Wnt/β-catenin signaling is involved in different stages of mammalian development and implicated in various cancers . Recent experimental and computational studies have revealed characteristics of the pathway, however a cell-specific spatial perspective is lacking. In this study, a novel 3D confocal quantitation protocol is developed to acquire spatial (two cellular compartments: nucleus and cytosol-membrane) and temporal quantitative data on target protein (e.g. β-catenin) concentrations in Human Epithelial Kidney cells (HEK293T) during perturbation (with either cycloheximide or Wnt3A). Computational models of the Wnt pathway are constructed and interrogated based on this data.A single compartment Wnt pathway model is compared with a simple β-catenin two compartment model to investigate Wnt3A signaling in HEK293T cells. When protein synthesis is inhibited, β-catenin decreases at the same rate in both cellular compartments, suggesting diffusional transport is fast compared to β-catenin degradation in the cytosol. With Wnt3A stimulation, the total amount of β-catenin rises throughout the cell, however the increase is initially (~first hour) faster in the nuclear compartment. While both models were able to reproduce the whole cell changes in β-catenin, only the compartment model reproduced the Wnt3A induced changes in β-catenin distribution and it was also the best fit for the data obtained when active transport was included alongside passive diffusion transport.This integrated 3D quantitation imaging protocol and computational modeling approach allowed cell-specific compartment models of the signaling pathways to be constructed and analyzed. The Wnt models constructed in this study are the first for HEK293T and have suggested potential roles of inter-compartment transport to the dynamics of signaling. The pThe computational-experimental model developed by Lee and colleagues  is stillRecent Wnt signaling models have progressed towards spatial multi-compartmental models ,28-31 taThe aim of this study was to interrogate the Wnt pathway in HEK293T cells . HEK293The 3D confocal microscopy protocol employed in this study was adapted from the confocal compartment cell volume measurement protocol established previously . SpecifiThree sets of independent experiments were performed for the two types of perturbation Wnt3A or CHX), each experiment having 4 time points . Figure A or CHX,0 in 4 hours, p-value,T0-T240 p = 0.02) whereas during CHX treatment β-catenin levels decreased . Under Wnt3A stimulation . After this initial rapid rise of nuclear β-catenin in the nuclear compartment, the rate of increase in the nuclear compartment concentration of β-catenin reduced  and appeared to be similar to the rate of change in the concentration in the cytosol-membrane compartment . For CHX inhibition, a decrease in β-catenin levels was observed in the nuclear compartment , with potentially an oscillation observed. For the cytosolic-membrane compartment, the decrease in β-catenin was non-significant during the duration of the experiment .Qualitatively, the 2D images Figure . Visual 0 (∆NCM) was plotted, indicating the shift in β-catenin between the compartments due to the specific perturbations in time (i.e. a greater ∆NCM implies a shift of β-catenin levels to the nuclei compartment as compared to T0). Wnt3A stimulation increased the ∆NCM by 0.22 ± 0.04  within the first hour and was followed by a steady rate of β-catenin increment in both compartments as indicated by a constant ∆NCM of approximately 1.2 . For CHX inhibition, the ∆NCM decreased over the 4 hours of CHX incubation , indicating a gradual shift of β-catenin to the cytosol-membrane compartment. At the onset of Wnt3A stimulation the analyses showed a rapid increase of β-catenin concentration in the nucleus and with CHX inhibition, a slow gradual decrease of β-catenin in both compartments. These observations demonstrated the ability of the protocol to successfully quantify and monitor (in 3D) the concentration of a specific target protein and its localization in intracellular compartments.This redistribution of β-catenin between compartments was quantified using the ratio of the β-catenin level in the nucleus (N) compartment and cytosol-membrane (CM) compartment (the N:CM ratio). An N:CM ratio > 1.0 implies greater nucleus β-catenin concentration than that in the cytosol-membrane compartment. In Figure 0-T240 p = 0.05). In the presence of CHX, total N-cadherin decreased 17 ± 10% after 1 hour and then remained stable (±1.7%) over the remaining 4 hours .Visual inspection of 2D images Figures  for endoTo validate the results of the above 3D quantification technique, quantitative Western blots were performed under the same perturbation and culture conditions (as described in the Methods section). Five sets of independent quantitative Western blot experiments were conducted with HEK293T cells. β-tubulin was used as the loading control in these experiments and as a reference for normalization between the samples. Extracts from cells incubated with either Wnt3A or CHX for 0, 1, 2 or 4 hours were loaded onto the gels as shown in Figure There are two types of experimental data presented in this study. First, we had whole cell data in which spatial information was hidden by summing up the changes in β-catenin within subcellular compartments .Here we show that once diffusion and active transport mechanisms between compartments are included (Refer to Table C and BN were the free β-catenin concentrations in either compartment (C or N), CC and CN were the bound β-catenin-ligand complex concentrations in either compartment (C or N) and LC and LN were the free ligand concentrations in either compartment (C or N). synthesis was the β-catenin synthesis rate, kdegradation was the β-catenin degradation rate, kdiffusion was the diffusion rate constant and included contributions of the nuclear membrane surface area, effective membrane thickness and diffusion coefficient on the mass flux between compartments by passive diffusion. Finally, active transport between compartments was described using the constants where: the superscripts denote either cytosol-membrane (C) or nuclear (N) compartments. At steady state the two compartment model requiredand that0), the total β-catenin concentration in the cytosol-membrane compartment was calculated as 465.5nM and in the nucleus as 515.8nM. Therefore, in the absence of active transporters, there was a lower bound β-catenin steady state concentration In the absence of active transport at steady state, i.e. prior to perturbing the system with Wnt3A or CHX, Equation (8) could only be satisfied if  studies , initialBsynthesis = 0 for CHX inhibition and kdegradation = 0 for Wnt3A stimulation. We tested both conditions with (active) or without (passive) active transport and attempted to calibrate the model in each case. Unlike the single compartment model, our optimal solutions  ,16 as weIn order to bridge the gaps in the existing data on the cellular distribution of protein components, a protocol using immunofluorescence, confocal microscopy was developed and validated for analysis of the 3D sub-cellular localization of target proteins in whole cells. This protocol allowed the concentrations of target proteins in different compartments of the cells to be determined and changes in concentrations to be analyzed under specific perturbations at different times.It is difficult to use transfection of fluorescently-tagged proteins (β-catenin in this case), as often the levels, distribution and turnover of the recombinant forms can be quite different to the endogenous proteins. Unless it can be validated that an introduced recombinant protein behaves in a similar manner to the endogenous protein, the use of live cell imaging has limited utility for the study of signaling systems. Therefore, individual formaldehyde fixed samples at specific time points were used. In this case it was essential to have a reference fluorescence probe to normalize the results between the different samples and different time points. We used the InSpeck™ Green beads in each sample as intensity markers.This imaging quantitation technique provided insights previously unavailable from the cellular extract experiments, in particular protein localization under different perturbations in intact cells. Critically, we observed a distinctly different dynamic behavior for the Wnt signaling proteins in HEK293T mammalian cells than for the Xenopus oocyte cell-free extract. In particular, the rate of β-catenin degradation in HEK293T cells was relative slow (15% decrease in 1 hour compared to a 50% decrease for Xenopus cell free extracts). From the analysis of the transient change in compartment β-catenin concentrations, Wnt stimulation increased the β-catenin concentrations in both the nucleus and cytosol-membrane compartments. Although over 4 hours the rate of increase appeared to be similar in both compartments, an initial rapid increase in β-catenin concentration was observed for the nuclear compartment. This was reflected in the observed rapid increase in the normalized N:CM ratio (∆NCM) in the first hour, before stabilizing at ~1.2 for the duration of the experiment. These results suggest that there is an active transport mechanism, stimulated by Wnt, which concentrates β-catenin in the nuclear compartment. One possibility is that Wnt stimulation increases the activity of potential transporter proteins  for HEK293T was available, the initial value (kdiffusion = 36 m∙min−1) was estimated using information from the literature and physics  whereas in the case of no active transport kdiffusion = 1354 m∙min−1 . This suggests that active transport is likely to be involved in determining the compartment distribution of β-catenin in HEK293T cells under Wnt stimulation. For both conditions of Model 2, the model’s initial response to Wnt3A was too slow. This was evidenced by both sets of simulations missing the nuclear β-catenin concentration data point at 60 minutes for Wnt3A stimulation. The other major change in parameters is that the β-catenin equilibrium constant in the nucleus for both simulations increases  that will modulate its levels in specific compartments. The simulations presented in this study suggest that a multi-compartment model, in contrast to the homogeneous Lee et al. whole cell model of Wnt signaling (that takes into account compartments and active transport mechanisms) was needed to understand the Wnt3A signaling pathway. Although Model 2 did not take into account the variety of potential binding proteins found in Model 1, using a generic binding partner instead, this simplified β-catenin model provides a good platform for advancing a Wnt signaling model which is more representative of real life systems.The schematic illustrating Model 2 is similar to that recently proposed by Schmitz et al. . SchmitzOne approach for constraining computational models of biological systems is to acquire and optimize for both spatial and temporal data for the signaling components. In most studies, either spatial data or temporal data are available, but seldom are both acquired together. This was one of the key objectives of the methods introduced in this study, to acquire both spatial (compartments) and temporal (time-course) data using immunofluorescence. Computationally, the typical approach has been to use ordinary differential equations and generic rate constants to approximate and optimize these data for simulating complex biological pathways. However, in a biological system, cell growth causes volume changes which could change the associated rate constants in time . As moreComputationally, the optimization process to find the best solution for a complex signaling network is not simple. As the number of parameters in a model increase, the number of permutations to be considered for optimization increases exponentially. As an example, in our study, each model includes four flux terms (two each for Axin and β-catenin) so four unique sequences would require 24 permutations. Approximating each simulation having 1000 random starting points, each search typically requires 100 iterations, a total of 2,400,000 simulation cycles are required. Thus the computational requirements and time will be considerable and constraining. The use of a topological analysis approach  for each cell in a monolayer or even an aggregate of cells.One potential limitation of our experimental system is the number of available confocal channels that can be used for each analysis. Current available microscopes had three to four fluorescent channels, which were shared among monitoring target proteins as well as markers for specific compartments. This study used three channels for both compartment probes and target protein identification. To monitor more target proteins, the procedure had to be repeated for each additional protein of interest. The number of compartments which can be analyzed depends not only on the availability of confocal channels, but also on the suitability/specificity of the marker probes which define the compartments. In this study, two compartments (the nuclei and cytosol-membrane compartment) were identified and analyzed quantitatively. For the study of epithelial cells monolayer or aggregates, it would have been ideal to be able to analyze the membrane compartments separately. Unfortunately, efficient isolation of the membrane fluorescence was limited by membrane marker suitability. Despite many attempts to effectively distinguish and isolate the membrane fluorescence from the cytosol, we were unable to resolve the boundaries between neighboring adherent cells at various depths We note that the compartment concentration distribution of β-catenin reported here does not correlate with our previously reported β-catenin distributions using sub-cellular fractionation technique . The subOther components of the cell also modulate the distribution and function of β-catenin. For example, the Cadherin family proteins are key adhesion partners of β-catenin in the cell-cell adhesion processes ,50. ThesIn this study, we have developed and applied an integrated experimental computational approach to investigate the spatial and temporal dynamics of Wnt signaling in HEK293T cells. A 3D quantitation protocol using 3D confocal microscopy, immunofluorescence and image processing was developed to acquire quantitative cellular compartment (nucleus and cytosol-membrane) and temporal data for target protein β-catenin concentrations under different signaling perturbations. Our results suggest that β-catenin diffusion between compartments is faster than β-catenin degradation in the cytosol and that Wnt stimulation increases total β-catenin throughout the cell. Initially (~first hour), there is a faster increment of β-catenin in the nucleus. This experimental data was used to parameterize mathematical models of Wnt signaling in HEK293T cells. Computational analysis comparing a single and a two compartments model were conducted. Both models were able to reproduce whole cell changes in β-catenin. However, only the two compartment model could reproduce the Wnt3A induced β-catenin changes, and the best reasonable fit was obtained with the inclusion of an active transport component alongside passive diffusion to account for the rate of transfer of β-catenin to the nucleus. This suggested a role of active transport and compartmentalization in regulating both the spatial and temporal dynamics of β-catenin when HEK293T cells are stimulated by Wnt3A.The antibodies used in this study were: mouse monoclonal anti-β-catenin  and mouse monoclonal anti-N-cadherin . The anti-β-catenin marks the target protein endogenous β-catenin in the cell while the N-cadherin was used both as a marker to outline the whole cell as well as to stain the endogenous N-cadherin protein in the cell. The fluorescent stain 4, 6-diamidino-2-phenyl indole Nucleic Acid Stain  was used to stain the nuclei. The secondary fluorescent dyes used were Alexa Fluor ®488 goat anti-rabbit IgG (H + L)  and Alexa Fluor® 546 goat anti-rabbit IgG (H + L) .Perturbation of the Wnt pathway was conducted using a known activator/stimulator of the Wnt signaling pathway , Wnt3A , were utilized as the intensity reference for confocal microscopy. 0.3% rated microspheres were identified for use in these experiments based on a selection criteria outlined in Additional file HEK293T, a human kidney epithelial cell line  was usedThe fixed and stained cell samples were mounted onto Sykes Moore Chambers  with an aliquot of InSpeck™ intensity microspheres PBS mixture and allowed to settle. As this was a fixed time-course system with independent samples of cells on a different cover slip, the InSpeck™ intensity microspheres were used as the intensity reference to facilitate comparisons between the samples ) to obtain the resultant β-catenin measurements for each sample. In this study, either Wnt3A or the inhibitor CHX was added to the cells and spatial 3D image stacks acquired 0, 1, 2 or 4 hours after adding the reagent.Note that for compartmental analysis, only two sub-cellular compartments (nuclei and cytosol-membrane) are defined. Experimentally it was not possible to distinguish the membrane compartment from the cytosol, primarily due to interferences from neighboring cells in 3D space. With improved image processing and membrane markers, subsequent studies will be aimed at progressing to a three compartment model.Quantitative western blots were conducted to validate the 3D microscope fluorescence intensity quantitation technique. Specifically, HEK293T cell cultures were plated in 6 well plates with DMEM + 10% FCS and cultured overnight (until about 70 ~ 80% confluent). The media was removed and replaced with new media containing Wnt3A (40% Wnt3a L-cell CM in DMEM + 10% FCS) or CHX (100 μg/ml) for respective wells at predefined times. The cells were incubated for the required time intervals . HEK293T cultures had been incubated with either Wnt3A or CHX for the 0, 1, 2 or 4 hours at 37°C .At the end of incubation, the cells were harvested, lysed in lysis buffer on ice containing 1% triton-X100 and 1% deoxycholate, and the debris spun down at 13000 rpm for 10 minutes at 4°C. The cell extract was collected as the whole cell lysate and stored at −20˚C. Quantitative SDS-PAGE was conducted with SDS sample buffer  and DTT using NuPAGE 4-12% Bis-Tris Gels. In the analysis, recombinant β-catenin proteins were loaded in the same gel to form the standard curve for quantitation.Proteins were transferred to Nitrocellulose or PVDF membranes and immuno-blotted with primary mouse monoclonal anti-β-catenin, mouse monoclonal anti-β-tubulin antibodies  and secondary antibody . The resulting blot was scanned using the Odyssey infra-red scanner at 800 μm and results analyzed using the LI-COR Odyssey™ application software , quantifying the protein band intensities and normalizing with the respective β-tubulin intensities (as control). The quantitative western blots results were compared to the confocal 3D-measurements.The computational models developed in this study are referred to as Models 1 and 2 Figure A and B. Model 1 extends the Lee et al. model to also account for additional potential permutations of the key proteins complexes . The following reactions had been added, V18: binding of GSK3β with APC  form and solved in Matlab version R2007a (The Mathworks Inc.) using the stiff solver ‘ode23s’ . Model os Figure C. Stage Each stage required two modeling phases  [03 [20]).The authors have declared that no competing interests exist.CT, YH and AWB conceived and designed the experiments. CT and YH performed the experiments. CT, YH and AWB analyzed and interpreted the experimental data. CT and BSG conducted the computational modeling. CT, BSG and DWS evaluated the computational model results. CT drafted the manuscript. CT, BSG, DWS and AWB participated in revising the paper. All authors have read and approved the manuscript.Supplementary Materials. This file provides further details of confocal imaging protocols and computational processing analysis utilized in this study. Documentation includes Supplementary Text sections 1.1-1.10, Tables S1-S7 and Figures S1-S5.Click here for fileSBML file for Model 1. The SBML file Model1.xml can be browsed using JDesigner of the Systems Biology Workbench. Please download JDesigner at http://sbw.sourceforge.net/, install it, and open the SBML file Model1.xml to browse the Wnt signaling model.Click here for fileSBML file for Model 2. The SBML file Model2.xml can be browsed using JDesigner of the Systems Biology Workbench. Please download JDesigner at http://sbw.sourceforge.net/, install it, and open the SBML file Model2.xml to browse the Wnt signaling model. Use the ‘Manage Parameter Sets’ option to switch between parameters for Model 2 (active) and Model 2 (passive).Click here for file


RMRP, the RNA component of mitochondrial RNA processing endoribonuclease, is a non-coding RNA (ncRNA) part of the RNase MRP complex functioning in mitochondrial and ribosomal RNA processing. Even though various mutations in the RMRP gene are linked to developmental defects and pathogenesis, its relevance to cancer etiology has not been well established. Here we examined the expression of RMRP and found a significant increase in colorectal and breast cancer patient tissues. So we tested whether the oncogenic signaling pathways, Wnt/β-catenin and Hippo/YAP pathways, are relevant to the enhanced expression of RMRP in cancer cells because of the predicted β-catenin/TCF and YAP/TBX5 elements in the upstream regions of the RMRP gene. As expected, Wnt signal activation significantly induced the RMRP transcription thru β-catenin and YAP transcription factors. More importantly, YAP protein was critical for RMRP transcription by association to the proximal site near the transcription start site of the RMRP gene, a Pol III promoter, along with β-catenin and TBX5 proteins. We propose that the interplay of Wnt and Hippo signaling pathways could regulate target genes, coding or non-coding, by the β-catenin/YAP/TBX5 transcription complex in cancer cells. RMRP), is well documented to be essential for normal development and growth, as shown by the incapacitating mutations in cartilage-hair hypoplasia [RMRP forms the RNase MRP complex required for pre-rRNA processing to 5.8S rRNA [RMRP is regulated by cellular components is not understood at the present time.Recent advances of transcriptome analysis envision multiple functions for various non-coding RNAs (ncRNAs), but their crucial roles in the survival, proliferation and growth of organisms remain elusive , 2. Howepoplasia , 4. RMRP.8S rRNA  and for .8S rRNA . In addiWnt signaling is critical for the development and homeostasis in metazoans with β-catenin being a critical transcription activator . In normThe Hippo signaling pathway is important for cell death induction, cell proliferation suppression and tissue growth control . The HipThe extensive interrelationship of Wnt and Hippo signaling pathways has emerged as an important tumorigenic signaling network in some cancer cells -21. In nRMRP as a common target gene of the Wnt and Hippo signaling pathways. Even though the RMRP gene harbors the RNA polymerase III (Pol III) promoter [RMRP transcription is elevated in cancer cells by β-catenin and YAP activation. Especially, the YAP protein is a key transcription factor in this process by associating to the RMRP promoter very close to the transcription start site, TATA box. In addition, β-catenin also forms the transcription complex along with YAP and TBX5 at the same site. So it is possible that the interplay of Wnt and Hippo pathways could enhance the tumorigenic potential, probably by RMRP-mediated proliferative functions. Moreover, common target genes activated by YAP and β-catenin, such as RMRP as shown here, could provide novel oncotargets for cancer therapeutics.Here we have identified promoter , here weRMRP expression level in patient tissues and cancer cells by RT-PCR as well as by real-time qRT-PCR. RMRP level was low in nonmalignant lung epithelial Beas-2B cells immortalized by adeno12/SV40 virus. In comparison, higher levels of RMRP were detected in embryonic kidney epithelial HEK293 cells, transformed HEK293T cells, and many malignant cancer cell lines, such as colorectal cancer HCT116, SW480 and HT-29, lung cancer H1299 and cervical cancer HeLa  mRNA, which is frequently overexpressed in various cancer patient tissues. Interestingly, enhanced level of RMRP was correlated with that of β-catenin mRNA in breast cancer tumor tissues , (A/T)(A/T)CAA(A/T)G, were noticed between 3-4 kb upstream to the transcription start site of the RMRP gene  assay was performed for three TBEs in the upstream of the RMRP gene. ChIP experiments clearly showed the FLAG-β-catenin recruitment to TBEs in HEK293 cells  with 10 different proteins and plays essential roles during development and growth [RMRP seem to be relevant to the regulation of two distinct functions [RMRP or heterozygous null of RMRP in mouse clearly demonstrates a developmental lethal phenotype [RMRP gene have been reported, some of which are in the promoter region of the RMRP gene [RMRP mutations cause the reduction in RMRP level and associate with cartilage-hair hypoplasia, reduced functions of the RNase MRP complex contribute to the pathogenesis [RMRP is increased upon Wnt activation, it is not clear at this moment whether additional RNase MRP is assembled upon Wnt stimulation. However, the increase of mitochondrial and ribosomal processing activities indirectly points to the fact that functional RMRP is likely to be generated by Wnt activation. Recent studies propose that RMRP can play a critical role as the alternative RNP complex with hTERT (Telomerase Reverse Transcriptase) acting as a RNA-dependent RNA polymerase [RMRP can be induced by Wnt stimulation [d growth . Consideunctions , 37. ConMRP gene . Since mogenesis . Even thlymerase , 39. It mulation .RMRP contribute to proper growth in humans so that the tight regulation of the expression level and localization of RMRP might be linked to normal human development [RMRP and other ncRNA enhancement via nuclear β-catenin and the YAP protein, all of which could be relevant to tumor cell formation and progression.Diverse sets of non-coding RNAs, either short or long, have been discovered recently . Some ofelopment . Therefo2 incubator. Embryonic kidney epithelial HEK293 and HEK293T  were cultured in MEM medium (Hyclone). SW480 and HT-29 , HCT116  and H1299  were cultured in RPMI 1640 medium (Hyclone), HeLa cells  were cultured in DMEM high glucose medium (Hyclone) with 10% FBS and 1x antibiotics (Hyclone) at 37°C in 5% CO2 incubator. Matched pairs of primary tumor and adjacent normal tissue samples were obtained from the Liver Cancer Specimen Bank, supported by the National Research Resource Bank Program of the Korea Science and Engineering Foundation in the Ministry of Science and Technology. Consent to use the individual tissue specimens for research purposes was obtained from the patients, and the utilization of the specimens for this research was authorized by the Institutional Review Board of College of Medicine, Yonsei University, Korea (provided from Dr. Hogeun Kim)Beas-2B lung transformed cells  were cultured in defined keratinocyte serum-free medium (Gibco) at 37°C in 5% COTM real time PCR systems (Life Technologies) was used for real-time PCR. Primers for PCR were listed in RNA was isolated using Trizol reagent . For reverse transcription, 2 ug of total RNA was used. StepOneCells were lysed in RIPA lysis buffer . After loading proteins on the 8-12% SDS-PAGE gel, they were transferred to PVDF membrane (BMS). Antibodies used for this research were as listed as below: β-catenin (Epitomics #1247-1 and BD biosciences) used with 1:3000, YAP  used with 1:500, phospho-YAP (Ser127)  used with 1:1000, Lamin B1 (Abcam ab16048) used with 1:5000, β-actin (Abcam ab6276) used with 1:10000, Myc-tag (Millipore 05-724) used with1:3000, FLAG (Sigma F3165) used with 1:3000.RMRP (100 ng/ml), 5.8S ribosomal RNA (5 ng/ml), U6 small nuclear RNA (100 ng/ml) and 5S ribosomal RNA (6 ng/ml). List of probes is shown in DIG Northern Starter Kit (Roche) was used for non-radioactive northern blot analysis. RNA was loaded on the denaturing 7 M urea-6% polyacrylamide gel and transferred to the Nylon membranes (Roche) for 1 hour. After UV cross-linking, the probes were treated on membranes and hybridized overnight. Washing was as follows: 2xSSC and 0.1% SDS buffer at room temperature for 5 min twice under constant agitation, second washing was in 0.1xSSC and 0.1% SDS buffer at 68°C for 15 min three times under constant agitation. With anti-digoxigenin-AP antibody, AP reaction was carried out for more than 30 min. The probes used for this study were RMRP probe  was added onto the coverslip and the hybridization was carried out overnight. Washing was done in SSC buffer.After fixing, cells were permeablized with 0.5% Triton X-100 in 1x PBS buffer for 10 min. Blocking was carried out for 1 hour in 5% normal horse serum in 1x PBS, then primary antibodies were added onto the coverslips for immunocytochemistry. Secondary antibodies were treated onto coverslips for 1 hour. For FISH, TM real time PCR systems (Life Technologies) was used for real-time PCR of precipitated DNA.After fixing cells with 1% formaldehyde for 15 min, cells were treated by 125 mM glycine for 10 min. Cells were lysed in ChIP RIPA buffer  and sonication was carried out . Protein G sepharose beads  were mixed with cell lysates for pre-clearing and 500 ug proteins were mixed with 2-3 ug antibodies. Washing was carried out 5 times in low salt buffer , high salt buffer  once, LiCl wash buffer  once, TE buffer  twice. DNA was eluted in elution buffer  and RNA was removed. To revert the cross-linking, the samples were incubated at 65°C overnight. StepOne2, 1x protease inhibitor cocktail, RNase inhibitor 100 units/ml) and then 0.4 ug/ml digitonin was added to the tubes. After 10 min incubation in ice, the samples were centrifuged at 4,400 rpm for 8 min at 4°C. Cytoplasmic extract was obtained from the supernatant. RIPA lysis buffer  was added to the pellet and then the samples were incubated in ice for 15 min. They were centrifuged at 13,200 rpm for 15 min at 4°C. Nuclear extract was obtained from the supernatant.Cells were added in RSB buffer  was added to the cells, it was incubated in ice for 10 min. Homogenization was carried out using homogenizer with 5,000 rpm ten times. Cell lysates were centrifuged at 1,000 x g for 10 min at 4°C. The supernatant was centrifuged at same condition, followed by being centrifuged at 13,000xg for 20 min at 4°C. Homogenization buffer was added to the pellet and then it was centrifuged at 13,000 x g for 10 min at 4°C. Lysis buffer  was added to the pellet. After incubation at 55°C for 1 hour, phenol extraction was carried out three times. The supernatant was mixed with same volume of isopropanol and 30 ug glycogen, and centrifuged at 13,000xg for 15 min at 4°C. Mitochondrial DNA was obtained from the pellet.


The Wnt proteins are a family of 19 secreted glycoproteins that occupy crucial roles in the regulation of processes such as cell survival, proliferation, migration and polarity, cell fate specification, body axis patterning and self-renewal in stem cells. The canonical pathway has been implicated in a variety of cancers. As such, it is only fair to conclude that therapies targeting the Wnt pathway may play an essential role in the future of anticancer therapeutics, both alone or in conjunction with traditional therapies. The Wnt signaling pathways represent a group of pathways that comprise of proteins involved in the transduction of signals via cell surface receptors. First identified in 1982 by Nusse in mouse models of mammary cancer, these pathways can be broadly divided into two major groups: canonical and non-canonical, with the differentiating factor being the involvement of β-catenin (encoded by CTNNB1) in the former .The Wnt proteins are a family of 19 secreted glycoproteins that are 350–400 amino acids in length .While the non-canonical pathways are involved in functions such as cytoskeleton development and intracellular calcium homeostasis, the canonical pathway Figure  has greaThe earliest evidence of Wnt involvement in human cancers was with the discovery of the association between APC tumor suppressor gene and β-catenin . Loss ofInappropriate Wnt signaling has also been implicated in various facets of both benign and malignant breast tumors . ElevateHepatocellular carcinoma (HCC) has also been identified as a heterogeneous cancer with active Wnt signaling . The dysInappropriate Wnt activation is also critical in certain lung cancers . At leasDysregulation of the canonical pathway in endometrial carcinoma can be attributed to inactivating β-catenin mutations or downregulation of Wnt antagonists by epigenetic silencing . The wntIn view of the above discussion, it is only fair to conclude that therapies targeting the Wnt pathway may play an essential role in the future of anticancer therapeutics, both alone or in conjunction with traditional therapies. Recent reports have grouped potential Wnt inhibitors into three major categories: small molecules, antibodies and peptides . The firA number of compounds are undergoing clinical trials . For exaIn conclusion, with the current era of advancements in anticancer therapeutics, therapies targeting the Wnt pathway do carry some significance . HoweverThe authors declare that they have no competing interests.AS conceived the topic and was involved in drafting the initial manuscript. AKN, MZA, BI, SMS and HHK were involved in critically revising the manuscript, listed in decreasing order of their contributions. The authors have read and approved the manuscript. The authors did not receive any financial support/grant. All authors read and approved the final manuscript.


P = 0.018; formal interaction test P = 0.025). Among those who underwent complete resection , 5-year time-to-recurrence analyses were performed, and stage II patients with loss of cytosolic E-cadherin were identified as an independent high-risk subgroup . Nuclear β-catenin and SOX9 protein, regardless of intracellular location, were not associated with prognosis. In conclusion, the protein expression level of membranous or cytosolic β-catenin and E-cadherin predicts CRC patient subgroups with inferior prognosis.Robust biomarkers that can precisely stratify patients according to treatment needs are in great demand. The literature is inconclusive for most reported prognostic markers for colorectal cancer (CRC). Hence, adequately reported studies in large representative series are necessary to determine their clinical potential. We investigated the prognostic value of three Wnt signaling-associated proteins, β-catenin, E-cadherin, and SOX9, in a population-representative single-hospital series of 1290 Norwegian CRC patients by performing immunohistochemical analyses of each marker using the tissue microarray technology. Loss of membranous or cytosolic β-catenin and loss of cytosolic E-cadherin protein expression were significantly associated with reduced 5-year survival in 903 patients who underwent major resection  independently of standard clinicopathological high-risk parameters. Pre-specified subgroup analyses demonstrated particular effect for stage IV patients for β-catenin membrane staining ( As of 2008, 1.2 million patients were diagnosed with colorectal cancer (CRC) annually, and only about half of them were alive 5 years after their initial diagnosis . Also, sIn the early 90s, the hereditary cancer syndrome familial adenomatous polyposis (FAP) was discovered to be directly linked to mutations within the adenomatous polyposis coli gene (APC) , 7. Two Briefly, canonical Wnt signaling  is initiated when Wnt proteins are released by stromal cells and Paneth cells in the intestinal crypt, and these proteins bind to heterodimeric receptor complexes on the surface of intestinal stem cells (Frizzled/Lrp6) and their immediate descendants . A signaThe prognostic potential of various components of the Wnt/β-catenin pathway in CRC has been explored in many datasets over the last decade, both on the genetic and the protein level. In particular, deregulation of APC, β-catenin, and E-cadherin has received much attention –19. FromSOX9 is a transcription factor and a downstream target of Wnt/β-catenin signaling with possible roles in β-catenin regulation –42. DereHere, we used a tissue microarray (TMA) constructed from a large consecutive, population-representative single-hospital series of primary CRCs to explore the prognostic significance of the protein expression of β-catenin, E-cadherin, and SOX9 by immunohistochemistry. We have attempted to report the study according to the REMARK guidelines   and primA population-representative consecutive series of 1290 CRC patients admitted to Oslo University Hospital – Aker 1993–2003) was analyzed. This hospital treated all CRC patients from a geographically defined catchment area, including most relapses. Of these, 929 patients underwent major resection, and DNA was extracted from corresponding formalin-fixed paraffin-embedded (FFPE) tissue from which a TMA was constructed  and the Norwegian Data Protection Authority, and the patients were enrolled after informed consent. The research conformed to the Declaration of Helsinki and the research biobanks have been registered according to national legislation.Cores from FFPE tissue from 670 colonic, 233 rectal, and 26 synchronous carcinomas  from patients treated at Oslo University Hospital – Aker (1993–2003), were organized into a TMA according to the original method described by Kononen and colleagues in 1998 .The immunohistochemical analyses were done on 3–4 μm thick TMA sections on microscope slides, and were performed as previously described . BrieflyFor immunohistochemical analysis, mouse monoclonal anti-β-catenin (Clone 14) antibodies were obtained from BD Biosciences , recognizing a C-terminal epitope between residue 571 and residue 781 of β-catenin. Mouse monoclonal anti-E-cadherin (Clone 36) antibodies were obtained from BD Biosciences , recognizing a C-terminal epitope between residue 735 and residue 883 of E-cadherin. Rabbit polyclonal anti-SOX9 antibodies were obtained from Atlas antibodies AB , recognizing the 117 amino acid residue C-terminal end. The antibodies were employed at dilutions of 1:800, 1:2000, and 1:500, respectively.The staining of β-catenin, E-cadherin, and SOX9 was scored according to the proportion and intensity categories proposed by Allred et al. . The proFor β-catenin the interobserver agreement, as measured by the intraclass correlation coefficients (ICC)  were 0.8Five-year overall survival (OS) and time-to-recurrence (TTR) plots were generated using the Kaplan–Meier method in the SPSS 18.0 software . TTR and OS were defined according to the guidelines given by Punt et al. . Brieflya priori association with the three biomarkers, reducing the need for rigorous correction. Clinically relevant subgroup analyses were therefore pre-specified and additional subgroup analyses labeled as exploratory. Interaction tests were integrated in the Cox models to assess whether effects were different between subgroups, but must be interpreted carefully due to the low power of such tests. All P-values were two-sided and derived from statistical tests using SPSS, and considered statistically significant at P < 0.05. To correct for multiple testing in the correlation analyses, we set a significance threshold of P < 0.001. Correlation between expression of protein markers and standard clinicopathological variables was evaluated using Spearman’s rho test.The correlation and survival analyses involve multiple tests and false positive findings are to be expected with a 5% significance level. However, several of the clinicopathological parameters, such as stage and differentiation, can be assumed to have some Among 903 stained histospots, 722 (80%) were evaluable for β-catenin, 720 (80%) for E-cadherin, and 761 (84%) for SOX9 . Further exploratory subgroup analyses of β-catenin membrane staining by standard clinicopathological variables showed that the initial association to survival was also particularly significant for females . However, interaction tests were not significant.Stratification according to stage demonstrated valid significance for OS only within stage IV  demonstrated that cytosolic β-catenin expression was an independent prognostic marker Table . MultivaP = 0.0010, n = 246; 69–77 years of age, P = 0.62, n = 227, 78–96 years of age, P = 0.26, n = 249). This finding was supported by significant interaction tests (P = 0.0025 for β-catenin and P = 0.038 for the interaction test). The strata had unfortunately too few patients and events to perform an adequate TTR analysis.Exploratory subgroup analyses (OS) showed a particular age-related effect for younger patients  and P = 0.033, n = 279 with 74 events (TTR) . In our study, strong versus weak β-catenin membrane expression showed the clearest stratification of patients into poor and good prognostic groups (Table or less intensive treatment depending on their health condition. Cytosolic E-cadherin on the other hand, might have some prognostic value for stratification of stage II patients. Independent validations are warranted to confirm these results. If validated, we believe β-catenin and E-cadherin may serve as valuable biomarkers in a panel of biomarkers. They are not likely to separate high- and low-risk patient groups with sufficient precision for a clinical test as sole biomarkers.TNM-stage classification is well-established to assess CRC prognosis. Survival varies considerably between stages, at about 90% for localized disease compared to about 10% for metastatic disease , althougps Table . UnfortuIn summary, nuclear β-catenin protein expression lacks prognostic value for CRC, while decreased expression of both membranous and cytosolic E-cadherin and β-catenin are associated with worse outcome among primary CRC patients, having potential to serve as biomarkers in stage II and IV CRC, respectively.Jarle Bruun participated in the study design, performed IHC experiments and scoring, interpreted all results, performed all statistics, and drafted the manuscript. Matthias Kolberg participated in IHC experiments and in the statistical analyses and did an independent scoring of results. Arild Nesbakken collected the patient samples and provided the clinical data. Jahn M. Nesland performed quality control of the IHC analysis and carried out independent scoring of a subset of the data. Aud Svindland performed morphological identification of qualified tumor areas for TMA construction. Ragnhild A. Lothe conceived and coordinated the study, was responsible for the study design, and participated in discussion of results and in the drafting of the manuscript. All authors participated in manuscript writing and in scientific discussions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Journal/10.3389/fonc.2014.00118/abstractThe Supplementary Material for this article can be found online at Click here for additional data file.


Background: This paper describes the shaping of freestanding objects out of metallic structures in the nano- and submicron size. The technique used, called nanoforging, is very similar to the macroscopic forging process.Results: With spring actuated tools produced by focused ion beam milling, controlled forging is demonstrated. With only three steps, a conical bar stock is transformed to a flat- and semicircular bent bar stock.Conclusion: Compared with other forming techniques in the reduced scale, nanoforging represents a beneficial approach in forming freestanding metallic structures, due to its simplicity, and supplements other forming techniques. The macroscopic world offers a large variety of three-dimensional forming processes, for example casting, forging or cutting. Producing three-dimensional mechanical resilient structures in the submicron- and nanoscale is a very challenging task. Generally, when size is reduced it becomes more and more difficult to position nanoobjects and to control their geometry in three individual dimensions. Adhesive effects become dominant and forces such as gravity are negligible. Other macroscopical versatile processes like casting or cutting are not reproducible to the nanoscale, at the moment. For example, studies on chip formation in the nanoscale are a wide field of research at present –2. But t3Al-particles were compressed, investigating the true strain-true stress response of the freestanding nanoparticles. High degrees of deformation (true strain φ > 1) were observed without any signs of cracking . Fo. FosB/w0iii) The third forging tool allows the bending of a flat bar to a semicircle. A semicircular forging die and a cylindrical upper spring arm defines the contour of the flat bar. The lower side is stretched and the upper side is compressed resulting in a semicircle geometry of the flat bar. In summary we have given an overview of the forging process in the submicron- and nanoscale. A tungsten bar stock with a taper diameter of 2 μm at the forging end was first cut, than stretched and broadened and finally bent to a semicircle. On the basis of these three techniques it is exemplary shown that forging offers a great potential in shaping of three-dimensional structures in the submicron- and nano-range. The forging tools, produced here by focused ion beam milling of a single crystalline silicon wafer, can be individually designed and adjusted to the needs. Therefore, forging parts with a great variety of shapes are possible. With little constraint much of the macroscopic forging technique is realizable in the nanoscale. Typical parts produced by forging could be axle-shafts, discs, flat or spiral springs. Furthermore, bending and cutting of nano wires could be also possible applications of nanoforging. In contrast to structures produced by ion- or electron beam induced deposition, homogenous metallic structures can be manufactured with a high mechanical load capacity. Nanoforging plays out its strength when a small quantity and flexible and individual control of shape is required as well as mechanical properties of metals are of interest.File 1Tungsten bar stock trimmed by a cutting tool.File 2Tungsten bar stock flattened by forging between even surfaces.File 3Tungsten flat bar bent to a semicircle geometry.


Myf5 early enhancer at the early stage of stem cell differentiation, and its HAT activity is important for the recruitment of β-catenin to this early enhancer. In addition, histone H3-K27 acetylation, but not H3-K9/14, is intimately connected to the p300 HAT activity. Thus, p300 is directly involved in the regulation of the Myf5 early enhancer, and is important for specific histone acetylation and transcription factor recruitment. This connection of p300 HAT activity with H3-K27 acetylation and β-catenin signalling during myogenic differentiation in vitro offers a molecular insight into the enhancer-elements participation observed in embryonic development. In addition, pluripotent stem cell differentiation is a valuable system to dissect the signal-dependent regulation of specific enhancer element during cell fate determinations.Skeletal myogenesis is an intricate process coordinated temporally by multiple myogenic regulatory factors (MRF) including Myf5, which is the first MRF expressed and marks the commitment of skeletal muscle lineage. The expression of Myf5 gene during early embryogenesis is controlled by a set of enhancer elements, and requires the histone acetyltransferase (HAT) activity of transcriptional coactivator p300. However, it is unclear as to how different regulatory signals converge at enhancer elements to regulate early Myf5 gene expression, and if p300 is directly involved. We show here that p300 associates with the  However, less is known as to how different regulatory signals and transcription factors converge at enhancer elements to regulate Myf5 gene expression during different phases of skeletal myogenesis [Skeletal myogenesis  lineage ,2. The e lineage –5. The gin vivo. Knock-out of p300 abrogates MRF gene expression and skeletal muscle formation in mouse and ES cell systems [The transcriptional coactivator p300 has an intrinsic histone acetyltransferase (HAT) activity, and is essential for a myriad of cellular processes, including myogenesis ,7. The H systems . More im systems . Nonethe systems . Interes systems . Thus ge systems . HoweverMyf5 gene expression at the onset of myogenesis, and Myf5 is a direct target of β-catenin [Canonical Wnt/β-catenin signalling is also important for embryonic development and skeletal myogenesis. Wnt signalling regulates -catenin ,11. Whil-catenin . Further-catenin –15. The -catenin ,17, and -catenin .in vitro, to differentiate into cell types of all three germ layers [in vivo, and their ease differentiation in large scale have made them a valuable differentiation system to study the mechanisms of lineage specification [Pluripotent P19 embryonal carcinoma (EC) cells, like embryonic stem (ES) cells, respond well to development cues m layers –23. Pax3m layers ,23. Thisfication .Myf5 enhancer during the early stage of P19 cell differentiation. Our studies have demonstrated that p300 is directly involved in the early regulation of Myf5 enhancer. Furthermore, the p300 HAT activity is intimately connected with specific histone acetylation and β-catenin signalling in the regulation of Myf5 early enhancer.In this study, we have examined the requirement of p300 HAT activity for early Myf5 gene expression with respect to β-catenin occupancy and histone acetylation at the trans retinoic acid (RA) significantly enhanced the development of skeletal myocytes, which also exhibited a more intensive staining of myosin heavy chain as revealed by the immune fluorescence microscopic analysis  during RA-enhanced myogenic differentiation [Myf5 early enhancer, which appears not to be a direct target of RAR binding, increases moderately (about 2-fold) following RA treatment  were cultured in minimum essential medium α (Invitrogen) supplemented with 5% fetal bovine serum, 5% bovine calf serum (PAA), and Penicillin/Streptomycin (Invitrogen) at 37°C with 5% CO2. Cells were differentiated as described previously . Brieflyeviously . All reaFollowing EB formation, the cells were grown on cover slips coated with 0.1% gelatin for 5 days without treatments, fixed with methanol at 4°C, and rehydrated in PBS at room temperature. Incubation with primary antibodies was carried out overnight at 4°C, and with fluorescent labelled secondary antibodies at room temperature for 30 minutes . DNA wasVisualization and quantification were performed with Axiovert 200 M microscope, 16AxioCam HRM camera  and AxioCells were differentiated and harvested at the indicated time points, and then lysed in whole cell extract buffer containing 10 % glycerol, 50 mM Tris-HCl pH 7.6, 400 mM NaCl, 5 mM EDTA, 1 mM DTT, 1 mM PMSF, and 1 % NP-40 for 30 minutes on ice . ProteinCells were treated as indicated during the EB formation and then harvested on day 4 to prepare the whole cell extracts. Endogenous p300 protein was then immunoprecipitated and evaluated for its HAT activity by using a HAT assay kit following the manufacturer’s instruction (BioVision). Sample immunoprecipitated with protein A agarose only was used as a negative control to subtract the background. Quantitative Western blot analysis of input p300 served as an internal control.® Green PCR Master mix on a 7500 Fast Real-Time PCR System (Applied Biosystems). Results were normalized to GAPDH, and analyzed by threshold cycle (Ct) comparative method [Myf5fwd- GGCATGCCTGAATGTAACAGC;Myf5 rev- CAATCCAAGCTGGACACGGA;GAPDH fwd- TCGGTGTGAACGGATTTG;GAPDH rev- GGTCTCGCTCCTGGAAGA.Total RNA was isolated using the Total RNA Kit I (Omega) according to the manufacturer’s protocol. Equal amounts of RNA were reverse-transcribed with High capacity cDNA Reverse Transcription kit . Real-tie method . The 2-ΔMyf5 early enhancer:EE fwd- AGAAGCGGCACACGTTGTA;EE rev- TGGAGAAGAGTGAACATCCTTGChIP assay was performed as described previously ,45. Brie


Gastric and duodenal pseudomelanosis is a rare endoscopic mucosal finding, characterized by the accumulation of iron in macrophages of the lamina propria of the stomach and duodenum. The clinical significance and long term sequelae have not been clarified yet. However, this benign condition is associated with a variety of clinical conditions, such as essential hypertension, chronic renal failure, diabetes mellitus, long term intake of iron supplements, and furosemide. Duodenal pseudomelanosis appears to be more common than gastric pseudomelanosis given the fact that a few cases of gastric pseudomelanosis have been reported in the literature so far. We report a case of 88-year-old lady with ESRD who is maintained on hemodialysis and presented with abdominal pain. An upper GI endoscopy showed discoloration of the antrum of the stomach and most portion of her duodenum. Histopathology report confirmed the presence of iron laden macrophages in the lamina propria of both stomach and duodenum. Gastric and duodenal pseudomelanosis (GDP) is a benign and infrequent condition of uncertain clinical significance and it is associated with a variety of clinical conditions and intake of several medications , 2. The  Helicobacter pylori organism was absent. Therefore, the diagnosis of gastric pseudomelanosis and duodenal pseudomelanosis was confirmed. The iron therapy was discontinued and subsequently patient did fairly well.This 88-year-old lady with ESRD on hemodialysis, anemia, GERD, hyperlipidemia, COPD, and CHD was admitted to our hospital with complaints of abdominal pain, odynophagia, and vomiting for 4 days. Pain was diffused and dull in nature with no radiation. Vomitus was nonbilious and nonbloody. She reported similar episodes over the last three months. She has been taking ferrous sulfate for her anemia, for the last one year. Previous endoscopy, which was done two years back, was unremarkable, although endoscopy, which was done three months back, showed discoloration of mucosa of duodenum without any discoloration of the stomach mucosa. Upper endoscopy at this time revealed scattered moderate mucosal abnormality characterized by discoloration of the antrum and part of her corpus of the stomach. No ulcers, erosions, or any other causes for bleeding were identified. Also patchy mild inflammation characterized by erosion, erythema, and friability was found on the lesser curvature of the stomach. A diffuse moderate mucosal abnormality of the duodenum characterized by discoloration was found in the entire duodenum extending to the fourth part . HistopaAlthough etiology and clinical significance of this condition have not been established yet, GDP is associated with a variety of clinical conditions including hypertension, hemochromatosis, diabetes, chronic renal disease, gastrointestinal bleeding, and intake of several medications including iron sulfate and certain antihypertensive medication such as furosemide, hydralazine, propranolol, and hydrochlorothiazide , 5. ClinIt has been suggested that such pigmentation may cause problems with intestinal absorption of nutrients , 7. AlthHistologically, pigments are deposited in the lysosomes of macrophages in the lamina propria . The pigThe predominant pigments deposited are iron oxide and hemosiderin. These pigments are detected by both the Prussian blue and Fontana-Masson stains of macrophages in the lamina propria. It has been suggested that in duodenal pseudomelanosis iron metabolism may be impaired and iron is pooled within macrophages . One casLong term prognosis and sequelae of this condition are not known and no specific protocol for treatment and follow-up has not determined yet.Gastric along with duodenal pseudomelanosis is a very rare, benign condition of unknown clinical significance. This condition is asymptomatic per se, but the patient may present with dyspeptic symptom. In such cases, it is important to exclude other causes of dyspepsia by endoscopic and laboratory evaluation. Proper knowledge and understanding of the common associations of this condition may help in avoiding unnecessary workup. The longer duration of iron therapy in the setting of ESRD might be responsible for extensive pigmentation involving both stomach and duodenum in our case. However, further studies are needed to prove this association.


PLOS Computational Biology is celebrating its 10th birthday. Such a milestone provides an excellent occasion to reflect on the transformation that the field has undergone during the journal’s lifetime, and on the challenging question of where we may expect it to go next. As the leading journal in computational biology, PLOS Computational Biology encompasses the entire discipline and is therefore well placed to narrate this remarkable story. The evolution of the journal tells a rewarding story of success and accomplishment.This year, PLOS Computational Biology, a more apt question is, “Is there any area of biology that doesn’t involve computational biology?” Indeed, nowadays most life science departments search for and hire faculty whose research embraces sophisticated approaches to computational biology. Increasingly, graduate training programs include computational modeling and data analysis as integral parts of the curriculum. This reflects not only the scientific merit of a mature field; it also points to a wider recognition that a background in computational biology is becoming mandatory. Students need to have solid training in computations.Computational biology has forged ahead, branching into every field of the biological sciences, and has become an integral part of basic biological research from molecules to ecosystems. We are no longer asking, "What is computational biology?” Instead, glancing over recent publications in BD2K grants launched in 2012.What will we be able to accomplish in the next ten years that we were not able to achieve over the last ten? The explosion in computational biology has been driven by the rapid increase in the availability of experimental data and computational power. Both were, and will continue to be, revolutionized; both promise to keep transforming cutting-edge computational biology. “Big data” is a recurring theme, and as the volume of data continues to increase, there will be a growing need for computational tools and techniques to convert “big data” into biological knowledge, a challenge supported by the National Institutes of Health via the Firstly, the next decade will very likely be more data-intensive than the last decade. Biological data will increasingly be quantitative, substituting and supplementing traditional, descriptive biology. Examples (chosen for illustrative purposes) include identifying, cataloging, and analyzing microbes populating different tissues, ages, and health states, as well as microbes residing in the soil or in the ocean in specific environments where they can be useful, for example, against oil spills; discovering and classifying parallel cellular pathways that activate overlapping (or distinct) functions, for example, those emanating from the same family of receptors that may take over in cancer; and unveiling the combinatorial post-translational modifications (PTMs) codes, in which distinct PTM combinations spell specific (though related) functions for the same protein. There are hundreds of PTM types; each has specific chemistry, shape, and unique structural consequences. The number of possible combinations is immense. For example, in transcription factor p53, there are at least 50 PTM sites; the FoxO family of forkhead transcription factors is regulated by specific combinations of PTMs, including phosphorylation, acetylation, and ubiquitylation, in which distinct FoxO PTM combinations act as a “FoxO code.” Seventeen possible PTM acceptor residues were described in FOXO3a (Forkhead box O3) alone, and it was estimated that single and binary multiple modifications could give rise to thousands of different PTM isoforms. The vast amounts of data span ecosystems, diagnostic codes, diverse tumor clone phenotypes, and mapped mutations—single as well as combinations in which each mutation on its own has not been observed to drive a phenotypic change but together they do—can open new horizons and permit new analyses not possible before. When bolstered by a repertoire of patient-centered data, they may lead to more informed, knowledge-based correlations of physiological traits and predictive disease markers.At the same time, data on such huge scales will also require development of methods to derive meaningful conclusions that are robust with respect to measurement error and noise, inherent biological variability and statistical biases. We anticipate that data accuracy and consistency will be major hurdles that will need to be overcome. These include background effects, noise such as that resulting from expression levels, which is mostly Poisson-like for high expression levels but more complex at low expression levels. Other sources of noise include differential binding strengths for different probe-target combinations; for example, the brightness of a spot depends on the binding strength between the probe and the target, which is itself a function of the specific sequence at the binding site. Noise sources also include lack of observed correlation between mRNA levels and protein levels, which may be due to the fact that some proteins are regulated after translation, insufficient spatial resolution, and more. These problems are compounded by lack of standardized procedures across experiments, which may lead to different levels of random noise, unsynchronized cell cycles in the sample and tissue inhomogeneity, which may obscure cell-specific data. In addition to noise induced by measurement error, the inherent variability and rich structure of biological systems poses challenges for data acquisition and analysis. These challenges are paramount in mapping structure and function of complex systems such as the brain.PLOS Computational Biology launched.Capturing population heterogeneity and associating it with function, whether for single molecules or for cells, poses yet another special challenge. One example of the multitude in this category is how to model the heterogeneous three-dimensional genome population across cell diversity, time, and space based on data including genomic sequences, function, and epigenetic information. This presents a daunting goal for the next ten years, but one that would have been virtually impossible when Computational neuroscience gives us another key example of how computational methods are transforming the traditional landscape of the biological sciences.Current neuroscience databases can provide information relating to gene expression, neurons, macroscopic brain structure, and neurological or psychiatric disorders. Some databases contain descriptions of neuronal morphologies or interconnections, some record the activity of neurons or brain regions in relation to behavioral or cognitive functions, and others comprise volumetric imaging data, such as stacks of postmortem brain sections or 3-D MRI and fMRI images. This cornucopia of data is helping scientists understand and model the structure and function of the brain, widely recognized as one of the most challenging scientific frontiers of the 21st century. As the number of neuroinformatics resources that seek to disseminate information about the structure, development, and function of the brain has grown, so has the need to exploit them to advance basic scientific insight. Development of capable computational methods that can be applied across diverse types of data is critical and forms an integral part of computational (neuro)biology. These require not only advanced computing power; they also increasingly demand integration of data across different domains  and levels of biological organization  into a single analytic or modeling framework.On their own, neither computations nor experiments can overcome these pressing challenges. Our current computational power can only simulate a small fraction of the time scale of biological processes, and it is limited to a tiny portion of the real molecular complex in a cellular environment. To mention just one example, the complete conformational change of the R to T transition in hemoglobin occurs in tens of microseconds. Using current resources, our large-scale simulations of a small protein can only account for processes covering microseconds. To effectively complement experimental studies, the time and spatial scales covered in computational biological models must continue to grow, beyond our current capabilities. Only exascale computing can meet the pressing challenges facing the biological sciences. Exascale computing, capable of at least one exaFLOPS, or a billion billion calculations per second, represents a 1,000-fold increase over the first 2008 petascale computer. While the National Institutes of Health is currently considering the possibilities of insights to be gained based on future computational resources, exploiting such power, estimated to become available in 2018 (at the earliest), may—under the best of circumstances—only be realized a decade from now.PLOS Computational Biology is a community journal, championing the open access paradigm. Within this framework, we believe in data and software sharing. This has always been of paramount importance; with the unprecedented advancement of the field and its diversified disciplines, open data and software will become even more critical to drive further revolutions and to enlist participation from across the globe. No one knows what the future will bring—but it seems certain that the future is not going to be less computational; instead it is likely that computational analysis, tools, and models will become seamlessly integrated into the wider field of biology. In parallel with the inevitable expansion of biological data, computational biology will be indispensable for developing tools and approaches that are robust against noise and statistical biases, for devising testable and predictive models, and for delivering quantitative insight into fundamental biological processes within and across scales of organization. PLOS Computational Biology has been part of the field from its infancy, when questions such as, "What is computational biology?" were common, through to today, when, "What isn't computational biology?” seems far more appropriate. Together with the International Society for Computational Biology (ISCB) community and our broad and increasing readership, the journal will continue to serve as a forum for all aspects of computational biology, leading the discipline into its next decade.Finally,


To support the high rates of proliferation, cancer cells undergo the metabolic reprogramming: aerobic glycolysis and glutamine addiction. Though glucose regulated protein 78 (GRP78) is a glucose-sensing protein and frequently highly expressed in tumor cells, its roles in glucose and glutamine metabolic regulation remain poorly unknown. We report here that glucose deficiency-induced GRP78 enhances β-catenin signaling and consequently promotes its downstream c-Myc-mediated glutamine metabolism in colorectal cancer cells. Mechanistically, GRP78 elevates intracellular free β-catenin level via disruption of adenomatous polyposis coli (APC)-β-catenin and E-cadherin-β-catenin protein complexes. Notably, overexpression of GRP78 causes APC protein downregulation in proteasome- and lysosome-independent manners. Further mechanistic studies reveal that GRP78 facilitates the extracellular release of APC, thereby rendering the liberation of β-catenin from APC. Furthermore, GRP78 acts through both hindering E-cadherin expression and impairing the interaction of E-cadherin with β-catenin to indirectly and directly influence E-cadherin-β-catenin complex stability. Our study reveals that GRP78 is a novel molecular link between metabolic alterations and signal transduction during tumor progression. Compared with normal differentiated cells, cancer cells display ten typical cancer hallmarks, among which an emerging hallmark is the metabolic reprogramming . Cancer Tumor cell metabolism is sophisticatedly regulated by signal transduction pathways that are affected by genetic mutations or the alterations in tumor microenvironment . PI3K/AkGlucose regulated protein 78 (GRP78), a molecular chaperone in the endoplasmic reticulum (ER), is also found to be present in tumor cell plasma membrane, cytoplasm, mitochondria, nucleus as well as cellular secretions , 18. GRPIn the present study, we identified GRP78 as a molecular link between glucose metabolism and glutamine metabolism. Upon glucose deprivation, the increased levels of GRP78 lead to elevated c-Myc expression and concomitantly to enhanced glutamine metabolism and cell survival from glucose deprivation. These effects of GRP78 are attributed to the elevated β-catenin protein levels, mediated by interference with the adenomatous polyposis coli (APC)-β-catenin and E-cadherin-β-catenin complexes. Specifically, overexpression of GRP78 impairs the expression of E-cadherin and its interaction with β-catenin to influence E-cadherin-β-catenin complex stability. Interestingly, GRP78 is able to facilitate the extracellular release of APC via protein-protein interaction, consequently resulting in the liberation of β-catenin from APC. Our results thus reveal a novel complementary pathway between glucose metabolism and glutamine metabolism. It may hold important implications for strategies aiming at targeting tumor metabolism.Previous studies demonstrated that glioblastoma cells required the glutamate dehydrogenase (GDH) to drive glutamine metabolism to the TCA cycle and survived glucose metabolism impairment . To deteWe next observed that the mRNA levels of both glutaminase 1 (GLS1) and glutamine transporter SLC1A5 (ASCT2) but not GDH were increased in response to glucose deprivation Fig.  and S1E.Given that c-Myc is a direct target gene of the β-catenin pathway , the expGRP78 protein is an important glucose-regulated protein and contributes to the acquisition of several cancer hallmarks by tumor cells , 22. BotIn epithelial cells, β-catenin is mainly localized in the cytoplasm, complexed with APC and Axin, and in cell-cell junction in conjunction with E-cadherin . How wouThe downregulation of APC protein in Gd-treated cells led us to test whether it occurred at transcriptional level or posttranscriptional level. The APC mRNA levels in Gd-treated cells were moderately increased in comparison to cells grown in high glucose medium, while its protein levels were decreased at the corresponding treatment time points Fig. . TherefoThe proteasomal and lysosomal pathways are the two main routes of protein and organelle clearance in eukaryotic cells . To furtGiven that GRP78 has been reported to be secreted from solid tumor cells into the tumor microenvironment , we specBesides in the β-catenin-destruction complex, β-catenin is also localized at the adherens junction complexing with E-cadherin and α-catenin . We nextWe next asked whether GRP78 could directly interfere with the β-catenin-E-cadherin complex. Immunocytochemical staining showed an obvious colocalization of GRP78 with E-cadherin Fig. . Co-IP aGlucose and glutamine are abundant in plasma, and supply the carbon, nitrogen, energy, and reducing equivalents required for tumor cell growth and division . TargetiDeregulation of Wnt/β-catenin pathway usually occurs in colorectal cancer, which leads to a constitutively stable and active β-catenin and induces aberrant cell proliferation . Under hThere is solid evidence that GRP78 plays a role in resisting stressful microenvironment and facilitating cell survival, and the underlying mechanism involves blockage of pro-apoptotic pathways or activation of pro-survival pathways . Our datGRP78 is multifunctional protein, and what makes GRP78 more special is that GRP78 can be present on cancer cell surface or secreted into extracellular microenvironment. The present data demonstrate that these features are intertwined with the modulation of β-catenin-E-cadherin and β-catenin-APC protein complexes. High expression of GRP78 can reduce E-cadherin expression through induction of epithelial-mesenchymal transition Fig. . FuthermFull-length APC is a β-catenin binding protein and can down-regulate β-catenin-mediated transcription. However, different APC truncations freguently happen in colorectal cancer , which uAPC is a large protein, with multi-functional domains, implicated in a wide range of biological processes. The COOH-terminal domain of APC (C-APC) interacts with its NH2-terminal domain (N-APC), and this interaction can cause changes in the protein interactions of N-APC . Our datRPMI-1640 medium and fetal bovine serum (FBS) were from GIBCO . Trizol, PrimeScript RT Master Mix and SYBR green PCR master mix were from Takara . Antibodies for E-cadherin and β-actin were from Bioworld Technology . Antibodies for β-tubulin, GFP, GRP78 and APC were from Abcam . Antibodies for β-catenin and CD63 were obtained from Abmart  and Santa Cruz Biotechnology , respectively. FITC-, TRITC-, Cy5-, and HRP-conjugated secondary antibodies were obtained from Invitrogen .2. MDCK cells stably expressing the truncated N1-APC (aa 1-448), N2-APC (aa 449-781) and N3-APC (782-1082) were the gift from Prof. Inke Näthke . The viability of cells was assessed by the MTT assay as the literature described [Human colon carcinoma HT29, SW480 and DLD1 cell lines were obtained from the American Type Culture Collection and cultured in RPMI-1640 medium containing 10% FBS at 37 °C in humidified tissue culture incubator containing 5% COescribed .Total RNA extraction, reverse transcription, and real-time PCR were performed as previously described . The priThe nuclear and cytosolic proteins were extracted using a nuclear protein extraction kit . The protein concentrations of nuclear, cytosolic and whole cell lysates were determined by the BCA protein assay. An appropriate amount of protein from each sample was subjected to SDS-PAGE and subsequent Western blotting as previously described .Cells were lysed in Western and IP buffer  containing protease inhibitor cocktail (Thermo scientific). The whole cell lysates (WCL) were pre-cleared by incubating with 1.0 μg of control IgG corresponding to the host species of primary antibody, together with 20 μl Protein A/G PLUS-Agarose (Santa Cruz) at 4 °C for 2 h, followed by centrifugation at 4°C for 5 min at 2, 500 rpm. The supernatant was subjected to immunoprecipitation by addition of 2 μg immunoprecipitation antibodies and incubated overnight at 4 °C followed by incubation with Protein A/G PLUS-Agarose for 2 h. After washing with cell lysis buffer, the beads were boiled in 2×SDS loading buffer and the supernatants were resolved by SDS-PAGE. For the GST-pulldown assays, GST-GRP78 or GST immobilized on Sepharose 4B-glutathione beads  were incubated with pre-cleared WCL at 4 °C for 12 h followed by washing with the lysis buffer. The bound proteins were dissolved in 2×SDS loading buffer, separated by 10% SDS-PAGE, and immunoblotted with the APC antibody.Cells were plated on 6-well glass slides. After the required treatments, the cells were then fixed in 4% paraformaldehyde in PBS for 30 min, and permeabilized with 0.3% Triton X-100 in PBS for 10 min. Next, the slides were blocked in 2% goat serum for 1 h and incubated with the primary antibodies at 4 °C overnight. The slides were then washed and incubated with the corresponding secondary antibodies. After three PBS washes, the slides were mounted in gelvatol for confocal immunofluorescence analysis.For tissue samples, paraffin-embedded sections were deparaffinized in xylene and rehydrated through descending concentrations of ethanol. After routine antigen retrieval procedures, sections were treated with 10% FBS for 30 min to block the nonspecific binding. Sections were then incubated with the appropriate diluted primary antibodies and secondary antibodies as described above. The sections were finally counter stained with DAPI and mounted in gelvatol for immunofluorescence analysis.7 formaldehyde crosslinked DLD1 cells were sonicated for DNA shearing to achieve DNA fragments of about 200-1000 bp. Immunoprecipitation was carried out using anti-β-catenin antibody, anti-pol II antibody (positive control) and the control IgG (negative control) overnight at 4°C. The primers used to amplify the DNA fragments were depicted in CHIP assay was performed using a chromatin immunoprecipitation kit  following the manufacturer's instruction. Briefly, 1×10Human GRP78 cDNA was subcloned into the lentiviral expressing vector pLVX-AcGFP1-N1 Vector (Clontech). GRP78 shRNAs constructed in pLKO.1-Puro were purchased from Sigma (Mission shRNA). GRP78 overexpression/shRNA plasmid, pCMVdR8.91 and pCMV-VSV-G were co-transfected into 293T cells using the Calcium Phosphate method at 15:10:5 g (for a 10-cm dish). Media containing virus was collected 48 h after transfection and then concentrated using 100 kDa ultrafiltration membranes (Millipore). DLD1 cells were infected with the viruses in the presence of polybrene (8 μg/ml) for 48 h, and then subjected to selection by 5 μg/ml puromycin for 72 hours. Hairpin sequences in these shRNA constructs are depicted in Data are expressed as the mean ± SEM. Differences among groups were tested by one-way analysis of variance (ANOVA). Comparisons between two groups were evaluated using Student's t-test. A value of p<0.05 was considered statistically significant.


RH genes expression remain poorly studied. Here we addressed the expression regulation of one of these factors, RHBG. We identify HepG2 hepatocellular carcinoma cells and SW480 colon adenocarcinoma cells as expressing RHBG and show that its expression relies on β-catenin signaling. siRNA-mediated β-catenin knockdown resulted in significant reduction of RHBG mRNA in both cell lines. Pharmaceutical inhibition of the TCF4/β-catenin interaction or knockdown of the transcription factor TCF4 also downregulated RHBG expression. We identify a minimal RHBG regulatory sequence displaying a promoter activity and show that β-catenin and TCF4 bind to this fragment in vivo. We finally characterize the role of potential TCF4 binding sites in RHBG regulation. Taken together, our results indicate RHBG expression as a direct target of β-catenin regulation, a pathway frequently deregulated in many cancers and associated with tumorigenesis.Ammonium is a metabolic waste product mainly detoxified by the liver. Hepatic dysfunction can lead to cytotoxic accumulation of circulating ammonium and to subsequent encephalopathy. Transmembrane ammonium transport is a widely spread process ensured by the highly conserved proteins of the Mep-Amt-Rh superfamily, including the mammalian Rhesus (Rh) factors. The regulatory mechanisms involved in the control of  This correlation appears to hold true in a normal mouse liver context as transgenic models enabling targeted inactivation or activation of β-catenin signaling show downregulation or upregulation of RHbg expression, respectively [Ammonium, hereafter referring to the sum of the NHd plants . It is hd plants . Hepaticentified . Sequencentified . Non-eryentified –14. Miceentified –17. Of n-catenin , suggestectively ,20. The ectively –23. The ectively ,22. Wnt ectively . The resectively ,23.RH genes expression and the signaling pathways potentially implicated are so far poorly documented. Here, we sought to identify human cancer cell lines expressing the RHBG gene to study its expression regulation and address the potential direct influence of the Wnt/β-catenin signaling. We show that RHBG is highly expressed in HepG2 hepatoma cells and relies on β-catenin signaling. Similarly, the RHBG expression revealed in SW480 colon cancer cells is dependent on β-catenin, further supporting the role of β-catenin signaling in RHBG regulation. Promoter analysis and chromatin immunoprecipitation assays are consistent with a direct involvement of TCF4/β-catenin in RHBG up-regulation in HepG2 cells.The regulatory mechanisms involved in the control of human The human hepatocellular carcinoma (HepG2 and Hep3B) and human embryonic kidney (HEK293T) cell lines were kindly provided by Professor Claude Szpirer, Université Libre de Bruxelles, Belgium. The human colon adenocarcinoma (SW480) cell line was purchased from CLS (Germany). HepG2, Hep3B, SW480 and HEK293T cells were cultured in advanced DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (Biowest), 2 mM L-glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin. Cells were maintained in an incubator with humidified air (5% CO2) at 37°C. PFK118-310 (#K4394) was purchased from Sigma and used at a 0,2 or 0,4μM concentration from a DMSO solution.RHBG promoter was amplified using polymerase chain reaction with the F-Pr- BG- and R-Pr-BG (DNA fragment (2492 bp) corresponding to the potential  R-Pr-BG  primers  R-Pr-BG . The PCRTotal cellular RNA was extracted using TRIzol reagent (Invitrogen) according to manufacturer instructions. DNase treatment was done using a DNA Removal Kit . One μg of total RNA was reverse-transcribed to cDNA using the SuperScriptIII First-Strand Synthesis SuperMix (Invitrogen) according to manufacturer instructions. Realtime RT-PCR were performed on a StepOnePlus Real-Time PCR System (Applied Biosystems) using GoTaq qPCR Master Mix (Promega) using the indicated primers  and normCells were cultured in Millicell EZ 8 chamber slide (Millipore). Cells were fixed with 4% paraformaldehyde (PFA) for 15 min and subsequently permeabilized with 0,1% TritonX-100 for 3 minutes and were next blocked with goat serum (5%) for 30 minutes. Cells were incubated with β-catenin antibody  overnight at 4°C. Slides were washed and incubated with anti-rabbit IgG (H+L), F(ab')2 Fragment  for 1 hour at room temperature. After washing with PBS, the slides were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen).Total proteins were extracted using RIPA  lysis buffer supplemented with cocktail of phosphatase and protease inhibitors (Roche). After centrifugation, proteins were quantified using Pierce Microplate BCA Protein Assay Kit—Reducing Agent Compatible assay (Thermo Scientific). Equal amounts (~20μg) of proteins were then separated by Mini-PROTEAN TGX gels (Bio-Rad). Proteins were transferred to nitrocellulose membrane . Membranes were blocked with 5% milk and incubated with anti-β-catenin , anti-TCF4 , anti-Glutamine synthetase  or anti-β-Actin . Primary antibodies were detected with horseradish-peroxidase-conjugated anti-rabbit or anti-mouse-IgG secondary antibodies  followed by measurement of chemoluminescence .Cells were reverse transfected with pre-designed silencer select non-targeting control  or siRNAs targeting β-catenin  or TCF4  with Lipofectamine siRNAMAX (Invitrogen). Cells were incubated at 37°C for 72 or 96 hours and the indicated mRNA and proteins level were examined by qRT-PCR and western blotting, respectively.RHBG promoter constructs for 48h using the Viafect transfection reagent (Promega). Luciferase activity in total cell lysates was measured using the Dual-Glo luciferase reporter assay (Promega) according to manufacturer instructions. Luminescence was measured using GloMax-96 Microplate Luminometer (Promega).Cells were transiently co-transfected with the pTK-Renilla luciferase reporter vector (Promega) and empty plasmid  or the plasmid containing the indicated CHIP assay was done using SimpleChIP Enzymatic Chromatin IP Kit  according to manufacturer instructions. Briefly, cells were fixed with 1% formaldehyde solution to cross-link histone and non-histone proteins to DNA. Nuclear chromatin was digested with Micrococcal Nuclease for 20 min at 37°C and then incubated overnight at 4°C with either anti-β-catenin , anti-TCF4  Normal Rabbit IgG . Following washing with low and high salt ChIP buffers, the protein-DNA complexes were eluted and cross-links were then reversed. After proteinase K digestion, DNA is purified and quantified by Real time-PCR as described earlier using primers listed in t-tests using Graph Pad Prism version 5.00 software (Graph Pad Software). Differences were considered significant when the p value is below 0.05 , n = 3, except for Chip experiments where n = 2.Data are expressed as means ± S.E.M. Statistical comparisons were assessed by Student’s RHBG gene was found to be overexpressed in a subset of hepatocellular carcinoma [RHBG, we evaluated its expression levels in the HepG2 and Hep3B hepatoma cells. HepG2 cells bear a heterozygous deletion in the exon3 of the β-catenin CTNNB-1 gene, producing a truncated β-catenin protein lacking key residues for its phosphorylation by the destruction complex and thus resulting in its cellular accumulation [The human arcinoma , howevermulation –28. Hep3mulation ,30.RHBG gene appeared highly expressed in HepG2 compared to Hep3B cells, the latter showing slightly higher expression level compared to that of HEK293T cells taken as a normal cell model  was also upregulated in HepG2 cells compared to Hep3B  was directionally subcloned into the pGL3-basic firefly luciferase reporter vector. To test whether this fragment possesses a promoter activity, the RHBG promoter construct (pGL3-RHBG) and the native pGL3-basic vector were used for transient co-transfection of HepG2 cells together with the pTK-Renilla luciferase reporter vector as transfection control. 48 hours after transfection, the luciferase activity in pGL3-RHBG transfected cells was about 30 fold higher than with the pGL3-basic plasmid indicating that the cloned RHBG sequence contains an active promoter  assays to determine whether TCF4 and β-catenin are capable of binding the -60/+142 segment of promoter . Consistreported ,53.RHBG promoter, and likely enhances RHBG expression in HepG2 cells.These results indicate that TCF4/β-catenin specifically binds to the -60/+142 region of the RHBG gene encoding an ammonium transport protein. We show that in these cells RHBG expression is largely dependent on β-catenin function. We perform a functional analysis of the RHBG upstream regulatory sequence, revealing a minimal region bearing a promoter activity. Our data indicate that the RHBG regulatory sequence is a TATA-less GC-rich promoter. We show that β-catenin and TCF4 are both able to bind the minimal promoter region in vivo and characterize potential TCF4 binding motifs important for the promoter activity. Our data support a direct role of β-catenin/TCF4 in the regulation of RHBG expression in this cell line, and further indicate that RHBG could serve as a direct reporter of the Wnt/β-catenin pathway in specific cancer cell contexts.This study identifies the hepatoma HepG2 cells as expressing the RHBG is expressed in the colon cancer SW480 cells bearing an APC mutation and that its expression is also dependent on TCF4/ β-catenin. In contrast, the expression of the gene encoding the second non erythroid ammonium transport protein, RhCG, is independent of β-catenin signaling in either HepG2 or SW480 cells.Hepatocellular carcinoma is the most common adult liver malignancy and many lines of evidence associate hyperactivation of the Wnt/β-catenin pathway to its initiation and development . AbnormaRHBG overexpression observed in hepatocarcinoma obtained from surgical resections and bearing activating mutations in the β-catenin gene CTNNB-1 [RHBG was up-regulated in 9 over 10 HCC of the latter resections compared to normal liver, while it was slightly overexpressed in only one over 15 HCC showing wild-type CTNNB-1. RHBG overexpression was strongly correlated with upregulation of GLUL, but also of SLC13-A3 encoding a sodium-dicarboxylate (including α-ketoglutarate and succinate) transporter and GPR49, also known as LGR5, a coreceptor of Wnt signaling. A correlation between HCC with activated β-catenin pathway and upregulation of GS, of ornithin amino transferase (involved in glutamate synthesis), and of the Glt1 glutamate transporter, were also reported [+ V-ATPase which is supposed to drive NH3 efflux by favouring urinary trapping of NH4+ [E. coli where the GS activity is strictly required to drive substrate uptake via the Rh orthologue AmtB [in vivo, as plasma levels of glutamine and urea appear normal in mice lacking Rhbg [Our data are consistent with the  CTNNB-1 . RHBG wareported . The uprreported ,20. Thisreported . HepG2 creported . Howeverreported . Of notereported . Addictireported . Prolifereported . In condreported ,63. Rhcgof NH4+ ,13,17. Tgue AmtB . Howevering Rhbg . Althouging Rhbg .3 production. The latter molecule was shown to act as an autocrine and paracrine diffusible signal that triggers a specific autophagic program, in turn enabling survival and proliferation of cancer cells deep in a tumour mass [Ammonium was recently proposed to play a particular role in a tumoral context . Up-reguour mass ,68. Whet