLC system consisted of LaChrom instruments : pump L7100, UV detector L-7400 and interface D-7000; an on-line radioisotope detector ; and a computerised data acquisition system.The radiochemical purity was determined by reversed-phase HPLC , centrifuged at 3,900 × g for 3 min at 4°C, and filtered through 0.45-μm Minispike filter (Waters Corporation). The filtrate was analysed by radio-HPLC.The D, of the 68Ga-DOTA-peptides was determined using the following procedure. Approximately 5 kBq of 68Ga-labelled peptide in 500 μl of phosphate-buffered saline  was added to 500 μl of 1-octanol. After the mixture had been vortexed for 3 min, it was centrifuged at 12,000 × g for 6 min, and 100-μl aliquots of both layers were counted in a gamma counter . The test was repeated three times. The logD was calculated as = log10 (counts in octanol/counts in PBS).The octanol-water distribution coefficient, logAll animal experiments were approved by the Lab-Animal Care & Use Committee of the State Provincial Office of Southern Finland and carried out in compliance with the Finnish laws relating to the conduct of animal experimentation.n = 14) were purchased from Harlan, Horst, The Netherlands. Twenty-four hours before the PET studies, turpentine oil  was injected subcutaneously into their neck area in order to induce a sterile inflammation [in vivo metabolite analyses.Male Sprague-Dawley rats (ammation . Six rat68Ga-DOTAVAP-P1 (n = 3) and 68Ga-DOTAVAP-PEG-P1 (n = 3) in rats harbouring a sterile inflammation were studied with a high-resolution research tomograph . The rats were anaesthetised with isoflurane . Two rats were imaged at the same time, and they were kept on a warm pallet during the imaging procedure. Following a 6-min transmission for attenuation correction, the rats were intravenously (i.v.) injected with 68Ga-DOTAVAP-P1  or with 68Ga-DOTAVAP-PEG-P1  as a bolus via a tail vein using a 24-gauge cannula . Dynamic imaging lasting for 60 min started at the time of injection. The data acquired in list mode were iteratively reconstructed with a 3-D ordered subsets expectation maximisation algorithm with 8 iterations, 16 subsets and a 2-mm full-width at half-maximum post-filter into 5 × 60 s and 11 × 300 s frames. Quantitative analyses were performed by drawing regions of interest (ROI) on the inflammatory foci, muscle (hind leg), heart, kidney, liver and urinary bladder. Time-activity curves (TACs) were extracted from the corresponding dynamic images . The average radioactivity concentrations in the ROIs (kilobecquerels per millilitre) were used for further analyses. The uptake was reported as standardised uptake value (SUV), which was calculated as the radioactivity of the ROI divided by the relative injected radioactivity expressed per animal body weight. The radioactivity remaining in the tail was compensated.The whole-body distribution and kinetics of After the PET imaging, the animals were sacrificed. Samples of blood, urine and various organs were collected, weighed and measured for radioactivity using the gamma counter . The results were expressed as SUVs.68Ga-DOTA-peptides into heparinised tubes . Radioactivity of whole blood was measured with the gamma counter . Plasma was separated by centrifugation , and plasma radioactivity was measured. The ratio of radioactivity in blood versus plasma was calculated. To determine plasma protein binding, proteins were precipitated with 10% sulphosalicylic acid, and the radioactivity in protein precipitate and supernatant was measured. The plasma supernatant was further analysed by radio-HPLC in order to evaluate the in vivo stability of the 68Ga-labelled peptides.Blood samples (0.2 ml of each) were drawn at 5, 10, 15, 30, 45, 60 and 120 min after injection of In vivo stability data were used in order to generate metabolite-corrected plasma TACs for 68Ga-DOTAVAP-P1 and 68Ga-DOTAVAP-PEG-P1, which were further used for the calculation of pharmacokinetic parameters. The area under curve (AUC) of the plasma TAC from 0 to infinity was calculated using a non-compartmental analysis employing the trapezoidal rule. The clearance (CL) of the 68Ga-labelled peptides after a single intravenous bolus dose was calculated by dividing the injected dose by the AUC. The plot of the natural logarithm of parent tracer concentration against time after bolus injection became linear in the end phase, as the tracer was eliminated according to the laws of first-order reaction kinetics. The elimination rate constant (kel) was calculated as the negative slope of the linear part of the plot. The plasma elimination half-life (t1/2) was calculated as t1/2 = ln(2)/kel. The metabolic half-lives of the 68Ga-DOTA-peptides were calculated according to the results of radio-HPLC, i.e. the time point when 50% of the total radioactivity is still bound to the intact peptide.ex vivo measurement values were evaluated using linear regression analysis. Inter-group comparisons were made using an unpaired t test. Statistical analyses were conducted using Origin 7.5 software . A P value less than 0.05 was considered as statistically significant.All the results are expressed as means ± standard deviation (SD) and range. The correlations between PET imaging and 68Ga-DOTAVAP-P1 and 68Ga-DOTAVAP-PEG-P1 were 97 ± 1% and 99 ± 1%, and specific radioactivities 2.27 ± 0.47 and 2.55 ± 0.45 MBq/nmol, respectively. The retention times for 68Ga-DOTAVAP-P1 and 68Ga-DOTAVAP-PEG-P1 were 6.6 ± 0.1 and 6.7 ± 0.1 min, respectively. The retention time for free gallium was approximately 12 min, and it eluted only with phosphoric acid. The in vitro stabilities of 68Ga-DOTAVAP-P1 and 68Ga-DOTAVAP-PEG-P1 were very similar. The amounts of unchanged peptide after the 4-h incubation in human or rat plasma were 88 ± 3% and 82 ± 11% for 68Ga-DOTAVAP-P1 and 89 ± 8% and 90 ± 6% for 68Ga-DOTAVAP-PEG-P1, respectively. Both peptides were highly hydrophilic; logD was -3.30 for 68Ga-DOTAVAP-P1 and -3.50 for 68Ga-DOTAVAP-PEG-P1.The radiochemical purities of 68Ga-DOTAVAP-P1 and 68Ga-DOTAVAP-PEG-P1, respectively, at 60 min after injection. Inflammation-to-muscle ratios at 60 min after injection were 6.7 ± 1.3  and 7.3 ± 2.1  for 68Ga-DOTAVAP-P1 and 68Ga-DOTAVAP-PEG-P1, respectively. The kinetics of 68Ga-DOTAVAP-P1 and 68Ga-DOTAVAP-PEG-P1 in inflammatory foci were quite fast, and the peak radioactivity was reached within 20 min for both peptides. On the average, the inflammation uptake of 68Ga-DOTAVAP-PEG-P1 was 59% higher than that of 68Ga-DOTAVAP-P1, and the difference was statistically significant (P = 0.047). According to the whole-body dynamic PET imaging, 68Ga-DOTAVAP-PEG-P1 showed slower renal excretion to urine but otherwise rather similar distribution kinetics as the original peptide 68Ga-DOTAVAP-P1 . When the tissue uptakes of 68Ga-DOTAVAP-P1 and 68Ga-DOTAVAP-PEG-P1 were compared, the inflammation, lung, small intestine, skin and urinary bladder radioactivities were significantly different. Although the PET and ex vivo methods correlate well, there are some discrepancies between the results. For example, in the PET image analysis, the urine and blood of kidney are included in the "kidney" ROI, whereas for ex vivo measurement, the excised tissue samples are dotted dry on a paper. Since the radioactivity of urine is extremely high, the in vivo kidney SUV is higher than that of ex vivo.The PET imaging results were verified by s Figure . Linear 68Ga-DOTAVAP-P1 and 1.3 ± 0.1 and 0.86 ± 0.02 for 68Ga-DOTAVAP-PEG-P1, respectively. The in vivo stability of 68Ga-DOTAVAP-PEG-P1 was better than that of 68Ga-DOTAVAP-P1. The proportions of unchanged peptides in rat plasma at 60 and 120 min after injection were 19 ± 4% and 4 ± 1% for 68Ga-DOTAVAP-P1 and 76 ± 18% and 49 ± 6% for 68Ga-DOTAVAP-PEG-P1, respectively , i.e. the fraction of total radioactivity in plasma that is unbound to plasma proteins, were 1.3 ± 0.1 and 0.84 ± 0.04 for 68Ga-DOTAVAP-P1, for PET imaging of inflammation in different rat models [68Ga-DOTAVAP-P1 is cleared very rapidly from circulation and its in vivo stability against degradation by enzymes is only moderate. In this study, we showed that the incorporation of a mini-PEG spacer in 68Ga-DOTAVAP-P1 enhanced its in vivo stability and improved the PET imaging of inflammation.Previously, we have reported the feasibility of the VAP-1-targeting peptide, t models . Howeverin vitro, ex vivo and in vivo experiments to evaluate the VAP-1 targeting, inflammation imaging efficacy and pharmacokinetics of 68Ga-DOTAVAP-PEG-P1 in comparison to the original 68Ga-DOTAVAP-P1. The incorporation of a mini-PEG spacer had no apparent effect on the in vitro properties of the VAP-1 binding peptide; both peptides were stable in plasma incubations and their solubility was very similar. However, when i.v. administered, 68Ga-DOTAVAP-PEG-P1 showed significantly longer metabolic and elimination half-lives and slower total clearance compared to 68Ga-DOTAVAP-P1. Furthermore, our results revealed that while both peptides were able to visualise experimental inflammation by PET imaging, 68Ga-DOTAVAP-PEG-P1 showed a higher inflammation-to-muscle ratio than the original 68Ga-DOTAVAP-P1. As regards 68Ga-DOTAVAP-P1, the results of this study are in line with our previous publications [68Ga-DOTAVAP-PEG-P1 was slower, resulting in a significantly lower urinary bladder radioactivity in comparison to 68Ga-DOTAVAP-P1. The liver uptake was rather high for both peptides, which is, at least in part, due to the high number of VAP-1 receptors in the sinusoidal endothelial cells in the liver [68Ga-DOTA-peptides, such as free 68Ga, also tend to accumulate in the liver [The animal model used in our experiments involves turpentine oil injection-induced subcutaneous inflammation as described previously . In thatications . The renhe liver . Some dehe liver . Althougin vivo kinetics of pharmaceuticals. However, the results of such modifications depend much on the nature of the lead compound and the choice of PEG linker [PEGylation has widely been used for improving the G linker -20. In mG linker .In this study, we incorporated an eight-carbon mini-PEG spacer between the DOTA and the VAP-P1 peptide in order to prolong its biological activity. The 8-amino-3,6-dioxaoctanoic acid contains the shortest ether structure possible of PEG with two ethylene oxide units. A similar spacer has previously been used in imaging agents by Burtea et al. , Ke et ain vivo imaging of inflammation suggesting that PEGylation had other highly pronounced in vivo effects beyond modification of pharmacokinetics. Although the modification with a mini-PEG spacer increased the target-to-background ratio, the SUV values in the inflamed area were still very low. Thus, further improvement of the tracer is warranted.Modification with a mini-PEG spacer increased metabolic stability of VAP-1-targeting DOTA-peptide. In addition, it also improved in vivo stability and pharmacokinetics of the VAP-1-targeting peptide, thus leading to higher target-to-background ratios and improved in vivo PET imaging of experimental inflammation. 68Ga-DOTAVAP-PEG-P1 warrants further investigations for its feasibility in PET imaging of inflammation.The incorporation of a mini-PEG spacer enhanced the HPLC, high performance liquid chromatography; H & E, haematoxylin and eosin; MW, molecular weight; PEG, polyethylene glycol; PET, positron emission tomography; VAP-1, vascular adhesion protein-1.The authors declare that they have no competing interests.in vitro and in vivo PET studies and drafted the manuscript. TH participated in the design of the study and drafted the manuscript. HJS performed the labelling chemistry and participated in in vitro studies. AR and SJ conceived the study, participated in its design and coordination and critically revised the manuscript. All authors read and approved the final manuscript.AA participated in the design of the study, carried out the


Food allergy is the most common cause of anaphylaxis in children and recent studies suggest increased prevalence of both food allergy and anaphylaxis. Among foods, fruits are rarely implicated as the cause of anaphylaxis. Furthermore, anaphylaxis to fruit in the first months of life is rare. Although banana allergy has been well described in adults, there are only two case reports of anaphylaxis to banana in children.A 4-month-old Hispanic baby boy with a history of eczema presented to our emergency room with vomiting, urticaria and cyanosis following first exposure to a banana. He improved with administration of intramuscular epinephrine. Skin prick tests showed positive results for both fresh banana  and banana extract .Banana is not considered a highly allergenic food. However, as food allergy becomes more common and solid foods are being introduced earlier in babies, banana may become an important allergen to consider in cases of babies presenting with anaphylaxis. Food allergy is the most common cause of anaphylaxis in children, with the usual foods implicated being: peanut, tree nuts, shellfish and milk . Recent There have been several other case reports of allergic reaction to banana in babies ranging from 5 to 7 months of age. Three cases involved generalised urticaria only -6, whereIn the adult population banana allergy is well documented. It is often associated with latex allergy (fruit-latex syndrome).A 4-month-old Hispanic baby boy with a history of eczema presented to our emergency room with vomiting, urticaria and cyanosis within 5 minutes of eating banana. This was his first known exposure to banana. On emergency room triage, he was tachycardic at 190 beats per minute, but his other vital signs were stable . A physical examination was significant for diffuse urticaria and moderate perioral cyanosis. In our emergency room he was treated with intramuscular epinephrine  and oral diphenhydramine  with gradual improvement. He remained stable during a 6-hour observation period following the administration of epinephrine and was discharged home with a prescription for injectable epinephrine and out-patient consultation to an allergist  for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.AOK performed the literature review and wrote the first draft of the manuscript. MBS collected the patient data and edited the manuscript. Both authors read and approved the final manuscript.


PLOS Computational Biology, making it a good time to reflect on the journal, reassess the needs of our community, and, broadly, the direction of computational biology within the framework of the biological sciences. The rapid growth in computational power, the masses of data that need to be understood, and the advances in the biological sciences all point to a need to reevaluate directions, merging these with the fast pace of discoveries made by experimental studies. Are we, as computational biologists, contributing as much as we can to the advancement of the sciences in key areas? Since PLOS Computational Biology is a broad, community-based open-access journal, the areas of focus that we deem central to the field may impact the future of computational biology.A year has passed since I took over as the Editor-in-Chief of PLOS Computational Biology can, and, I believe, should, foster a highly stimulating and interactive environment among computational and experimental scientists in our community toward these aims.The biological sciences increasingly shift to research projects that aim to understand the causes and the mechanisms of diseases and to discover therapeutics. This shift over recent years is fueled both by the desire to alleviate human suffering and by the preferences of funding agencies. Within this framework, computational biology can effectively contribute to the understanding of a broad range of biological processes under normal physiological conditions and in disease; and it can do this across a range of scales, from the molecular and biochemical to the organismal and population levels. As a leading journal in the computational sciences, Computational biology increasingly gains the scientific center stage. It is an exciting interdisciplinary field that draws scientists from different fields: physics, chemistry, mathematics, engineering, computer science, and biology. Computational biology aims to organize and make sense of huge amounts of data and large-scale cellular processes and pathways, and, at the same time, to understand biological phenomena on the atomic scale. Experiments obtain information at multiple levels. Computational biology is a quantitative field. The goals of computational biology are to distinguish between noise and signals, obtain and quantify trends, and put these together, so that we are able to figure out how the information flows, how the processes are regulated, and what goes wrong in disease. We would like to understand the mechanisms through which mutations lead to cell proliferation, how the signals transmit in the cellular network and how external stimuli translate to cellular differentiation and to turning genes on and off, and how viruses enter cells. However, beyond these, computational biology aims to use the information to make predictions and obtain experimentally testable models.PLOS Computational Biology embraces all areas of computational biology. However, I believe that to help the advancement of the biological sciences, the journal should consider papers that address biologically relevant questions that are of broad interest and provide new concepts that can guide the design of experiments. This has been in our scope since the inception of the journal, and we shall continue to follow these guidelines. It is emboldening to think of open questions in the biological sciences, particularly those relating to diseases, where computational biology can drive progress.PLOS Computational Biology is highly regarded by the scientific community. We should aim to not only retain this appreciation but to further it. In response to requests by our community, the first step that I took after taking over as the Editor-in-Chief was to introduce a Methods section, led by Thomas Lengauer. Under Thomas' leadership, this section is flourishing, and is fast becoming highly popular, with an increasing number of submissions. The section strives to publish only the most outstanding methods, aiming to consider only those that are of exceptional quality. We are now also actively engaged in reducing the response times to authors, publication processing, and in simplifying the submission process.PLOS Computational Biology is important not only for scientific advancement; it also bears on our students who should be exposed to a variety of ways to define, analyze, question, and solve relevant scientific research problems computationally, relying on experimental data. For PLOS Computational Biology to help and inspire the biological sciences, we, as a community, should remain tuned-in to research trends and new data, and take action. These goals are shared with the International Society for Computational Biology (ISCB), with whom we are proud to partner. Together, we hope to make a difference.The content of PLOS Computational Biology would not have been possible had it not been for the devoted journal staff, who are always there to help, guide, suggest, and follow on initiatives, and our extended Editorial Board. Armed with these, and embracing our community, our open-access PLOS Computational Biology journal will continue to thrive.Finally, the achievements of


They harbor a mutated Apc allele and develop intestinal adenomas and carcinomas during the first months of life. This phenotype is caused by the mutation of the second Apc allele and the consequent accumulation of nuclear β-catenin in the affected cells. Here we describe that vitamin D receptor (VDR) is a crucial modulator of nuclear β-catenin levels in colon cancer in vivo. By appropriate breeding of min/+Apc mice and +/−Vdr mice we have generated animals expressing a mutated Apc allele and two, one, or none Vdr wild type alleles. Lack of Vdr increased the number of colonic Aberrant Crypt Foci (ACF) but not that of adenomas or carcinomas in either small intestine or colon. Importantly, colon ACF and tumors of min/+Vdr-/-Apc mice had increased nuclear β-catenin and the tumors reached a larger size than those of min/+Vdr+/+Apc. Both ACF and carcinomas in min/+Vdr-/-Apc mice showed higher expression of β-catenin/TCF target genes. In line with this, VDR knock-down in cultured human colon cancer cells enhanced β-catenin nuclear content and target gene expression. Consistently, VDR depletion abrogated the capacity of 1,25(OH)2D3 to promote the relocation of β-catenin from the nucleus to the plasma membrane and to inhibit β-catenin/TCF target genes. In conclusion, VDR controls the level of nuclear β-catenin in colon cancer cells and can therefore attenuate the impact of oncogenic mutations that activate the Wnt/β-catenin pathway.Aberrant activation of the Wnt/β-catenin pathway is critical for the initiation and progression of most colon cancers. This activation provokes the accumulation of nuclear β-catenin and the induction of its target genes.  APC/adenomatous polyposis coli, CTNNB1/β-catenin or AXIN genes which activate Wnt/β-catenin pathway are responsible for the initiation of most colon cancers Colon cancer is the second most common cause of death from cancer in developed countries min/+Apc mice harboring a germ line inactivating mutation in one Apc allele develop multiple intestinal adenomas and carcinomas after three months of age Apc allele (loss of heterozygosity) and the activation of the Wnt/β-catenin signaling pathway. This mouse model has become the gold standard of intestinal cancer initiation.adherens junctions, as the free protein is rapidly phosphorylated by casein kinase-Iβ (CK-Iα) (Ser45) and glycogen synthase kinase-3 (GSK-3β)  in a complex formed also by the tumor suppressor proteins APC and Axin. Phosphorylation labels β-catenin for ubiquitination and degradation by the proteasome Wnt/β-catenin pathway regulates the ability of β-catenin protein to drive the regulation of specific target genes 3 , its most active metabolite 1α,25-dihydroxyvitamin D3 2D3) and several analogs protect against colon cancer 2D3 inhibits proliferation and promotes epithelial differentiation of human colon cancer cells by inducing the expression of E-cadherin and by antagonizing the Wnt/β-catenin pathway. The latter is the result of two main mechanisms: a) the induction of direct binding of vitamin D receptor (VDR) to β-catenin, which precludes the formation of transcriptionally active β-catenin/TCF complexes, and b) the induction of β-catenin nuclear export and relocalization at plasma membrane adherens junctions as a consequence of E-cadherin upregulation 2D3 increases the expression of DICKKOPF-1 (DKK1), a secreted protein that inhibits Wnt signaling from its plasma membrane receptors VDR gene is a direct target of SNAIL1 and SNAIL2/SLUG transcription repressors, and that VDR expression in colon cancer patients is reduced at advanced stages of the disease associated to the upregulation of these factors Many epidemiological and experimental studies indicate that vitamin Dmin/+Apc mice with 1,25(OH)2D3 or analogs reduces tumor load β-Catenin has a wide range of pleiotropic effects that cannot probably be explained solely by the modulation of TCF/LEF action. Thus, β-catenin has been recently described to bind several transcription factors of the nuclear receptor superfamily and homeobox proteins K-RASIt is important to highlight that the level of β-catenin in the nucleus define the strength of the Wnt signal and in consequence the fate or behavior of several types of normal and tumoral cells VDR deficiency on the initiation and development of intestinal cancer driven by the activation of Wnt/β-catenin pathway. The effect of VDR absence has been analyzed both in vivo and in vitro: in tumors generated in min/+Vdr-/-Apc mice and in cultured human colon cancer cells in which VDR expression was knocked-down by means of shRNA. In addition, we have studied the effect of VDR reconstitution in VDR-negative human colon cancer cells. Our results establish a direct link between VDR function and nuclear β-catenin levels that is crucial to control the activity of Wnt/β-catenin signaling in colon cancer in vivo.In this study we evaluate the consequences of min/+Apc and +/−Vdr mice we generated by appropriate crossings min/+Vdr+/+, Apcmin/+Vdr+/−Apc and min/+Vdr-/-Apc animals. Histological analysis at five months of age revealed that Vdr deficiency did not affect the total number of tumors in min/+Apc mice, either in the small intestine or in the colon  staining, we observed a net increase in nuclear and cytosolic β-catenin levels in the complete absence of Vdr 2D3 to induce E-cadherin expression and β-catenin relocation from the nucleus to the plasma membrane 2D3 that leads to the formation of compact epithelioid islands  was attenuated in shVDR cells 2D3 target genes. The induction of both genes was drastically inhibited in shVDR cells 2D3 to induce its target gene CYP24A1  in colon ACF and carcinomas of min/+Vdr+/+Apc, min/+Vdr+/-Apc and min/+Vdr-/-Apc mice. Accordingly with the data from cultured cells, a significant increased expression of both gene products was found in the lesions of min/+Vdr-/-Apc mice  and villin1 as markers of epithelial differentiation were used 2D3 decreases the synthesis and secretion by THP-1 macrophages of interleukin-1β, a cytokine that activates the Wnt/β-catenin pathway in colon cancer cells through the blockade of β-catenin phosphorylation by GSK-3β in vivo remained unknown.Very little is known about mechanisms to diminish the level of β-catenin within the nucleus. We have described that 1,25(OH)VDR deficiency alters β-catenin nuclear content and Wnt/β-catenin pathway in the most commonly used animal model for colon cancer, the min/+Apc mice. Our results show that Vdr deficiency in min/+Apc mice increases nuclear β-catenin levels and expression of Wnt/β-catenin target genes and, in line with these effects, enhances total colon tumor load. Consistently, knocking-down VDR by shRNA in human colon cancer cells enhances the nuclear content of β-catenin, its transcriptional activity, and the expression of Wnt/β-catenin target genes. Furthermore, transient restoration of wild type VDR expression in VDR-negative human SW620 colon cancer cells decreases nuclear β-catenin level, whereas VDR-ΔAF2, VDR-L417S or VDR-E420Q mutants, unable to bind classical coactivators and activate gene transcription Vdr-/- mice rescues alopecia but not rickets phenotype In this study we examined whether Our data show that VDR knock-down in SW480-ADH cells does not affect β-catenin phosphorylation by CK-Iα or GSK-3β, discarding a role of VDR regulating total β-catenin accumulation. Whereas nuclear β-catenin level increases in the absence of VDR, the total cellular amount of β-catenin protein is not altered . Unexpecin vivo function of VDR as crucial modulator of Wnt/β-catenin signal strength in colon cancer. The finding that VDR deficiency does not change the number of tumors but increases tumor load indicates that VDR does not block the initial mutations that provoke the early activation of the Wnt/β-catenin pathway, but that it preferentially has a long term protective effect on tumor growth by limiting the strength of the Wnt/β-catenin oncogenic signal. Concordant with our results, a very recent report has shown that min/+Vdr-/-Apc mice present larger intestinal tumors than min/+Vdr+/+Apc mice, although the molecular mechanisms behind this phenotype was not investigated Our results reveal a novel SNAI1 and SNAI2 genes that code for SNAIL1/SNAIL2 transcriptional repressors 2D3 compounds that would potentially reduce Wnt/β-catenin oncogenic function. However, when VDR expression is lost at advanced stages, tumors will not only be refractory to this type of treatment but will also acquire higher levels of nuclear β-catenin and enhanced Wnt/β-catenin signaling.The results of our study are relevant for the clinic, as VDR expression is downregulated in approximately two-thirds of advanced colon tumors associated to the upregulation of Animal work within this article was carried out in strict accordance with the recommendations by the European Union  and all the experiments were approved by the Ethical Committee for Animal Experimentation of the Consejo Superior de Investigaciones Científicas . All efforts were made to minimize animal suffering. The work involving human samples was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the Research Ethics Board of Vall d'Hebrón Institute of Oncology 79/2009). All patients provided written informed consent for the collection of samples and subsequent analysis.2D32D3 were performed in DMEM supplemented with charcoal-treated FCS to remove liposoluble hormones. Cells were treated with 100 nM 1,25(OH)2D3 or the corresponding vehicle/isopropanol concentration for the indicated times.Human SW480-ADH, HT29, HCT116 and SW620 colon cancer cells were grown in DMEM supplemented with 10% FCS and 2 mM L-glutamine . SW480-ADH cells are a subpopulation derived from SW480 cell line that express substantial VDR levels and are thus responsive to 1,25(OH)VDR expression, cells were infected with lentiviral particles containing a shRNA targeting VDR  and selected by puromycin treatment as previously described To knock-down Renilla reniformis luciferase gene (RLuc) under the control of the thymidine kinase promoter and with the TOP-Flash or FOP-Flash plasmid containing four multimerised copies of wild type (TOP-Flash) or mutated (FOP-Flash) TCF/LEF-binding sites upstream of a minimal c-fos promoter driven the expression of the Firefly luciferase gene (Luc) . Luc and RLuc activities were separately measured 48 h after transfection using the Dual Luciferase Kit (Promega). Luc activity was normalized to RLuc and the TOP-Flash/FOP-Flash ratio calculated.For analysis of β-catenin/TCF transcriptional activity, shControl and shVDR SW480-ADH cells were infected with lentiviruses containing the 7xTCF/LEF-eGFP-SV40-mCherry vector +/−Vdr and min/+Apc mice  were previously described min/+Vdr+/+Apc, min/+Vdr+/−Apc and min/+Vdr-/-Apc mice were obtained by appropriate crossings. All mice were kept in a virus- and parasite-free barrier facility. They were exposed to a 12 h light and dark cycle and allowed free access to autoclaved water and chow. At five months of age mice were sacrificed. The mice were dissected and the small intestine and colon removed and flushed with PBS. Intestines were then wound into “Swiss” rolls which were subsequently fixed O/N in 10% neutral-buffered formalin solution (Sigma-Aldrich). Then, tissues were embedded in paraffin, sectioned, deparaffinized, hydrated and stained with hematoxylin/eosin for microscopic tumor scoring. The number of tumors was counted and the size (diameter) of each tumor was measured blindly by two independent researchers using a conventional light microscope.The Cells growing on coverslips were rinsed once in PBS, fixed in cold methanol for 1 min and rinsed in PBS. Non-specific sites were blocked by incubation with PBS containing 5% BSA for 1 h at RT. Then, cells were incubated with the primary antibodies  diluted 1/100 in PBS O/N at 4°C. After four washes in PBS, cells were incubated with secondary fluorophore-conjugated antibodies (Invitrogen) and Hoechst 33342 (Sigma-Aldrich) for 1 h at RT, washed and mounted in Vectashield . Cell imaging was performed on a Leica TCS SP5 DMI6000 microscope  using argon ion (488 nm), HeNe (543 nm) and violet diode (405 nm) lasers. Images were acquired sequentially by direct register using LAS AF Leica Confocal Software.For tissue immunofluorescence, formalin-fixed paraffin-embedded sections of mouse small intestine and colon or human colon tumors were prepared and immunolabelled as described elsewhere vs blue channel) was calculated in cells transfected with empty vector or with wild type or mutant VDR (green channel). One hundred cells expressing exogenous VDRs (green positive) or the empty vector (green negative) were estimated in each condition and values were represented as arbitrary units. All quantifications were performed blindly by two independent researchers.For the quantification of the fluorescent signal of tissue immunofluorescences we used the MacBiophotonics ImageJ software. In brief, we defined Regions of Interest (ROI) and obtained the integrated density of the ROI for the green and the red channels corresponding to specific antibody staining. In parallel, we measured the Hoechst integrated density of each ROI and divided it by a standard nucleus signal. The standard nucleus signal was calculated as the average integrated density of ten nuclei taken at random. The resulting values corresponded to the number of nuclei and, therefore, to the number of cells present in each ROI. We finally divided the integrated density of the proteins of interest in the ROI by the number of cells present in the same ROI. This value indicates the level of expression of the proteins of interest per cell. To measure nuclear β-catenin levels we previously used MacBiophotonics ImageJ software to generate an image where the positive staining corresponded to coincident Hoechst (blue channel) and β-catenin (red channel) signals. To quantify the level of nuclear β-catenin in SW620 cells ectopically expressing different VDR variants, we adapted the above mentioned method to single cell measurements. Nuclear β-catenin level  in order to exclude dead, apoptotic or clumped cells. Acquisition was performed using Beckman-Coulter Moflo Cytometer  and the data obtained was analyzed with FCS Express Version 3 Software.SDHA) and TATA box binding protein (TBP), following the recommendations by Vandesompele et al. VDR, CDH1 and SDHA were previously reported AXIN2 , LEF1 , c-MYC  and TBP . Primers were designed to recognize regions of the mRNAs codified by different exons to avoid genomic DNA amplification. In addition, control samples in which reverse transcription was performed without reverse transcriptase were always included in the quantitative PCR runs. The level of CYP24A1 and DKK1 RNA were measured in relation to that of 18S rRNA using the comparative CT method and TaqMan probes (Applied Biosystems). The quantitative PCR reaction was performed in an ABI Prism 7900 HT thermal cycler using SYBR or TaqMan Gene Expression Master Mixes . Thermal cycling consisted of a denaturing step at 95°C for 10 min and 40 cycles of denaturing at 95°C for 15 s and annealing and elongation at 60°C for 60 s.RNA was extracted from cultured cells using RNeasy Mini Kit  and retrotranscribed using the High-Capacity cDNA Archive Kit . Target gene RNA expression was measured by quantitative RT-PCR in relation to the geometric average of two reference housekeeping genes, succinate dehydrogenase complex subunit A  plus phosphatase- and protease-inhibitor mixture  for 15 min on ice followed by centrifugation at 13,000 rpm for 10 min at 4°C. Western blotting of cell lysates was performed by electrophoresis in SDS-PAGE and protein transfer to Immobilon P membranes (Millipore). The membranes were incubated with the appropriate primary and secondary horseradish peroxidase-conjugated antibodies, and the antibody binding was visualized using the ECL detection system . The following primary antibodies were used: mouse monoclonal against E-cadherin, β-catenin (BD Biosciences) and active-β-catenin (anti-ABC) (Millipore); rat monoclonal against VDR (Millipore); and rabbit polyclonal against phospho-β-catenin-Ser552, phospho-β-catenin-Ser45, phospho-β-catenin-Ser675, and phospho-β-catenin-Ser33/Ser37/Thr41 . Quantification was done by densitometry using ImageJ software.Whole-cell extracts were prepared by washing the monolayers twice in PBS and cell lysis by incubation in RIPA buffer . Differences were considered significant when p<0.05.Statistical significance was assessed by unpaired Figure S1β-Catenin expression and localization is heterogeneous in colon carcinomas from min/+Apc mice and in human colon tumors. (a) Representative immunofluorescence image showing β-catenin expression and localization in colon carcinomas from min/+Apc mice. Numbers indicate regions with different pattern of β-catenin expression and localization: 1) high cytosolic and nuclear; 2) low membrane and cytosolic. Scale bar, 500 µm. (b) Representative immunofluorescence images showing β-catenin expression and localization in human colon tumors. Numbers indicate regions with different pattern of β-catenin expression and localization within the same tumor: upper panel: 1) membrane and cytosolic; 2) very high cytosolic and nuclear; 3) high membrane and cytosolic; lower panel: 1, membrane; 2, high cytosolic and nuclear. Scale bar, 100 µm.(TIF)Click here for additional data file.Figure S2VDR knock-down in human HCT116 and HT29 colon cancer cells increases β-catenin nuclear levels and transcriptional activity. (a) Analysis by qRT-PCR of CYP24A1 and CDH1/E-cadherin mRNA expression in shControl and shVDR SW480-ADH cells treated with 1,25(OH)2D3 or vehicle for 48 h. Data correspond to three independent experiments. (b) Representative immunofluorescence images showing β-catenin expression and localization in HCT116 and HT29 cells infected with lentiviruses expressing shVDR or shControl. Scale bar, 100 µm. (c) Cells were transfected with the wild type (TOP-Flash) or mutated (FOP-Flash) β-catenin/TCF reporter plasmid and luciferase activity was measured 48 h later. Values were represented as the TOP-Flash/FOP-flash ratio and related to shControl cells. Data correspond to five independent experiments. The p-values for unpaired t test with Welch's correction are indicated. (d) Analysis by qRT-PCR of VDR, CYP24A1, AXIN2 and DKK1 mRNA expression in shControl and shVDR HCT116 and HT29 cells treated with 1,25(OH)2D3 or vehicle for 48 h. Data correspond to three independent experiments.  The geometric average of the expression of SDHA and TBP housekeeping genes  or the expression of 18S rRNA (for CYP24A1 and DKK1) was used for RNA expression normalization as indicated in (TIF)Click here for additional data file.Figure S3Exogenous wild type VDR expression reduces nuclear β-catenin levels in SW620 cells. (a) Representative immunofluorescence images showing β-catenin and VDR expression and localization in SW620 cells transiently transfected with wild type (WT) VDR, or the VDR-ΔAF2, VDR-L417S or VDR-E420Q mutants for 48 h. Scale bar, 50 µm. (b) Quantification of nuclear β-catenin level in SW620 cells transiently transfected with empty vector (pSG5), wild type VDR or the indicated VDR mutants for 48 h. One hundred cells per condition were studied. Each dot corresponds to one cell analyzed and the horizontal line indicates the mean. The p-value for one-way ANOVA analysis is shown. Asterisks indicate significant differences between groups upon Bonferroni multiple comparison post-test.(TIF)Click here for additional data file.Figure S4Effect of VDR absence on β-catenin phosphorylation by several kinases. (a) Western blot analysis of β-catenin phosphorylation in residues targeted by GSK-3β , CK-Iα (Ser45), PKA (Ser552 and Ser675) or AKT (Ser552) in SW480-ADH shControl and shVDR cells treated with 1,25(OH)2D3 or vehicle for 72 h. Antibodies that specifically recognize each type of phosphorylated β-catenin or the unphosphorylated protein in Ser37 or Thr41 (active-β-catenin) were used. VDR and E-cadherin expression in the same extracts was shown as a control. A representative experiment of three performed is shown. (b) Quantification of the data obtained in three independent experiments performed as in (a). Phosphorylated β-catenin levels were normalized to total β-catenin.(TIF)Click here for additional data file.Figure S5Pan-cytokeratin and villin1 are differentiation markers in mouse colon tissue. Representative immunofluorescence images showing β-catenin and pan-cytokeratin (a) or β-catenin and villin1 (b) staining of mouse normal colon tissue. Both pan-cytokeratin and villin1 markers stain the differentiated cells located at the upper part of the normal mouse colon crypts. Scale bar, 50 µm.(TIF)Click here for additional data file.


The Wnt (Wg/Wnt) signaling cascade plays an important role in tumorigenesis. Our previous report indicated that aberrant localization of β-catenin proteins was a feature of pterygia. Therefore, this study aimed to analyze the association of β-catenin protein and expression of a downstream gene, cyclin D1, in pterygial tissues.Using immunohistochemistry, β-catenin and cyclin D1 protein expression was studied, in 150 pterygial specimens and 30 normal conjunctivas.Seventy-three (48.7%) and 60 (40.0%) pterygial specimens tested positive for β-catenin and cyclin D1 protein expression, respectively. Cyclin D1protein expression was significantly higher in β-catenin-nuclear/cytoplasmic positive groups than in β-catenin membrane positive and negative groups (p<0.0001). In addition, cyclin D1 expression was significantly higher in the fleshy group than in the atrophic and intermediate groups (p=0.006).Our study demonstrated that β-catenin expressed in nuclei/cytoplasm increases cyclinD1 protein expression, which invokes pterygial cell proliferation. Pterygium has long been considered a degenerative disease. However, the p53 protein was found to be abnormally expressed in epithelium; consequently, pterygium is now considered to be a UV exposure-related uncontrolled cell proliferation that is similar to a tumor -7.Several adhesion molecules, including cadherin, cell-cell adhesion molecules (CAMs), selectin, and integrin, maintain tissue architecture . E-cadheIn the absence of a mitotic signal, free cytoplasmic β-catenin is degraded by the ubiquitin-proteasome system following phosphorylation by the protein complex, which is composed of adenomatous polyposis coli (APC) tumor suppresser protein, Axin, and serine threonine glycogen synthetase kinase (GSK-3β) . This meIn addition, reduced expression of E-cadherin leads to both the decomposition of the E-cadherin-catenin complex and an increase in free cytoplasmic β-catenin. E-cadherin also mediates epithelial cellular adhesion. When the free cytoplasmic β-catenin increases, it eventually translocates into the nucleus and binds with transcription factors LEF (Lymphoid Enhancer Factor) and TCF (T Cell Factor), resulting in the activation of target genes ,18 such 1-phase by forming active holoenzymes with CDK (cyclin-dependent kinase) 4 and CDK6 and leads to phosphorylation of pRb (retinoblastoma protein) [1–S transition and S-phase [Cyclin D1 is a well known cell cycle control gene that promotes cell cycle progression through the Gprotein) . Phospho S-phase .Our previous report indicated that promoter hypermethylation of E-cadherin may be involved in the reduction of E-cadherin protein expression in pterygium . We alsoIn the present study, we hypothesized that β-catenin expressed in nuclei/cytoplasm could increase cyclin D1 protein expression to involve the formation of pterygia. To test these hypotheses, we analyzed both the expression of β-catenin and cyclin D1 in pterygium and the relationship between β-catenin protein localization and cyclin D1 protein expression.Pterygial samples were harvested from 150 patients undergoing pterygium surgery and the patients were asked to submit a written informed consent approved by the Institutional Review Board. Patients in whom the apex of the pterygium had invaded the cornea by more than 1 mm were included in this study. The pterygia were classified into grades 1, 2, or 3 based on slit-lamp biomicroscopy evaluation. Grade 1 (“atrophic”) had clearly visible episcleral vessels under the body of the pterygium; grade 2 (“intermediate”) had partially visible episcleral vessels under the body of the pterygium; grade 3 (“fleshy”) had totally obscured episcleral vessels underlying the body of the pterygium. The controls included normal conjunctival samples collected from the superior conjunctiva of 15 patients and the medial conjunctiva of 15 patients without pterygium and pinguecula; all patients were undergoing cataract or vitreoretinal surgery. There were 87 males and 63 females in the pterygium group , and there were 15 males and 15 females in the control group . Normal conjunctival samples were collected from bulbar conjunctivas. All pterygial specimens came from primary pterygia. All specimens were fixed in formalin and paraffin embedded.All sections were deparaffinized in xylene, sequentially rehydrated through serial dilutions of alcohol and washed in phosphate-buffered saline. Sections used for β-catenin and cyclin D1, detection were heated in a microwave oven twice for 5 min in citrate buffer (pH 6.0). Mouse anti-β-catenin and a cyclin D1 monoclonal antibody  were used as the primary antibodies. The visualization was performed by DAB  using the DAKO LSAB/HRP kit . Negativ2 test were applied for statistical analysis. A p<0.05 was considered to be statistically significant.A statistical analysis was performed using the SPSS statistical software program . The Fisher’s exact test and χWhen the free cytoplasmic β-catenin increases, it ultimately translocates into the nucleus, resulting in the activation of cyclin D1 and c-myc, which are responsible for cell proliferation and neoplastic transformation ,20. We fTo understand the relationship between the β-catenin and cyclinD1 protein expression, the correlations of β-catenin localization and cyclin D1 protein expression were analyzed . As showTo understand whether the cyclin D1 protein expression was correlated with the pterygium development, the correlation of cyclin D1 protein expression and the grade of pterygium was analyzed. As showed in Pterygium, previously considered a degenerative process of the corneal limbus, pterygium is characterized by the invasion of a fleshy triangle of conjunctival tissue onto the cornea. Shimmura et al. demonstrated an increased activity of telomerase in pterygial epithelial cells, indicating their hyperproliferative nature . Satoru , such as cyclin D1 and c-myc [Previous reports indicated that the nuclear β-catenin associated with TCF/LEF proteins activates target genesnd c-myc ,20,27, wnd c-myc ,20,27,28nd c-myc . A prevind c-myc . In addind c-myc ,31. Thesnd c-myc . We alsond c-myc . In the Studies of cyclin D1 expression in pterygium are very few in number. Kase et al.  found thIn conclusion, our study is the first to provide evidence showing that the cyclin D1 protein may be activated through β-catenin protein nuclear/cytoplasmic expression. These data in our study provided evidence that changes in β-catenin signaling may contribute to cell proliferation in pterygium.


In their widely noticed study, Gergely, Bekkering, and Király (2002) showed that 14-month-old infants imitated an unusual action only if the model freely chose to perform this action and not if the choice of the action could be ascribed to external constraints. They attributed this kind of selective imitation to the infants' capacity of understanding the principle of rational action. In the current paper, we present evidence that a simpler approach of perceptual distraction may be more appropriate to explain their results. When we manipulated the saliency of context stimuli in the two original conditions, the results were exactly opposite to what rational imitation predicts. Based on these findings, we reject the claim that the notion of rational action plays a key role in selective imitation in 14-month-olds. It is a demanding task for infants to filter relevant information from the enormous amount of input they receive from their environment. Nevertheless, one-year-old infants already selectively use this information to guide their own action production. A prominent example is the phenomenon of selective imitation. In a widely recognized study, Gergely, Bekkering, and Király The authors proposed an intriguing explanation for this phenomenon, referring to a rational action account This work is since regarded as evidence that infants draw rational inferences which influence whether they imitate a model's behavior or not e.g. . In the In both conditions, the model put on a blanket before illuminating the lamp. In the hands-free condition, she only loosely put it over her shoulders. In the hands-occupied condition, however, she pretended to be cold and wrapped herself in the blanket, holding it together from underneath in front of her torso. The blanket thus completely covered her upper body, forming an eye-catching outfit. On top of the unusual head action performed in both conditions, there was thus an additional unusual and therefore salient feature in the hands-occupied condition. This feature may, as a perceptual distractor, have competed with the head action, drawing the infant's attention away from it. Accordingly, we propose a perceptual distraction approach to account for infants' selective imitation. While the importance of directing the infant's perception towards the model's action is widely acknowledged in imitation paradigms e.g. , systemahands-occupied familiarization condition), we aimed to reduce perceptual distraction from the strange and unexpected appearance of the model by familiarizing the infants with the sight of this appearance during the 5-minute warm-up phase which in all conditions preceded the imitation task. Second, in a new version of the hands-free condition (hands-free distraction condition), we aimed to enhance perceptual distraction by placing red smileys on the table.We tested the hypothesis of perceptual distraction from the head action due to the presence of salient context stimuli in the modeling phase by introducing two manipulations in addition to the original conditions see . First, For these two additional conditions, opposing outcomes can be hypothesized from the two alternative approaches, rational action and perceptual distraction. According to the rational action approach, infants will imitate the head action in the hands-occupied familiarization condition as infrequently as in the original hands-occupied condition. They should infer that the model performed this unusual action due to situational constraints . If anything, the fact that the model was already wrapped in the blanket and could not use her hands during the warm-up phase should make these situational constraints even more obvious. By contrast, according to the perceptual distraction approach one would predict the opposite – that infants will show more imitation of the head action in the hands-occupied familiarization condition than in the original hands-occupied condition. Once familiarized with the sight of the covered model, they should be in a position to focus on the unusual head action in a manner similar to in the hands-free condition.Following the same logic, the two approaches also lead to opposite predictions in the hands-free distraction condition. Rational action predicts that infants will imitate the head action because the model's action is not constrained in any obvious way. By contrast, perceptual distraction predicts that the smileys are salient context stimuli which will distract the infants from the target action, leaving less capacity for encoding, and hence imitating it. The likelihood of imitation will thus be reduced.M = 13 months, 27 days; range 13.15 to 14.15) participated in the experiment. They were randomly assigned to one of four experimental groups . Ten additional infants were tested, but had to be excluded from the final sample due to parental interference, fussiness, procedural errors, or lack of interest. The experimenter was a female adult. Infants were recruited from a database of parents who had agreed to participate in infant studies. Parents gave informed written consent prior to the experiment. The study was approved by the local ethics committee of the University of Leipzig, and conducted in accordance with the Declaration of Helsinki.Fifty-eight 14-month-olds  which was fixed on a black panel and could be illuminated by touching, and a blue blanket (145×190 cm) served as material. Additionally, in the hands-free distraction condition, two black boxes , each with a red smiley centrally attached, were used. The infants' and experimenter's behavior were recorded by two cameras.The experimenter entered the test room together with the infant and one parent. All conditions started with a 5-minute warm-up phase during which the experimenter played with the infant using a soft ball. The infant was then seated on the parent's lap on one side of a table, and the experimenter sat on the opposite side with the lamp in front of her. Experimenter and infant continued playing with the ball for a minute. In the following experimental phase, the experimenter called the infant by his or her name and said: ‘Look what I am doing!’ She then bent down, illuminated the lamp for 2 seconds using her forehead and returned to the upright position. This sequence was repeated three times. Subsequently, the experimenter put the lamp in front of the infant, said: ‘Now you can play with it!’ and left the room. The child was given 60 seconds to explore the lamp At the beginning of the experimental phase, the experimenter pretended to be cold and wrapped herself in a blanket. Her hands were occupied holding this blanket while she illuminated the lamp.At the beginning of the experimental phase, the experimenter put a blanket loosely around her shoulders. Her hands were visible and free while she illuminated the lamp.At the beginning of the warm-up phase, the experimenter pretended to be cold and wrapped herself in a blanket. From this moment on, her hands were occupied holding the blanket. Like in the other conditions, she played with the infant, but in this condition without using her hands . In the experimental phase, she continued to hold the blanket. Her hands were thus occupied while she illuminated the lamp.At the beginning of the experimental phase, the experimenter put a blanket loosely around her shoulders. She then put two smilies to her left and right on the table. Her hands were free and visible while she illuminated the lamp.In all conditions, the experimenter laid the blanket aside after she had performed the head action and before she put the lamp in front of the infant.2χ = 5.14; p<.05.All infants touched the lamp with their hands  = 5.81; p<.05, and did not differ from the original hands-free condition, 2χ = 0.01, p = .91.More notably, the additional manipulations had significant effects as well see : Althoug2χ = 1.8; p = .18). The difference to the original hands-occupied condition was not significant either, 2χ = 1.0; p = .32). However, if the conditions were ranked in the following order, hands-free, hands-free distraction, hands-occupied, there was a significant correlation between the performance of the head touch  and condition: Across the three conditions, there was a significant increase in the likelihood of imitation, d = .32; p<.05, Somer's d coefficient.Furthermore, although the model's hands were free in the hands-free distraction condition, only 8 out of 15 infants (53.3%) imitated the head touch. In the ranking of conditions, the likelihood of imitation thus fell between the two original conditions. The difference to the original hands-free condition, however, failed to reach significance, In previous work, selective imitation in 14-month-olds has been explained by the infants' ability to apply the principle of rational action Up to now, many studies have demonstrated that the manipulation of saliency affects whether and what kind of information infants perceive, and that selective perception in turn influences action performance Another alternative explanation for selective imitation in infants has recently been proposed by Paulus, Hunnius, Vissers, and Bekkering With some adaptations, the paradigm used by Gergely et al. To sum up, the present study demonstrates that the phenomenon of selective imitation in 14-month-olds does not require the demanding rational action approach, but can be comprehensively explained by a perceptual distraction approach. More importantly, our findings actually rule out the possibility that the rational action approach can serve as an explanation for the observed effects. In contrast to that approach, infants nonselectively imitate new and unusual means for goal achievement – provided that perceptual distraction by context stimuli present in the modeling phase is controlled in a way that allows them to encode these means. The rational action approach should thus be rethought in debates on infant imitation.