The role of ß-catenin signaling in mesodermal lineage formation and differentiation has been elusive.Dermo1(Twist2)Cre/+ line to target a floxed β-catenin allele, throughout the embryonic mesenchyme. Strikingly, the Cre/+Dermo1; f/−β-catenin conditional Knock Out embryos largely phenocopy −/−/Pitx2−/−Pitx1 double knockout embryos, suggesting that ß-catenin signaling in the mesenchyme depends mostly on the PITX family of transcription factors. We have dissected this relationship further in the developing lungs and find that mesenchymal deletion of β-catenin differentially affects two major mesenchymal lineages. The amplification but not differentiation of Fgf10-expressing parabronchial smooth muscle progenitor cells is drastically reduced. In the angioblast-endothelial lineage, however, only differentiation into mature endothelial cells is impaired.To define the role of ß-catenin signaling in these processes, we used a ß-catenin and PITX, as important regulators of mesenchymal cell proliferation and differentiation.Taken together these findings reveal a hierarchy of gene activity involving  During development and in adult tissues, mesenchymal cells serve as precursors to diverse cell lineages, including smooth muscle cells (SMCs), endothelial cells, pericytes, lipocytes and stromal fibroblasts. The proper generation of these cell types likely relies on the controlled amplification of lineage-restricted and non-restricted mesenchymal precursors followed by their timely differentiation into the appropriate progeny.N-myc expression N-myc results in premature differentiation and reduced epithelial cell proliferation Bmp4 and Fgfr2b expression in distal lung epithelium The developing lung provides a good system for studying the regulators of epithelial and mesenchymal cell lineage formation Ptch and respond to epithelially-derived SHH, while transient fate analysis studies using an Fgf10-LacZ reporter line (here after termed LacZ/+)Fgf10, show that the sub-mesothelial cells express high levels of Fgf10 and serve as progenitors to parabronchial smooth muscle cells (PSMCs). The PSMC progenitor status is maintained by mesothelialy-derived FGF9 We and others have studied the sequential development of several lung mesenchymal lineages. The distal lung contains two distinct mesenchymal cell populations: sub-epithelial; sub-mesothelial. Sub-epithelial cells express Fgf10 expression in the mesenchyme Hints that ß-catenin signaling is important for the development of the mesenchyme, in addition to the epithelium, have emerged from expression pattern studies and the analysis of TOPGAL and BATGAL reporter mice. TOPGAL and BATGAL alleles serve as LEF1/TCF mediated ß-catenin signaling reporters only Cre/+Dermo1-mediated conditional inactivation (CKO) of β-catenin to study the role of ß-catenin signaling in mouse embryonic mesodermal lineages. In these mutants, we find multiple mesenchymally-related defects that are remarkably reminiscent of a double knock out of Pitx1 and Pitx2 genes Fgf10-expressing PSMC progenitor cells but does not affect their differentiation. Yet, it is required for proper differentiation of endothelial cells. These findings reveal a critical requirement for ß-catenin signaling in the development of multiple mesenchymal lineages.We have carried out a Cre/+; β-cateninf/-Dermo1 conditional knockout (here on abbreviated to CKO) is lethal at embryonic day E13.5-E14.5 due to the severe cardiac  and vasculogenesis-related defects. CKO embryos show a set of phenotypes that are remarkably reminiscent of Pitx2 null embryos. These include: an arrest in turning of the body axis and defective body wall closure; partial right pulmonary isomerism; altered cardiac position with major cardiac outflow tract abnormalities classified as double outlet right ventricle (DORV) (90%) and Pulmonary truncus arteriosus (PTA) (10%) (n = 20); defective development of the mandibular and maxillary facial prominences and regression of the stomodeum  suggesting that the differentiation of the PSMC progenitors could occur normally in CKO lungs.We then compared the expression of β-catenin is specifically deleted in the mesenchyme but importantly, it also demonstrates that mesenchymal β-catenin signaling at E13.5 does not rely primarily on LEF1/TCF transcription factors.Next, we used an imported TOPGAL reporter allele to examine the level and distribution of LEF1/TCF-mediated β-catenin signaling. At E13.5, TOPGAL activity was restricted to epithelium, in both WT and CKO lungs, with no discernable difference in levels . This fiPitx2 expression itself, both at the level of transcription Pitx2 expression in CKO and WT lungs by in situ hybridization. Using a riboprobe that detects all three isoforms of Pitx2, we found that at E13.5, CKO lungs show an absence of Pitx2 expression  reported that inactivation of β-catenin in the distal lung epithelium, leads to a down-regulation of Fgfr2 receptor in the epithelium. By inactivating β-catenin in the lung mesenchyme, we might expect to observe a similar down-regulation of Fgfr2 in the mesenchyme. Using immunohistochemistry, we found that indeed FGFR2 expression is reduced in the mesenchyme but not epithelium of CKO lungs  compared to scrambled led to a corresponding downregulation in Fgfr2 expression of 40%±4   compared to scrambled led to a corresponding downregulation in Fgfr2 expression of 40%±6  (The observed reduction in KO lungs . Interes= 0.003) . Similar= 0.009) .β-catenin was deleted  . There was also a clear reduction in the number of mitotic cells, as determined by Phospho-Histone H3 (PH3) staining, in mesenchyme  (P = 0.03) of CKO, when compared to WT.Next, we set out to investigate if this relationship is reflected in functional assays. We quantified the distribution of P-ERK positive cells and found much fewer P-ERK positive cells in the mesenchyme of CKO lungs wherever  = 0.03)  arrows.  = 0.02)  and epitP = 0.2) , demonstrating that the effect of β-catenin deletion in CKO lungs on mesenchymal proliferation and ERK phosphorylation is cell autonomous.Interestingly, no significant differences in P-ERK or PH3 could be found in areas of CKO lung mesenchyme that lacked recombination  increase in P-ERK (P = 0.001) increase in P-ERK levels  indicating a possible redundancy with other PITX or LEF1/TCF transcription factors. Our results indicate that inactivation of β-catenin in the mesenchyme inhibits the differentiation of angioblasts into mature endothelial cells. However, it is important to note that ablation of β-catenin in mature endothelial cells using the CreTie2 driver line did not affect vasculogenesis and angiogenesis, or PECAM expression Tie2 expression starts later during endothelial cell differentiation and so our data suggests that ß-catenin signaling is an important regulator of early endothelial cell development So far, we have provided experimental evidence suggesting that CKO deletion of WT lungs . Close ax allele . This coO embryo  includinO embryo . AblatioO embryo . We alsoO embryo . LeakageFgf10-positive cells  and a 201 fragment of c-Myc (cloned by RT-PCR using primers c-Myc-Faccaacaggaactatgacctc and c-Myc-Raaggacgtagcgaccgcaac).WMISH was performed like previously described Mesenchymal cells from E13.5 WT and CKO lungs were isolated according to 2 fragments were purchased from Jackson Immunoresearch and slides were mounted with DAPI containing Vectashield®.E13.5 CKO and WT lung mesenchymal cells were grown on 8 well permanox Lab-Tek chamber slides in the presence of FGF9 (200 ng/ml) for 24 hours. The cells were fixed for 30 minutes with 4% paraformaldehyde and subsequently washed with PBS. Lungs were fixed in 4% PFA washed in PBS, dehydrated and paraffin embedded. Lung sections and slides were treated with a monoclonal anti α-smooth muscle actin antibody (Ab), clone 1A4, Cy3 conjugated (from Sigma®) at 1∶200, anti-β-catenin Ab (BD biosciences) 1∶200, anti-PH3 Ab  1∶100, anti-PERK Ab  1∶100, anti PECAM Ab (BD biosciences) 1∶50, anti FGFR2 (Bek) Ab (Santa Cruz) 1∶50, anti-Nkx2.1 Ab (TTF1) (Neomarkers), PITX1 and PITX3 were generated in Dr. Drouin's laboratory and were described previously Western blot and Immunoprecipitation studies using antibodies against P-ERK, total ERK, FGFR2 and PITX2 were carried out as previously described β-catenin and Pitx2 Dharmacon (ON-TARGETplus SMARTpool) were used. β-catenin and Pitx2 siRNA or scrambled siRNA were transfected into primary cultures of lung mesenchymal cells using Lipofectamine LTX (Invitrogen) according to the manufacturer's instructions. Mesenchymal cells at a density of 1×104 cells per well in 12-well plates were transfected in triplicate with 50 µM siRNA. The percentage of silencing of β-catenin and Pitx2 and the effect on Fgfr2c expression were detected by real-time PCR as previous described Pre-validated siRNA pools targeting mouse Proliferation and PERK assays were performed as previously described in Lung explants isolated from E12.5 WT and CKO embryos were cultured and treated with 200 ng/ml FGF9 (R&D systems) as previously described India ink was injected intracardially with custom made glass pipettes (12 µm opening) at E13.5. After injections, embryos were fixed in 4% formaldehyde for 12 hours, dehydrated and cleared in benzyl benzoate:benzyl alcohol (2∶1).Figure S1β-catenin in the mesenchyme resembles the phenotype of Pitx2 null embryos. (a) Frontal images of control and CKO embryos at E13.5. (b-c) Ectopic hearts and visceral organs in CKO's vs. WT with leftward displacement of ventricles (pseudo-colored in green).  β-galactosidase staining of WT or CKO embryos containing the Fgf10LacZ allele. CKO embryos display severe hind limb defects with no detectable LacZ/Fgf10 expression (inset in e).  Defective development of the mandibular and maxillary facial prominences and regression of the stomodeum.  Altered cardiac position with major cardiac outflow tract abnormalities in CKO heart (i) compared to WT (h). In WT, the pulmonary trunk (PT) rises from the right ventricle (rv) and is separated from the aorta (Ao), which rises from the left ventricle, by the aortic-pulmonary septum . (j-r) Histology of control (j-l) and mutant (m-r) at E13.5 . Most mutants display double outlet right ventricle or DORV , i.e., both the aorta and pulmonary trunk originate from the right ventricle. A subset of mutants demonstrates a single outflow tract rising from the right ventricle, i.e., they display Pulmonary truncus arteriosus or PTA (p-r). Leftward orientation of the heart is evident in all mutants; moreover the right ventricle is largely located above the left ventricle (m-r).Inactivation of (4.86 MB TIF)Click here for additional data file.Figure S2−/−Pitx2 phenotype. (a-b) Images of control and −/−Pitx2 embryos at E12.5. (c-d) Immunohistochemistry. Reduced expression for FGFR2 in E12.5 −/−Pitx2 lung mesenchyme and epithelium (d) compared to WT lungs (c). (e-f) Section RISH for c-Myc on E12.5 WT and −/−Pitx2 lungs. Expression of c-Myc is reduced in −/−Pitx2 lung mesenchyme (f) compared to WT lung mesenchyme (e). (g-h) Intracardiac India ink injection of E12.5 WT and −/−Pitx2 embryos. −/−Pitx2 embryos show defects in vasculogenesis and leakage of India ink from premature blood vessels is apparent (g) compared to WT embryos (h).Comparative analysis of the (10.05 MB TIF)Click here for additional data file.


P = 0.009). There was a trend towards an association between advanced tumour stage and loss of membranous expression of α-catenin or β-catenin, although these associations were not statistically significant. Univariate analysis revealed that advanced Dukes' stage, tumour de-differentiation, loss of membranous β-catenin expression, cytoplasmic β-catenin expression and widespread nuclear expression of β-catenin all correlated with short survival following apparently curative resection of the primary tumour. However, only Dukes' stage (P = 0.002), tumour grade (P = 0.02) and widespread nuclear expression of β-catenin (P = 0.002) were independent predictors of short survival. Disturbed growth signalling events in colorectal tumours are thought to result in nuclear accumulation of β-catenin. Consequently, tumours with widespread nuclear expression of β-catenin are likely to have severely abnormal growth characteristics, and which therefore might be predictive of short survival in these patients. © 1999 Cancer Research CampaignBoth cell adhesion and cell signalling events are mediated by components of the cadherin–catenin complex. Loss of expression of the components of this complex have been shown to correlate with invasive behaviour in many tumour types although their exact role in colorectal cancer remains unclear. Immunohistochemical analysis of the expression of components of the cadherin–catenin complex in colorectal cancers from 60 patients was undertaken. Loss of memberanous expression of E-cadherin, α-catenin and β-catenin was demonstrated in 52%, 85% and 40% of tumours respectively. Focal nuclear expression of β-catenin (< 75% of cells per section), usually associated with cytoplasmic expression, was clearly demonstrated in 19 (32%) tumours while widespread nuclear expression (> 75% of tumour cells per section) was seen in 11 (18%) tumours. Loss of membranous α-catenin expression significantly correlated with tumour de-differentiation (


The steroid receptor RNA activator is a functional RNA suspected to participate in the mechanisms underlying breast tumor progression. This RNA is also able to encode for a protein, Steroid Receptor RNA Activator Protein (SRAP), whose exact function remains to be determined. Our aim was to assess, in a large breast cancer cohort, whether levels of this protein could be associated with outcome or established clinical parameters.Following antibody validation, SRAP expression was assessed by tissue-microarray (TMA) analysis of 372 breast tumors. Clinical follow-up and parameters such as steroid receptor and node status were available for all the corresponding cases. Immunohistochemical scores were independently determined by three investigators and averaged. Statistical analyses were performed using standard univariate and multivariate tests.P < 0.05) higher in estrogen receptor-alpha positive , in progesterone receptor positive  and in older patients . When considering ER+ tumors, PR+ tumors, or younger patients (≤ 64 years), cases with high SRAP expression had a significantly  worse breast cancer specific survival (BCSS) than those with low SRAP levels. SRAP also appeared as a very powerful indicator of poor prognostic for BCSS in the subset of ER+, node negative and young breast cancer patients .SRAP levels were significantly (Mann-Whitney rank sum test, Our data suggest that SRAP levels might provide additional information on potential risk of recurrence and negative outcome in a specific set of patients with otherwise good prognosis when considering only estrogen receptor and nodal status. Breast cancer remains the second leading cause of cancer-related deaths in women worldwide and is one of the most frequently diagnosed cancers with an estimated 1,000,000 new cases detected each year worldwide . More reThe steroid receptor RNA activator (SRA) was originally identified as a functional non-coding RNA increasing the transcriptional activity of ligand-bound steroid receptors . It is cIt has now been confirmed that coding SRA transcripts co-exist in breast cancer cells, with the previously described non-coding transcripts -17. The Overall, accumulated data raise the intriguing possibility that SRAP levels could be associated with ER activity and/or expression, and could also potentially reflect on the response of breast cancer patients to endocrine therapy. It has recently been reported that the relative proportion of coding and non-coding SRA transcripts varies from one breast tumor to another and might characterize particular tumor subgroups . AltogetHela and Michigan Cancer Foundation (MCF)-7 cells  cells were cultured in DMEM  medium supplemented with 5% FBS , penicillin (100 units/ml), streptomycin (100 μg/ml) , and 0.3% glucose. Cells were grown in a 37°C humidified incubator with 5% carbon dioxide. Cells were transfected with empty vector or plasmids containing either the full SRA coding sequence and leading to the production of a SRAP-V5 tagged protein , or a pSTotal proteins were extracted from cells pellets as previously outlined . Frozen For neutralization experiments, 2 μg of SRAP-180-237 aa blocking peptide  was pre-incubated with 1 μg of 743A antibody for two hours at room temperature. A peptide, corresponding to 69 to 89 aa from the Small Breast Epithelial Mucin [Hela cells cultured on cover-slips were transfected with control empty vector or plasmid expressing V5-Tagged-SRAP or SRA-RNAi (see above). Twenty-four hours post-transfection, coverslips were washed with PBS and cells fixed with 4% formaldehyde  and 4% sucrose  for 15 minutes at room temperature. Fixed cells were then rinsed with PBS and permeabilized with 0.25% Triton-X100  in PBS for five minutes. After rinsing with PBS twice, non-specific binding sites were blocked with 10% BSA  in PBS for 30 minutes at 37°C. Cells were then incubated overnight at 4°C with 743A Anti-SRAP antibody diluted at 1/200 in 3% BSA/PBS. After washing with PBS for 15 minutes, cells were incubated with anti-rabbit secondary antibody-Cy3 conjugate  at a 1/1000 dilution in 3% BSA/PBS for one hour at room temperature. Cell nuclei were stained with 1 μg/ml Hoechst , and coverslips were mounted onto microscopy slides with FluorSave™Reagent . Fluorescent images were captured and visualized with a Nikon Eclipse E1000 epifluorescent microscope at wavelengths of 552 to 620 nm (CY3), 440 to 450 nm (Hoechst) using ACT-1 software .All invasive breast cancer and normal breast samples used in the current study were obtained from the Manitoba Breast Tumor Bank, which operates with the approval of the Faculty of Medicine, University of Manitoba, Research Ethics Board . The resMatched frozen and paraffin-embedded sections corresponding to 20 invasive breast tumors and 6 reduction mammoplasties were first selected and evaluated for protein expression by western blot and immunohistochemistry, respectively. Tumor ER and PR levels (determined by ligand binding assay (LBA)) ranged from 2.3 to 180 fmol/mg protein and 4.5 to 42 fmol/mg protein, respectively. The age of patients ranged between 58 and 78 years, and tumor size varied from 15 to 50 mm.TMAs corresponding to 450 ER positive  and 255 ER negative  cases were constructed from primary invasive breast carcinomas, as described before . DuplicaBreast tumor tissue sections and TMAs were stained with an anti-SRAP antibody  using an automated tissue immunostainer  at a dilution of 1:250, as described previously . Slides SRAP protein expression was assessed using a previously described semi-quantitative scoring consisting of an assessment of both staining intensity  and the percentage of positive cells (0 to 100%), which, when multiplied, generate an H-score ranging from 0 to 300 ,26. TMA Differences between SRAP expression in different subgroups  were tested using the Mann-Whitney rank sum test, two sided. Box and whiskers representation was performed with boxes at 25 to 75% and whiskers at 10 to 90%. Potential distributions of low and high SRAP cases in other clinical-pathological variables were tested using contingency methods (Fisher's exact test). Survival analyses were performed using the Mantel-Cox log-rank test to generate Kaplan-Meier curves. Breast Cancer Specific Survival (BCSS) was defined as the time from initial surgery to the date of death attributable to breast cancer only. Recurrence-free survival (RFS) was defined as the time from initial surgery to the date of clinically documented local or distant disease recurrence or death attributed to breast cancer. Deaths caused by other known diseases were censored. Statistical analyses were carried out using GraphPad Prism 5  and PASW Statistics 17 .Cox proportional hazards model was used to test the statistical independence and significance of the different predictors on BCSS (events modeled: deaths attributable to breast cancer only) and RFS (events modeled consisting of recurrences or deaths attributed to breast cancer). The predictors considered in the model were: age at diagnosis ; ER-alpha status ; node status ; SRAP ; all two, three and four-way interactions involving SRAP.The ability of the 743A anti-SRAP antibody  to specifically recognize SRAP was first assessed by western blot analysis of total protein extracted from MCF-7 breast cancer cells, previously shown to express this protein -17. A doin situ analyses, similar experiments were performed by immunofluorescence in Hela cells. As shown Figure To further validate the use of 743A anti-SRAP antibody for SRAP expression was subsequently assessed by western blot in a small series of 20 breast tumors and 6 normal breast samples as detailed in the Materials and Methods section. A signal, which varied in intensity between tumor samples and corresponding to SRAP of 30 kDa was detected in most cases  H-score values are higher in ER-positive than in ER-negative tumors . Similarly, SRAP staining is significantly stronger in PR-positive than in PR-negative cases . Tumors with higher Nottingham grade had a significantly lower SRAP staining than both low and medium grade lesions . When cases were divided into two groups using the median age at surgery (median H-score 64), older patients had a significantly higher SRAP expression detected in their tumors than younger patients . No difference in SRAP staining was observed between node-positive and node-negative tumors or between small and bigger lesions.As illustrated in Figure To further investigate potential non-random distributions of SRAP H-score staining in different tumor subgroups, we have arbitrarily divided the cohort into low and high SRAP expressors by selecting the median H-score value as cut-off point . When considering PR-positive cases, patients whose primary tumors expressed low SRAP also had a better BCSS and RFS . In younger patients  Low SRAP levels were similarly associated with a longer BCSS and RFS . No significant differences between low SRAP and high SRAP were found in the whole cohort or any other subgroups defined in Table When analyzing the cohort as a whole and most of the case subgroups detailed in Table P = 0.007) and higher rate of recurrence . In this subgroup, interaction between SRAP and age approached and reached significance, when considering BCSS and RFS, respectively (P = 0.060 and P = 0.030). Looked at in isolation, the interaction becomes clear , but not in older or node positive patients  have been grouped according to their age . Patients whose age fell in the first and second quartiles (24 to 55 and 56 to 64 years old), but not older patients, had a significant decrease in survival when their primary tumors expressed high SRAP levels Figure .In the present study, we have established that several SRAP-like peptides were detectable in breast tumor tissues. Interestingly, in ER positive, and more predominantly within node negative and younger patients, higher levels of the corresponding signal were associated with poor survival.No data reporting specific detection of SRAP employing the antibody used in our experiments (cat # A300-743A) has been published. It was therefore critical to first validate the use of this antibody for western blot and immunohistochemistry analyses. A doublet migrating at the apparent size of about 30 kDa is detected by western blot in MCF-7 as well as in multiple other cell lines . Sumoylation of this residue could potentially participate to the shift in migration from 30 kDa to 40 kDa ,32. FurtAlthough only six normal breast cases were analyzed, they all expressed only 30 kDa SRAP at a level lower than that seen in most tumors. This is in agreement with the previous observation that SRA RNA is overall expressed at lower levels in normal compared with matched adjacent tumor tissue . This alUsing 743A antibody, significant higher levels of SRAP are found in ER-positive tumors, in PR-positive tumors, and in lower grade tumors. This fits with the pattern of expression of a known activator of steroid receptor, the steroid receptor co-activator 1 (SRC1). Indeed, high SRC1 protein levels have recently been shown to positively correlate with ER, PR levels and low grade in a large cohort of 426 breast cancer patients . In theiAll 271 women whose tumors were ER positive had been treated with tamoxifen following surgery. Further analyses are needed to establish whether SRAP should be considered as a prognostic factor, a predictive factor, or both.Interestingly, the potential indicator power of SRAP appears limited to younger patients whose tumors are ER positive and node negative. It should be stressed that the age cut-point of 64 years (corresponding to median age at time of surgery) has been chosen to divide the cohorts in subgroups with comparable number of cases. A cut-point of 51 years, average age for spontaneous menopause ,36, woulThe particular group of younger patients identified in this cohort  usually benefits from better prognosis and is considered as having an increased likelihood of responding to tamoxifen therapy. Our data, however, suggest that among this group, women with higher levels of SRAP might need additional treatment compared with women with lower SRAP levels.We have previously reported that the detection in breast tumor extracts of 30 kDa SRAP by western blot, but not 25 kDa SRAP, might correspond to a better outcome for ER-positive/node-negative patients . There iAltogether, we have found that several SRAP-like peptides are expressed in breast tumors and that their detection by immunohistochemistry could be used as a new prognostic/predictive marker in younger patients with ER-positive/node-negative breast cancer. Additional studies, performed with other antibodies are warranted to confirm this observation and further establish whether specific SRAP-like peptides have additional predictor values.BCSS: Breast Cancer Specific Survival; DMEM: Dulbecco's modified Eagle's medium; ER: estrogen receptor alpha; FBS: fetal bovine serum; HER: human epidermal growth factor receptor; HR: hazard ratio; LBA: ligand binding assay; PBS: phosphate-buffered saline; PR: progesterone receptor; RFS: recurrence-free survival; SRA: steroid receptor RNA activator; SRAP: steroid receptor RNA activator protein; SRC1: steroid receptor co-activator 1; TMA: tissue micro-arrays.The authors declare that they have no competing interests.YY and GPS equally contributed to this work by performing antibody validation and TMA. CP scored the slides. ZN performed the statistical analysis. SC-K, CC and AB performed western blot analysis and immunofluorescent experiments. PHW, YM, LCM and EL conceived, designed and coordinated the study. All authors drafted, criticized and approved the manuscript.


Since the transcriptional functions of β-catenin are more often implicated in various disease processes, there is much interest in the development and use of reagents to interrogate spatial and temporal evidence of β-catenin nuclear signaling in cells and tissues. An important study demonstrated that the signaling form of β-catenin is specifically unphosphorylated at residues S37 and T41, and suggested that this form exhibits a propensity for cytosolic/nuclear accumulation relative to the total pool of β-catenin.We show that monoclonal antibody, 8E7, which recognizes the signaling form of β-catenin specifically unphosphorylated at S37 and T41 , also cross-reacts with a widely expressed, variably accessible nuclear antigen that is not β-catenin. In cell types commonly used to study Wnt activation, this non-specific nuclear staining can be robust, obscuring the ABC signal. Definitive detection of nuclear localized ABC can be confirmed through an ability of classical cadherins to sequester ABC to cell junctions. In tissues, milder antigen retrieval methods can reduce the accessibility of mAb 8E7 to this cross-reacting nuclear antigen.These findings reveal that interpretation of nuclear, signaling active β-catenin using monoclonal antibody 8E7 should be considered judiciously, and in conjunction with independent methods.This article was reviewed by Frank J. T. Staal (nominated by Rachel Gerstein), Jyoti M. Sen (nominated by Avinash Bhandoola) and Manabu Sugai. When β-catenin interacts with cadherin adhesion receptors, it serves to critically link these receptors to the cytoskeleton (reviewed in ). In thenot phosphorylated . This aillipore .As part of our own efforts to understand how phosphorylation of β-catenin's N-terminus alters its nuclear signaling activities, we discovered that while mAb 8E7 indeed recognizes cytoplasmic/nuclear ABC, this antibody also cross-reacts with a nuclear antigen in a number of cell types. Because nuclear staining persists in a cell line where the β-catenin gene is deleted by homologous recombination , we knowThrough our efforts to understand how the N-terminally, hypophosphorylated form of β-catenin is regulated, particularly in the context of fibrotic disorders where this pathway has been recently shown to play a causal role , we discTo interrogate the specificity of mAb 8E7, we subjected two cell lines that express no β-catenin protein to immunofluorescence and Western blotting analysis Figure . Mouse LWhile 8E7 non-specifically detects nuclei, this signal is relatively weak compared with cell lines that show strong activation of β-catenin signaling, such as the widely used human colon cancer cell line, SW480 Figure &3B. To pLacZ/+ reporter mouse . Taken altogether, the variable accessibility of this cross-reacting nuclear antigen may explain differences in detecting a specific nuclear ABC signal over this background antigen , in addition to providing evidence for increases in cytosolic and N-terminally unphosphorylated β-catenin by immunoblot analysis.We show that mAb 8E7, in addition to specifically recognizing an N-terminally hypophosphorylated form of β-catenin as previously demonstrated , also cr++). Cells were fixed in ice-cold anhydrous methanol for 10 minutes followed by three washes with PBS++. Cells were permeabilized and blocked for 30 minutes in PBS with 0.3%Triton-X100 and 0.3%BSA. Monoclonal mouse anti-activated β-catenin  and monoclonal mouse anti-β-catenin  were diluted in PBS with 0.3%Triton-X100, 0.3%BSA and 5% normal goat serum (NGS) and centrifuged briefly before use. Slips were incubated with primary antibody for 60 min at room temperature, followed by washing 10 dips each in 300 ml of PBS with 0.3%Triton-X100 and PBS alone. Cells were then incubated with a 1:500 dilution of goat anti-mouse or rabbit IgG-conjugated Alexa Fluor 488 or 568  in PBS with 0.3%Triton-X100, 0.3%BSA and 5% NGS centrifuged briefly before use for 60 min at room temperature. Slips were washed 10 dips each in a large volume of PBS with 0.3%Triton-X100, PBS alone, and finally distilled water. Coverslips were mounted under Gelvatol containing 0.1% DABCO and air dried one hour before storage at -20°C until viewing. Slides were viewed and imaged with a Zeiss Axioplan epifluorescence microscope equipped with a 63× objective.300,000 HEK293T, L cells or L cells stably expressing Wnt 3a were seeded onto sterile 22 × 22 mm glass coverslips within a 35 mm Petri dish in DMEM-10%FBS and cultured for 48 hours. Media was then aspirated and slips were washed three times with PBS containing calcium and magnesium .Cells were lysed in standard RIPA buffer  containing protease and phosphatase inhibitors (Roche). Protein content was assayed using the Bradford method (Bio-Rad). Proteins were separated by SDS-PAGE and electrophoretically transferred to BioTrace NT nitrocellulose . Membranes were stained with Ponceau S solution to evaluate protein transfer. Membranes were blocked in 5% milk-TBS-Tween and incubated with primary and secondary antibodies diluted in 5% milk-Tris-Buffered Saline-Tween. The ABC antibody was used at 0.2 μg/ml; anti-β-catenin clone 14 (BD Transduction) was used at 0.25 μg/ml. Immunoblots were developed in ECL solution (GE Amersham) and exposed to chemiluminescent-compatible film .TCF: (T-cell factor); LEF: (Lymphocyte Enhancer Factor); ABC: (Active β-Catein); mAb: .The authors declare that they have no competing interests.MTM, ASF and AMH carried out immuno-fluorescence and -blotting experiments; SR, RB and ONP performed immunohistochemistry experiments; MTM, ASF and CJG wrote the manuscript. All authors read and approved the final draft.Jyoti Misra SenLymphocyte Development Unit, Laboratory of Immunology, NIH, National Institute on Aging. Baltimore, MDIn this study Maher et al. evaluate the specificity of an important antibody reagent 8E7, which was believed to identify unphosphorylated beta-catenin, generally believed to be the signaling form of the protein. Their analysis clearly demonstrates that the 8E7 antibody cross-reacts with a non-specific protein antigen in cells that lack beta-catenin expression.Specifically data shown in figure Frank J.T. StaalErasmus MC, Rotterdam, The NetherlandsThe 8E7 antibody, developed in the laboratory of Dr. Hans Clevers recognizes the dephosphorylated N-terminus of β-catenin, the signalling competent form of β-catenin. This reagent was developed in order to better follow the fate of canonical Wnt signalling at the protein level, rather than simply assaying for increased levels of total or nuclear β-catenin which can occur independently of active Wnt signalling (as for instance elegantly shown by M. Watermanís lab). The development of these types of reagents was first described by Mascha van Noort et al in JBC and further on one of the developed antibodies, 8E7 or anti-ABC (activated-beta-catenin) was shown to report active Wnt signalling and , but not increases in β-catenin by other means. It has been described before in the JBC paper that 8E7 also recognizes a ubiquitous, apparently non-Wnt regulated protein of higher MW  with the similar name of 8E4. Despite a clarification we published last year in Blood, the incorrect antibody still appears every now and then in the literature. Besides that, 8E7 recognizes this "variably accessible" nuclear antigen that is not β-catenin, but it also correctly recognizes dephosphorylated β-catenin, and thus far is still the best reagent we have.raised against a peptide antigen corresponding to β-catenin's unphosphorylated GSK3 sites, this antibody should not be confused with an antibody like 8E7, which was specifically screened to only recognize β-catenin proteins that are unphosphorylated at certain GSK3 residues. In other words, the "non-phospho" status of an antigen does not ensure that the antibodies that recognize this region will only recognize the non-phosphorylated protein. Rather, a rigorous screening method . With regards to 8E4, a recent clarification published by the Staal group showed that 8E4 recognizes an epitope that lies well outside the GSK3 regulatory region . This cl of 8E7, ) is requThat a similar epitope is recognized by an antibody can be a problem under conditions with low active Wnt signals, is obvious. The notion that there is some background staining in unstimulated 293T cells, was reported previously  and with strong activation signals a clear increase is seen. I think it would be helpful if authors would include an experiment with milder Wnt stimulation, for instance by transfecting Wnt1 into 293T cells.We concur with the reviewer's statement that all antibodies cross-react with epitopes from other proteins, and that it is the responsibility of the investigator to understand the specific "signal-to-noise" ratio in their particular system. With regards to the non-specific nuclear staining associated with 8E7, titrating the antibody to reduce this background (reducing "noise"), or robustly activating the pathway , can certainly help. But not all cell systems can be manipulated this way. In this regard, Staal et al. nicely demonstrate that activation of HEK cells with Wnt-1 revealed enhanced nuclear staining with mAb 8E7 over the uninduced condition . HoweverI fully agree that besides antibody staining other methods to measure Wnt activity should be used, but a cautionary note should be added that even the so-called universal Wnt target genes (such as Axin2) may a) be regulated by other signals as well b) not always show up in all tissues as target genes (as is our experience). If authors can correct these points I see no reasons to object to publishing a cautionary report that when inappropriately used or with low Wnt signals, care should be taken one is actually measuring canonical Wnt signalling. tend to be more generally expressed across different cell types, we acknowledge the reviewer's point that there may be no "universal" Wnt pathway target gene(s). Thus, Wnt-activated targets for a particular cell and tissue type must be determined on a case-by-case basis.While negative feedback regulators of Wnt pathway activation like Axin2, naked and DKK Manabu SugaiKyoto University, Center For Genomic Medicine, Graduate School of MedicineIn this study, the authors have shown that monoclonal antibody 8E7 recognizes not only β-catenin but also other nuclear molecule(s) in some cell lines. From these observations, they recommend milder antigen retrieval method for tissue section staining using this antibody. However, this condition is very similar as described in Dr. Clevers's lab . There are some novel findings that are of interest, but the significance of the findings is not clear and the proposed procedures in using this antibody are not so informative comparing to the previous one.To determine the significance of weak signals appeared in some staining experiments is not easy. Usually, we determine the meaning of it by considering the data from the other kinds of experiments. As shown in this manuscript, translocation of β-catenin to cell surface by enforced expression of E-cadherin is used for it Fig . HoweverFrom this point of view, the authors show the reasonable imunohistochemical staining data Figure  using 8EI speculate that what the authors want to say in this manuscript is the signals observed using 8E7 antibody would make some troubles in estimating actual Wnt activity. So I want to know such situations. If the amount is not enough to detect using any experimental procedures, non-specific signals will appear by enhancing the signals. Accordingly, I think that the author's idea will be easily accepted by the readers if the authors will show some examples of the difficulty in discriminating the signals of ABC and those of non-specific. However, the author performed their experiment mainly using L cells. In L cells, the amount of non-phosphorylated β-catenin is very low and slight induction can be seen by Wnt3a expression Fig , Fig 3B.Figure is similar in abundance to the non-specific band (*) in HEK cells. While we cannot say that the ~160 kDa species is the cross-reacting nuclear antigen , this blot does show that the "signal-to-noise" ratio in Wnt/LiCl-treated HEK cells is low . Unless a cell type has an abundance of ABC relative to this background nuclear antigen , it will be difficult to know whether a nuclear ABC signal is truly due to ABC, outside of the cadherin-sequestration experiment proposed in Figure We firmly agree with the reviewer's acknowledgment that most antibodies cross-react with epitopes present within other proteins. We also agree that we need to establish under what specific conditions this can be a problem. We now show in Figure At first, I thought that longer exposure might be the cause of nuclear signals appeared in HEK 293 cells without LiCl stimulation, because ABC signals appeared in Figure Furthermore, their data contain more information, which is not addressed so much in the text. Activation of Wnt signal can be estimated by total β-catenin staining in Wnt3a-expressing L cells, but not ABC, because of the weak induction of dephosphorylated β-catenin Figure , 3A, 3B.For these reasons, I think that this manuscript is a reasonable paper for publication in Biology Direct.We agree with Dr. Sugai's suggestion that the Abstract and Conclusions of our manuscript could be written more clearly, so as to explain our findings more succinctly and directly. The following additions have now been incorporated:"In cell types commonly used to study Wnt activation, this non-specific nuclear staining can be robust, obscuring the ABC signal. Definitive detection of nuclear localized ABC can be confirmed through an ability of classical cadherins to sequester ABC to cell junctions. In tissues, milder antigen retrieval methods can reduce the accessibility of mAb 8E7 to this cross-reacting nuclear antigen.""These findings demonstrate that the ability of 8E7 to accurately capture evidence of Wnt signaling in cells or tissues critically depends on the strength and/or duration of the Wnt signal, relative to the accessibility of this background antigen."


This paper presents two approaches to the individual-based modelling of bacterialecologies and evolution using computational tools. The first approach is a fine-grainedmodel that is based on networks of interactivity between computational objectsrepresenting genes and proteins. The second approach is a coarser-grained, agent-basedmodel, which is designed to explore the evolvability of adaptive behaviouralstrategies in artificial bacteria represented by learning classifier systems. Thestructure and implementation of these computational models is discussed, and someresults from simulation experiments are presented. Finally, the potential applicationsof the proposed models to the solution of real-world computational problems, andtheir use in improving our understanding of the mechanisms of evolution, are brieflyoutlined.


APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant hereditary predisposition to the development of multiple colorectal adenomas and of a broad spectrum of extra-intestinal tumors. Moreover, somatic APC mutations play a rate-limiting and initiating role in the majority of sporadic colorectal cancers. Notwithstanding its multifunctional nature, the main tumor suppressing activity of the APC gene resides in its ability to regulate Wnt/β-catenin signaling. Notably, genotype–phenotype correlations have been established at the APC gene between the length and stability of the truncated proteins encoded by different mutant alleles, the corresponding levels of Wnt/β-catenin signaling activity they encode for, and the incidence and distribution of intestinal and extra-intestinal tumors. Here, we report a novel mouse model, Apc1572T, obtained by targeting a truncated mutation at codon 1572 in the endogenous Apc gene. This hypomorphic mutant allele results in intermediate levels of Wnt/β-catenin signaling activation when compared with other Apc mutations associated with multifocal intestinal tumors. Notwithstanding the constitutive nature of the mutation, Apc+/1572T mice have no predisposition to intestinal cancer but develop multifocal mammary adenocarcinomas and subsequent pulmonary metastases in both genders. The histology of the Apc1572T primary mammary tumours is highly heterogeneous with luminal, myoepithelial, and squamous lineages and is reminiscent of metaplastic carcinoma of the breast in humans. The striking phenotype of Apc+/1572T mice suggests that specific dosages of Wnt/β-catenin signaling activity differentially affect tissue homeostasis and initiate tumorigenesis in an organ-specific fashion.Germline mutations in the adenomatous polyposis coli ( Apc tumor suppressor gene and showed that the differentiation potential of embryonic stem cells is dependent on the dosage of Wnt/β-catenin signalling these mutants encode for. Likewise, analysis of the two mutational hits occurring at the Apc gene in human and mouse intestinal tumors showed that these are selected to retain specific residual dosages of β-catenin downregulation. Here, we provide further support for this “just-right” signalling model by targeting a germline Apc mutation encoding for very low levels of Wnt signalling activation when compared with other mutants known to trigger intestinal tumorigenesis. Notwithstanding the constitutive nature of this mutation, heterozygous mice show a remarkable and highly penetrant predisposition to multifocal and metastatic mammary cancers without the GI tract tumor phenotype characteristic of the majority of Apc mouse models.Although signal transduction pathways are often described as “on–off” systems, the more quantitative aspects of signalling are likely to represent a very important means of regulation of the downstream biological outcomes. Mutations in members of the canonical Wnt signaling pathway represent among the most common defects in human cancers. However, it is yet unclear which factors determine tissue and organ specificity of the tumours arising upon Wnt constitutive activation. Previously, we have generated a series of hypomorphic alleles at the  In the gastro-intestinal tract, Wnt/β-catenin signaling regulates stemness and differentiation of epithelial cells along the crypt-villus axis APC tumor suppressor gene, the main negative regulator of the Wnt/β-catenin pathway, result in the constitutive activation of canonical Wnt signaling thus affecting stem cell differentiation and trigger tumor formation in the GI-tract and in other extra-intestinal tissues in a dosage-dependent fashion in man and mouse Epithelial malignancies such as colorectal and breast cancer are thought to arise and progress towards malignancy due to alterations in signal transduction pathways that regulate the balance between self-renewal and differentiation in adult stem cell compartments APC tumor suppressor gene is particularly suited to study the effects of specific dosages of canonical Wnt signaling on the multiplicity and tissue-specific distribution of the resulting tumors  domains. Stable truncation of the mouse Apc gene at codon 1638 as encoded by the Apc1638T allele, results in a protein retaining a sufficient number of functional domains to ensure wild type β-catenin regulation, namely 3 of the 7 β-catenin down-regulating domains and one Axin-binding SAMP repeat  repeat encompassed by Apc1638T  cells from pre-implantation blastocysts and compared them by TopFLASH reporter assays Apc+/+, Apc1638T/1638T, and Apc1638N/1638N ES lines Apc1572T/1572T ES cells encode for intermediate Wnt/β-catenin signaling levels, in between those characteristic of Apc1638N/1638N and Apc1638T/1638T. The latter are in fact very close to those of wild type (Apc+/+) ES cells, as previously reported Apc-mutant ES cell lines confirmed that, when compared with wild type (Apc+/+) cells, decreasing amounts of Apc-bound β-catenin are observed in Apc1638T/1638T, Apc1572T/1572T, and Apc1638N/1638N ES cells  and other Apc-mutant ES cells. In line with their intermediate level of constitutive Wnt/β-catenin signaling activation, Apc1572T/1572T teratomas show a more heterogeneous spectrum of ecto-, meso-, and endodermal lineages than Apc1638N/1638N   cells to differentiate towards specific lineages in a dosage-dependent fashion S cells) . In agreal layer . Hence, Apc+/1572T mice were generated from two independent ES clones previously obtained by targeting a hygromycin cassette at codon 1572 of the endogenous mouse Apc gene Apc1572T/1572T mice, heterozygous Apc+/1572T animals were interbred and four litters analyzed (n = 18 mice). None of the resulting animals was found to be homozygous for the targeted allele  indicating that the Apc1572T allele results in embryonic lethality, as previously observed for the majority of Apc-mutant mouse models with the only exception of Apc1638T1572T and Apc1638T truncated proteins, namely the Axin-binding SAMP motif pinpoints to a key role for this functional domain in Wnt signaling regulation during embryonic development.Apc+/1572T heterozygous animals was performed on a total of 69 mice and compared with wild type, Apc+/1638N and Apc+/Min on two different genetic backgrounds, namely inbred C57Bl6/J and F1 C57Bl6/J×129Ola  and males (30%), in sharp contrast with Apc+/Min and Apc+/1638N animals   analysis revealed that all Apc+/1572T mammary adenocarcinomas (n = 12) encompass luminal and myoepithelial cell types together with areas of squamous metaplasia  . These tctively) . Histolopatterns . Varyingpatterns , in sometaplasia . Heterogstaining .APC gene mutations in man and mouse, LOH analysis of DNA and protein samples from Apc+/1572T mammary tumors revealed allelic imbalance in more than 90% of cases (21/23)  . These o (21/23) .Apc+/1572T mice were identified with gross pulmonary alterations subsequently identified as metastases of the primary mammary adenocarcinomas by histological and IHC analysis. Similar to the primary mammary carcinomas, these lesions encompassed both luminal and myoepithelial cell types  only at more advanced progression stages Smad4 function underlies intestinal tumour formation in these animals through Tgf-β/BMP downstream effectors independent of Wnt signaling, the histology and molecular features of the Apc+/1572T/Smad4+/Sad GI polyps strongly suggest that the further increase of Wnt/β-catenin signaling conferred by the Smad4 mutation in the Apc-mutant background results in intestinal tumours in the compound mice without apparently affecting the mammary cancer phenotype.To provide additional experimental support for the “just right” signaling model for ad model . MoreoveAPC germline mutations lead to different spectra of extra-colonic manifestations depending on their localization along the gene and on the stability of the resulting truncated polypeptide APC mutants localized at the 5′ and 3′ ends of the gene result in atypical FAP phenotypes characterized by reduced intestinal adenoma multiplicities and enhanced tumorigenesis outside the GI tract (mainly desmoids and cutaneous cysts) Apc tumour suppressor gene results in a highly penetrant predisposition to mammary adenocarcinomas without the intestinal tumours characteristic of FAP patients carrying germline APC mutations and of most Apc-mutant mouse models reported to date Although the role of Wnt/β-catenin signalling has been established for a broad spectrum of cancers APC gene, only 5 residues downstream of the targeted Apc1572T allele Apc-mutant mouse models, characterized by a pronounced predisposition to multiple intestinal tumors Apc models show predisposition to mammary adenocarcinomas, though with considerably less penetrance than the GI tract tumors Apc+/Min mice into wild type recipient animals results in metaplastic adenocarcinomas Apc-mutant cells.Notably, DU4475, a human breast cancer cell line derived from a recurrent thoracic wall tumor following mastectomy due to a poorly differentiated invasive ductal carcinoma Apc alleles in the lactating mammary gland cells (by BLG-Cre) which resulted in multiple metaplastic growths which do not progress to neoplasia Apc-LoxP allele is specifically driven in mammary progenitor cells (by K14-Cre) and in lactating luminal cells (by WAP-Cre) K14-Cre-mediated Apc heterozygosity resulted into mammary adenocarcinomas with similar histological features to those observed in Apc+/1572T tumors, thus suggesting the early progenitor or stem cell of origin of these mixed lineage tumors. Notably, analysis of the wild type Apc allele in the K14-Cre;Apc+/CKO mammary tumors revealed the presence of specific somatic point mutations clustering in the codon 1521–1570 region, i.e. very close to residue 1572 where our own mutation was targeted Apc-mediated mammary tumor formation are strikingly similar between the tissue specific conditional knock-out K14-Cre;Apc+/CKO model and the constitutive Apc+/1572T mice: in both cases one allele is completely lost  whereas the other retains residual β-catenin downregulating activity . The final outcome of these selection processes is the retention of the dosage of Wnt/β-catenin signaling that is “just right’ to allow clonal expansion of mammary stem cells or early progenitors and their neoplastic transformation.Further confirmation of the validity of the “just right” signaling model has been more recently provided by the conditional inactivation of both Apc1572T mutation is expected to trigger intestinal tumor formation. Recently, it has been reported that Tgf-β signaling antagonizes canonical Wnt thus negatively regulating stem cell self-renewal Smad4 loss of function mutations, are expected to lead to a further increase of Wnt/β-catenin signaling in the Apc-mutant cellular background. Accordingly, Apc+/1572T/Smad4+/Sad compound heterozygous mice revealed a similar incidence of mammary adenocarcinomas as in Apc+/1572T, but were also characterized by multiple GI-tract tumors never observed in the parental strain, even when kept for longer than 1 year. These polyps arise from loss of the entire mouse chr. 18 where both the Apc and Smad4 tumor suppressor genes map, and are clearly different from the hyperplastic lesions characteristic of Smad4+/Sad mice which retain the wild type Smad4 allele Smad4 function underlies intestinal tumour formation in these animals through Tgf-β/BMP downstream effectors independent of Wnt signaling, the histology and molecular features of the Apc+/1572T/Smad4+/Sad GI polyps strongly suggest that the further increase of Wnt/β-catenin signaling conferred by the Smad4 mutation in the Apc-mutant background results in the observed predisposition to intestinal tumours in the compound mice without apparently affecting the mammary cancer phenotype.A second implication of the “just right” signalling model is that an increase of the Wnt/β-catenin signaling dosage conferred by the Apc1572T model. However, with the only exception of the SAMP motif, all the known functional domains located in the COOH third of the protein which are truncated by the targeted mutation at codon 1572 are also absent in the Apc1638T model, previously shown to be tumor free even when bred to homozygosity In view of the well known multifunctional nature of the APC tumor suppressor protein Apc+/1572T animals, especially as far as the heterogeneity of their histology is concerned. Among the broad spectrum of breast cancers observed in man, the histological subtype that most closely resembles the Apc1572T mammary adenocarcinomas is represented by metaplastic carcinoma of the squamous type, responsible for 1 to 5% of the total breast cancer burden CTNNB1 (β-catenin), APC, and WISP3 (Wnt1 Induced Secreted Protein 3) are relatively common among metaplastic carcinomas of the breast Several members of the Wnt signaling cascade including the Wnt1 ligand and β-catenin, have been shown to result in mammary hyperplasia and tumors when overexpressed in transgenic mice in a tissue-specific fashion Apc+/1572T mice without the concomitant presence of multiple intestinal polyps raise the possibility that a fraction of the hereditary breast cancer cases could be caused by germline APC mutations located in the proximity of codon 1572. We are currently testing this by sequencing the APC gene around codons 1500–1700 in hereditary breast cancer patients which tested negative for BRCA1/BRCA2 germline mutations. Also, we are searching whether FAP patients with APC germline mutations in the proximity of codon 1600 show any eventual predisposition to breast cancer. However, it is also possible that mutations in other members of the Wnt pathway may result in the “just right” signaling level and in tumor predispositions similar to the Apc1572T model.Apart from their resemblance with breast metaplastic carcinomas in man, the mammary adenocarcinomas observed in Apc mutation encoding for intermediate levels of Wnt/β-catenin signaling results in a highly penetrant predisposition to multifocal mammary adenocarcinomas without the intestinal tumors characteristic of most Apc-mutant mouse models and individuals carrying germline APC mutations. Our results, also supported by several mammary cancer studies in mouse and man suggests that only specific dosages of canonical Wnt signaling are “just right” to expand the mammary stem/progenitor cell and result into mixed lineage (metaplastic) tumorigenesis.In conclusion, we have shown that a targeted Apc+/1572T mice were generated from two independent 129Ola ES clones targeted at codon 1572 as previously described Apc+/1572T mice. To generate inbred C57Bl6/J Apc+/1572T mice, F1 (C57Bl6/J×129Ola) animals were backcrossed to inbred C57Bl6/J mice for at least 8 generations. Control Apc1638N and ApcMin animals were generated by crossing inbred C57Bl6/J mutants with 129Ola for comparative purposes. Heterozygous mice were employed for the phenotypic characterization, together with wild type littermates as controls. Compound Apc+/1572T/Smad4+/Sadin cis mice were generated as previously described ad-libitum and housed in SPF facilities. Animal experiments were performed according to institutional and national regulations.All animals were fed Apc1572T/1572T ES cells were derived from pre-implantation (3.5 dpc) blastocysts as previously described 6 cells into isogenic mice.5 ES cells were plated on dishes coated with MEFs (mouse embryonic fibroblasts) and subsequently transfected by Lipofectamine 2000 (Invitrogen) with either 500ng of the TOP-FLASH or FOP-FLASH reporter constructs 5×10Apc IP analysis was performed according to previously published protocol Tissues were fixed in PFA (4%) and embedded in paraffin. Five μm sections were mounted on slides stained by HE for routine histology. Antibodies employed for IHC analysis include: β-catenin , Troma1 which recognizes a Ck8 epitope , Sma , Ck14 , Ck6 . The Ck6 and Ck14 antibodies are employed to detect hair follicle and skin differentiation, respectively. However, when employed at a lower dilution (1∶1000), Ck6 also detects mammary epithelial cells. All IHC images presented here were obtained with higher dilution (1∶5000) aiming at the identification of squamous differentiation lineages. The same primary antibody dilutions were employed for IF analysis, rabbit anti-rat-FITC (Sigma) and goat anti-mouse-A594 (Invitrogen) were used for signal detection.Apc+/1572T mammary adenonarcinomas and stained by HE. Tumour areas were localized and microdissected by LCM , followed by DNA isolation. PCR-amplified fragments were resolved in a denaturing 6% polyacrylamide gel, dried on paper and the number of counts per allele was determined on a phosphor imager. Subsequently, the number of counts of the larger allele was divided by the counts of the smaller allele to obtain an allelic ratio. A mean allelic ratio was calculated for at least five normal controls. This value was used to generate a comparative ratio (CR) >1.0 by dividing the tumor allelic ratio by the mean normal allelic ratio. In this way, normalization of imbalances already observed in wild type controls due to preferential amplification of a specific allele, is not necessary. A CR ≥1.5 was interpreted as significant, i.e. indicative of loss of the wild type allele.LOH analysis for the Apc locus was performed as previously described These PCR-based observations were further confirmed by western blot analysis. Mammary tumors and control tail tissue samples were digested with Blendzyme3 (Roche Diagnostics) in DMEM medium supplemented with gentamycin. Protein lysates, separation and blotting were performed using the NuPage Gel System (gel Tris-Acetate 3–8%) according to manufacturer's protocol (Invitrogen). Apc detection was accomplished using the antibody Apc Ab1 .Smad4 gene was carried out exclusively at the protein level by IHC on tissue sections . In this case, antigen retrieval was performed with Tris-EDTA pH 8.0 and the signal was detected using Envision HRP-ChemMate Kit (DAKO).LOH analysis for the


Esophageal intramural pseudodiverticulosis is a rare condition characterized by the dilatation of the submucosal glands.We present a case of esophageal intramural pseudodiverticulosis in a 72-year-old Caucasian man who presented with dysphagia and with a background history of alcohol abuse. An upper gastrointestinal endoscopy of our patient showed an esophageal stricture with abnormal mucosal appearances, but no malignant cells were seen at biopsy. Appearances on a barium esophagram were pathognomonic for esophageal intramural pseudodiverticulosis.We demonstrate the enduring usefulness of barium esophagography in the characterization of abnormal mucosal appearances at endoscopy. Esophageal intramural pseudodiverticulosis is a rare condition characterized by the dilatation of submucosal glands. Based on approximately 250 cases reported to date, this condition is slightly more common in men than in women ,2. IntraA 72-year-old Caucasian man  presented with a 4-year history of mild dysphagia for solid foods. His medical history was notable for alcohol abuse and associated alcoholic hepatitis. An upper gastrointestinal endoscopy was initially performed on our patient. At endoscopy, a stricture of the mid-esophagus with numerous tiny erythematous macules on the mucosal surface was seen Figure  and 2B. Biphasic esophagography is generally recommended for the evaluation of an esophageal stricture. While a single contrast barium esophagogram provides maximal esophageal dilatation and is useful for the depiction of a stricture, a double contrast examination allows accurate imaging of the esophageal mucosa and wall. Barium esophagram is valuable for establishing this particular diagnosis as reports in the literature have suggested that the diverticular orifices are detected at endoscopy in only 20% of patients [This case demonstrates the enduring usefulness of barium esophagography in the characterization of peculiar mucosal appearances at endoscopy.Written informed consent was obtained from our patient 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.OJOC prepared the manuscript. MOR performed upper gastrointestinal endoscopy. AB and MM performed barium esophagography. FS and EQ medically managed our patient. All authors read and approved the final manuscript.


B. pseudomallei and B. mallei genomes containing a SNP that differentiates the two species. Sensitivity and specificity were assessed by screening BurkDiff across 469 isolates of B. pseudomallei, 49 isolates of B. mallei, and 390 isolates of clinically relevant non-target species. Concordance of results with traditional speciation methods and no cross-reactivity to non-target species show BurkDiff is a robust, highly validated assay for the detection and differentiation of B. pseudomallei and B. mallei.A real-time PCR assay, BurkDiff, was designed to target a unique conserved region in the  Burkholderia pseudomallei and B. mallei, though genetically very similar, have divergent lifestyles. B. pseudomallei is a soil saprophyte and facultative pathogen and the cause of melioidosis, while B. mallei is an obligate pathogen and the cause of glanders. Melioidosis is mostly a disease of humans and animals in Southeast Asia and Northern Australia, where B. pseudomallei is present in the environment; infection mainly results from percutaneous inoculation or inhalation or aspiration of the organism. Clinical manifestations of melioidosis can be asymptomatic, localized to virtually any organ, or disseminated, though the primary presentations are pneumonia and sepsis, where mortality rates are significant The bacterial species B. pseudomallei and B. mallei and the diagnosis of melioidosis and glanders currently depend on time-consuming culture of the organism. Confirmation by biochemical assays can add a week onto definitive species identification Identification of B. pseudomallei and B. mallei now exist that utilize various platforms: Sanger sequencing B. pseudomallei and B. mallei are relatively genetically promiscuous, making development of robust, specific single-locus assay diagnostics challenging Molecular methods to identify B. pseudomallei and B. malleiB. oklahomensis types as B. pseudomallei, and the B. thailandensis-like strain MSMB43 types as B. mallei (unpublished data).A single-reaction real-time PCR Taqman allelic discrimination assay was previously developed to identify and differentiate B. pseudomallei and B. mallei and simultaneous differentiation when necessary. Alternatively, as these two species often occupy disparate niches under normal circumstances, BurkDiff can be used as a single-probe assay for definitive identification of B. pseudomallei or B. mallei.Here we introduce a new more highly validated allelic discrimination assay, referred to as BurkDiff, to provide a higher level of specificity for accurate identification of B. pseudomallei and 10 B. mallei genomes to search for shared orthologous SNPs, then filtered them by mismatch value (the distance to the next SNP in bases). We further analyzed the resultant pool of SNPs and their flanking regions with a GenBank BLAST search, and finally chose one for assay development and validation.We used the methods described by Pearson et al. B. mallei allele (5′-FAM-CTGAAACGCGCAGCG-3′-MGB) and the other to the B. pseudomallei allele (5′-VIC-CTGAAACGCGAAGCG-3′-MGB). Real-time PCR was carried out in 10 uL reactions containing 900 nM of both forward (CGAGCGCATCGTACTCGTA-3′5′-) and reverse (5′- CAAGTCGTGGATGCGCATTA-3′) primers, 200 nM of each probe, 1X Applied Biosystems Genotyping Mastermix, and 0.5 ng template. Thermal cycling and endpoint analysis was performed on an AB 7900HT sequence detection system (Life Technologies) using the following conditions: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 58°C for 1 min.Using Primer Express 3.0 software , we designed a Taqman SNP dual-probe allelic discrimination assay in which one probe was designed to hybridize with the B. pseudomallei and B. mallei, we used the Taqman allelic discrimination assay to genotype a collection of human, animal, and environmental isolates of B. pseudomallei (n = 469) and B. mallei (n = 49) from a broad geographic range . Template amounts ranging from 106 to 100 genome copies per reaction were used for limit of detection determination.The limit of detection of the Taqman assay was assessed using a dilution series of DNA from isolates of B. mallei genomes that differ from the character state shared by all B. pseudomallei genomes. Filtering the 1,235 SNPs using a mismatch value of 100 resulted in a pool of 74 SNPs. The GenBank BLAST search revealed the exclusivity of one of the regions to B. pseudomallei and B. mallei, so it was selected for assay development and validation.Genome comparisons revealed 1,235 SNPs with shared character states among all B. pseudomallei strains were shown to contain the allele with the SNP state A, and all 49 B. mallei strains were shown to contain that with the SNP state C  to distinguish between B. pseudomallei and B. mallei, a preferred method to random identification of SNPs in conserved genes, as was done previously in silico analyses of the markers allowed for the design of a highly specific assay. The illustrated specificity of BurkDiff to B. pseudomallei and B. mallei suggests that insertion of the genomic region that this assay targets occurred during or subsequent to the B. pseudomallei/B. mallei evolutionary split from its close genetic relative B. thailandensisB. mallei lineage diverged from B. pseudomalleiB. pseudomallei and B. mallei isolates successfully genotyped using BurkDiff suggest the genomic insertion is evolutionarily stable and therefore a good target for identifying the species, while the point mutation could now be considered a canonical SNP (canSNP), a point mutation that marks a point of evolutionary divergence of two taxa and is inherently stable and thus definitive Our use of whole genome sequence data allowed for targeted identification of phylogenetically informative markers (B. pseudomallei and/or B. mallei. Using several of these assays in combination for definitive identification could be important, as the Burkholderiaceae are highly recombining organisms BurkDiff adds to the growing number of molecular based assays, especially real-time PCR, that have been designed to detect


Despite similar computational approaches, there is surprisingly little interaction between the computational neuroscience and the systems biology research communities. In this review I reconstruct the history of the two disciplines and show that this may explain why they grew up apart. The separation is a pity, as both fields can learn quite a bit from each other. Several examples are given, covering sociological, software technical, and methodological aspects. Systems biology is a better organized community which is very effective at sharing resources, while computational neuroscience has more experience in multiscale modeling and the analysis of information processing by biological systems. Finally, I speculate about how the relationship between the two fields may evolve in the near future. PLoS Computational Biology, which has attracted both in its editorial board and in the papers it publishes a representative mix of both fields. In this review I will explore what are the most likely reasons for the separation between the two disciplines and argue that this is to their detriment, as they have a lot to learn from each other. I will focus more on computational neuroscience, as I know this field best.As a computational neuroscientist, I was quite enthusiastic when the systems biology field appeared on the international scientific agenda of the late nineties. Both fields strongly emphasize the use of computational modeling to predict and investigate the properties of biological systems, and I hoped that they would interact closely and strengthen each other. In fact, some of the early initiatives in systems biology were led by computational neuroscientists http://cosyne.org). Alternatively, computational neuroscience is about the use of computational approaches to investigate the properties of nervous systems at different levels of detail http://www.cnsorg.org/) and user meetings of specific neural simulator packages. Although these two opposing views are often swept under the carpet, and many scientists attend both conferences mentioned, they are reflected in partially separate communities and sometimes lead to heated debate about how the field should be defined.Interestingly, for both fields the exact definition of what they are about and whether they are defined by computational methods is in dispute . They can be defined as either fields of study or as a computational paradigm. In the case of computational neuroscience, the term is often used to denote theoretical approaches in neuroscience, focusing on how the brain computes information http://www.systemsbiology.org/). For others, it is an approach using theory and computational modeling in close interaction with experimental verification to understand the dynamical behavior of biological systems http://www.icsb-2007.org/).Similarly, systems biology has also been described in multiple ways. For some it is the integrative study of the interactions between different components of biological systems, and how such interactions give rise to the function and behavior of a system. This approach is, for example, typified by the (Seattle) Institute for Systems Biology , summer courses , standard neural simulator software packages like GENESIS and NEURON The term computational neuroscience appeared in the second half of the eighties Traditionally it is assumed that systems biology originated in the late nineties. This neglects the fact that Mihajlo D. Mesarović, Ludwig von Bertalanffy, and their colleagues already proposed applying general systems theory to biology in the sixties The genomics revolution of the nineties The separate origins of the two fields, with computational neuroscience clearly being the more “old-fashioned” one, can explain several major differences in their scientific cultures. I will emphasize two: data-driven modeling and community based standards development.randomly connected networks to investigate their dynamics http://www.brain-map.org/, which is genomics). The reasons for this deplorable situation have been described elsewhere As already mentioned, systems biology mostly operates in a data-rich environment, where the challenge is more to isolate the important from the less important than to infer unknowns. This is very different from the situation in neuroscience where data is usually incomplete and a lot of guesswork is needed. An example to clarify this issue is the different approaches to networks. A very active area in systems biology is the application of graph theory http://www.genesis-sim.org/, and NEURON http://www.neuron.yale.edu/neuron/). Even worse, large communities, especially in neural network modeling, still use “homegrown” software that is simply not available electronically. Otherwise, the respective software landscapes may seem similar as one can find in either field both open source  and copyrighted packages . The differences become more obvious if one looks at some of the infrastructure supporting this software development, in particular the development of standards. For example, compare the terminology efforts of the Neuroscience Information Framework  with the Systems Biology Markup Language (SBML http://www.sbml.org/). The first is an attempt by the neuroinformatics field to set up data annotation standards for neuroscience, and also covers computational neuroscience. Despite the fact that several terminology workshops have been organized since 2004, it is very difficult to find any online information on this project. Moreover, it is unclear how it relates to the NeuroML (http://www.neuroml.org/) initiative, which is developing common data formats and associated metadata infrastructure for computational neuroscience. Contrast the top-down, secretive approach of NIF terminology with SBML, which has become a de facto standard of systems biology. This standard model description language encapsulates the full domain of biochemical reaction systems in a single mixed pool. An important reason for SBML's success is the bottom-up approach used in its development, involving all stakeholders in active discussions about the standard and making very effective use of Web-based collaboration tools. This community-based development model is of course copied from the open source movement (note the similarities with Linux) and has resulted in a much wider acceptance of SBML than the competing standard (CellML http://www.cellml.org/), which is based at a single institution. The success of SBML should be a model for  neuroscience in how to develop standards in a cheap and effective manner, but such a change will not come easy as it goes against the current insular culture of the field.Systems biology inherited the large-scale collaborative approaches common in “industrialized” genomics and benefited from the availability of more mature software development tools in the late nineties Of course SBML can also be improved; i.e., despite its wide implementation, few software packages support all of SBML and many limit SBML support to write-only mode. For application in neuroscience, the current version lacks provisions for defining geometry or spatial coordinates, which are necessary to simulate biochemical models of synapses that include detailed 3-D geometry at the submicroscopic level http://www.sbml.org/SBML_Software_Guide), exactly like what happened in computational neuroscience http://www.ebi.ac.uk/biomodels/). An interesting reuse has been to simulate hundreds of models to validate and benchmark simulators (http://www.biouml.org/biomodels.shtml). The situation is very different in computational neuroscience where only now, more than 20 years after their origin, the interoperability between two major neural simulators is being implemented http://senselab.med.yale.edu/modeldb/) SBML has not prevented a proliferation of software programs executing very similar tasks , into morphologically detailed neuron models A major strength of computational neuroscience is the accumulated knowhow in simulator software development, especially for multiscale modeling. The latter started when the cable equation introduced by Rall An interesting specialized simulator is Mcell http://www.nest-initiative.org/) was created to model very large neural networks consisting of fairly simple neuron models. The NEST developers have achieved a deep understanding of distributed event modeling, leading to very efficient, supralinear parallelization of their algorithms http://bluebrain.epfl.ch/), which aims to build an extremely detailed tissue model of a cortical column containing tens of thousands of complex neuron models In other cases the interest may not be so much to apply the simulator itself, but to learn from the technical software expertise developed in building it. For example, the NEST simulator  have been noticing a decrease in participants interested in cellular modeling and a shift toward networks and information coding. This has often been attributed to the increased use of cellular modeling techniques by experimental neurophysiologists However, I do not believe that the current situation will persist. Faced with the big disparity in funding levels, and even the abolishment of neuroscience specific programs like the Human Brain Project To prevent this outcome, the field of computational neuroscience will have to explicitly reach out to systems biology and to adapt to some of its conventions, as mentioned before. Eventually, it could then form a bridge between systems biology and neuroscience. A related question, of course, is the stability of the neuroscience field itself and whether it will become a data-driven discovery science


The role in which electronic endoscopy plays is important in EMR. It is useful in diagnosis and treatment of gastric cancer from a clinical viewpoint. EMR with use of electronic endoscopy allows bettercoordination between the operator and assistants, and thus improves the results further.


In this paper, we propose a one degree of freedom test for association between a candidate gene and a binary trait. This method is a generalization of Terwilliger's likelihood ratio statistic and is especially powerful for the situation of one associated haplotype. As an alternative to the likelihood ratio statistic, we derive a score statistic, which has a tractable expression. For haplotype analysis, we assume that phase is known.By means of a simulation study, we compare the performance of the score statistic to Pearson's chi-square statistic and the likelihood ratio statistic proposed by Terwilliger. We illustrate the method on three candidate genes studied in the Leiden Thrombophilia Study.We conclude that the statistic follows a chi square distribution under the null hypothesis and that the score statistic is more powerful than Terwilliger's likelihood ratio statistic when the associated haplotype has frequency between 0.1 and 0.4 and has a small impact on the studied disorder. With regard to Pearson's chi-square statistic, the score statistic has more power when the associated haplotype has frequency above 0.2 and the number of variants is above five. Haplotype analysis is a popular strategy to analyze multiple single nucleotide polymorphisms (SNPs) typed within one candidate gene ,2. Most λ) for the excess frequency of the associated allele in the cases. To test for association he proposed a likelihood ratio test, i.e. comparing the likelihood under the alternative to the likelihood under no association, for testing. Since it is unknown which allele is associated, the likelihood is a weighted sum of conditional likelihoods of the data given that the allele is associated over the observed marker alleles. As for weights Terwilliger [For multi-allelic markers, Terwilliger  proposedwilliger  proposedTLR) statistic under the null hypothesis with the 50 : 50 mixture of chi square distributions of null and one degrees of freedom. This distribution appeared to yield conservative p-values [λ evaluated at λ = 0 is a constant zero for any observed data for the weights that Terwilliger proposed. Hence the corresponding score statistic does not exist. Score statistics are often preferred over likelihood ratio statistics, because they have often a tractable expression which provides insight in the data and are robust against model deviations [The log likelihood function of Terwilliger's model also appears to have some unusual statistical features. For example, the allele frequencies that are used as weights are also unknown parameters in the conditional likelihood functions. Maximizing the log likelihood function appears not always straightforward. Further its distribution under the null hypothesis is unknown. Terwilliger proposed to approximate the distribution of the likelihood ratio , beta (FGB) and gamma (FGA)in the Leiden Thrombophilia Study (LETS) . In thesχ2 and the Terwilliger's likelihood ratio with weights equal to pj's (TLR). We also considered a likelihood ratio test with equal weights (LR). We first evaluated the type I error rates of the test statistics. For the score statistic we used the chi square distribution with one degree of freedom to approximate the distribution under the null hypothesis. For the LR and TLR statistics we used the 50:50 mixture of two chi squares with zero and one degree of freedom. We generated 10,000 samples of 200 case chromosomes and 200 control chromosomes from the multinomial distributions with probabilities p1⋯pm for m equal to 4, 5, 8, 10, 15 and 20 haplotypes. Similar to the simulation described by Terwilliger, the frequency of the most common haplotype, p1, was set to 0.5, whereas the remaining haplotypes were equally frequent (0.5/(m - 1)). The results are shown in the left columns of Table By means of a simulation study we compared the performance of the new score statistic m, the type I error rates of the score statistic m < 10, the type I error rates of Pearson's chi square corresponded to the nominal level, but became conservative for larger m due to sparse data. For all m considered, the type I error rates for the TLR statistic were conservative (< 0.03). The type I error rates for the LR statistic were also somewhat small (≈ 0.04), but were better than that of the TLR statistic.For all p1(1 - λ) + λ and pj(1 - λ) for j = 2⋯m for cases and pj j = 1⋯m for controls, respectively. First, we considered the model used by Terwilliger [p1 in controls was again set to 0.5 and this haplotype was more frequent in cases. The parameter λ was fixed to 0.5 which corresponds to a haplotype frequency of 0.75 in the cases. The number of haplotypes m was again set to 4, 5, 8, 10, 15 and 20. The results are shown in the right columns of Table To study the power of the statistics, we generated 10,000 samples of 100 case chromosomes and 100 control chromosomes from the multinomial distributions with probabilities williger . The mosm < 8, the power of Pearson chi square statistic was good. The LR statistic with equal weights. The power of the score statistic TLR statistic, for m ≤ 8. For m > 8, the TLR statistic had higher power than m > 8, the power of the score statistic m is small and the frequencies of the non associated haplotypes are too small, yielding a large variance of the score statistic (see formula 3). Because the LR statistic appeared to perform worse than both TLR, we did not consider this statistic in the following simulations.For TLR as a function of the excess frequency λ for various values of the frequency of the associated haplotype p1 = 0.1, 0.2, 0.3, 0.4 and 0.5. The remaining haplotypes were again equally frequent. We restricted ourselves to the number of observed haplotypes m of 5 and 8, because most of the genes can be characterized by up to 8 common variants. The parameter λ was varied between 0 and 0.5. The number of chromosomes n1 and n2 were 200. We used a nominal significance level of 0.05. The results are depicted in Figure Second, we studied the power of the Pearson's chi square, p1 = 0.5, the score statistic TLR performed similarly, both better than Pearson's chi square. For m = 5 and p1 = 0.4 or 0.3 and for m = 8 and p1 = 0.4, 0.3 or 0.2, the score statistic TLR especially for small λ. For p1 = 0.2 and m = 5 all three statistics had similar power. For p1 = 0.1 and m = 5 or m = 8, Pearson's chi-square performed similar to TLR and both statistics performed better than λ ≤ 0.1 and p1 = 0.1 and m = 5. Note that for p1 = 0.1, the pooled frequency of the associated haplotype is around m = 5, the pooled frequency is equal to λ = 0.2 and for m = 8 the pooled frequency is equal to λ = 0.06. Thus when the associated haplotype has frequency around For p1 of 0.3 or 0.2 and a small impact on the disease (λ ≤ 0.1), the score statistic performed better than TLR statistic. For m = 5 and m = 8 and p1 = 0.3, the gain in power of the score statistic compared to TLR statistic was about 4% and 8% for λ of 0.05 and 0.1 respectively. For m = 5 and p1 = 0.2, both statistics performed similar. For m = 8 and p1 = 0.2 the gain in power of the score statistic was large, namely 7% and 12% for λ of 0.05 and 0.1 respectively.Especially for common variants with frequency We applied the three statistics to a study on haplotype association of fibrinogen alpha (FGA), beta (FGB) and gamma (FGG) with risk of deep venous thrombosis in LETS ,16. FiftFor all genes, haplotype H2 appeared to be more frequent in the cases than in the controls. For FGG, FGA and FGB the allelic odds ratios of presence of H2 versus the rest was 1.34, 1.29 and 1.28 respectively. Note that these odds ratios were rather similar while the p-values of the corresponding chi square statistics were different namely, 0.005, 0.034 and 0.090 respectively. The difference in p-values was caused by the difference in degrees of freedom of the chi square statistics and the frequencies of other haplotypes. From the results of the standard chi-square statistics we concluded that only FGG was significantly associated to thrombophilia.TLR were respectively 0.003, 0.012 and 0.072. These p-values were in line with the estimates of λ, namely 0.08, 0.07 and 0.05 respectively. Since FGA and FGB both had 5 variants, the frequency of the associated haplotype H2 was 0.3 and 0.2 respectively and the λ's were rather small, the score statistic should have more power than TLR for these genes. On the other hand TLR may perform better on the FGG gene, because of the larger difference in frequency between cases and controls and because the frequencies in the pooled set are around 1/m. For this situation, the score statistic The p-values of the TLR statistic and the score statistic had no power for FGA. Based on the results of the score statistic, FGA and FGB were significantly associated with thrombophilia risk.The p-values of the score statistic Although for each subject we were able to derive the pair of haplotypes, we also estimated the p-values of the statistics by 1000 permutations using TAGSNPS to estimate the haplotype counts in each permutation step. The results were similar.λ small), five to eight observed variants, and a frequency of the associated variant in the range of 0.2 to 0.4, the new statistic performs better than the Terwilliger's LR statistic. For larger frequencies (around 0.5) or for a large number of variants (m > 8), Terwilliger's LR statistic appears to perform better, because the weight corresponding to the associated haplotype is larger. Compared to Pearson's chi-square statistic, the score statistic has more power when the associated haplotype has frequency larger than 0.2 and the number of observed variants m is larger than 5. Especially when the frequency of the associated haplotype is small (< 0.2), Pearson chi-squared statistic performs similar or better than the score statistic.In this report we have derived a new score statistic to test for association between a candidate gene and a binary trait. Score statistics are easy to compute, locally most powerful, and robust to small model deviations under the alternative . From ouWe used a mixture of two chi-square distributions to approximate the distribution of Terwilliger's LR statistic under the null hypothesis. It is known that this distribution appears to give conservative p-values . PermutaWhen the sampled population is not in Hardy Weinberg equilibrium, the considered statistics are not valid since they assume that the haplotypes of a subject are independent. For Pearson's chi-square statistic on haplotypes, a multi-allelic version of Armitage's trend test on pairs of haplotypes is available ,18. UndeIn this paper we assumed that the haplotypes could be derived from the typed tagging SNPs. When uncertainty in phase exists the haplotype frequencies have to be estimated using for example an EM algorithm  and the  be used . In anot be used .We conclude that by choosing alternative weights, in particular constant weights, in the likelihood of Terwilliger, a set of new powerful and robust statistical tests was derived. For genetic association studies aiming to identify common associated variants, the new statistic should be used in addition to the standard Pearson's chi square statistic. By using both statistics more insight in the data can be obtained. A program  which com be the number of haplotypes describing most of the genetic variation in a gene. Assume that the haplotype frequencies are in Hardy-Weinberg equilibrium proportions. Let p =  be the vector of haplotype frequencies in controls. Assume that only one haplotype denoted with index i is over-represented in the cases, then the haplotype frequencies in the cases can be modelled as qi = pi + λ(1 - pi) and qj = pj - λpj for j ∈ . Let x =  and y =  be vectors of haplotype counts in the cases and the controls, respectively, and let n1 and n2 be the total number of case chromosomes and of control chromosomes, respectively, and n = n1 + n2. Then the conditional likelihood Li given that haplotype i carries the mutation is given byLet and the likelihood proposed by Terwilliger is equal toLj given in formula (1).with It is easy to see that likelihood function (2) can be generalized to the following likelihood function:Lj given in formula (1) and w =  a vector of known positive weights restricted by l = log) to λ evaluated in λ = 0 is equal towith pj, the distribution of the Uw under H0 can be approximated by the normal distribution with zero mean and variance Uw = 0 when for all j wj = pj.For known allele frequencies Uw is given byOften the haplotype frequencies are unknown and have to be estimated from the data. Under the null hypothesis we can estimate the frequencies from the combined sample of cases and controls by H0, under n2/n fold compared to the variance VAR(Uw) because the allele frequencies are estimated from the data. Now the score statistic Note that the variance pj by its estimate where i, the expectation of Under the alternative hypothesis of the presence of one positively associated haplotype qi = pi + λ(1 - pi). Note that with LR: Likelihood ratio with equal weightsTLR: Terwilliger's likelihood ratioFGA: fibrinogen alphaFGB: fibrinogen betaFGG: fibrinogen gammaSNP: single nucleotide polymorphismLETS: the Leiden thrombophilia studyREG participated in method development, wrote a C code, carried out the simulation study and data analysis, participated in interpreting results, and in drafting of the manuscript. SUdeW and MCHdeV provided the data and participated in interpreting the results. QH designed the web interface and extended the C code to account for phase uncertainty. LH participated in method development and in interpreting results. JJH-D participated in method development and in interpreting results, and participate in drafting of the manuscript. All authors read and approved the final version.


Mutational analysis for the entire coding region of β-catenin cDNA was also undertaken in 20 lung, 9 breast, 5 kidney and 6 cervical carcinoma cell lines. Deletion of most β-catenin coding exons was confirmed in line NCI-H28 (lung mesothelioma) and a silent mutation at codon 214 in exon 5 was found in HeLa . A missense mutation at codon 19 and a silent mutation at codon 28 in the NH2-terminal regulatory region of the γ-catenin gene were found in H1726 (squamous cell lung carcinoma) and H1048 , respectively. Neither deletions nor mutations of these genes were detected in the other cell lines examined. These results suggest that β- and γ-catenins are infrequent mutational targets during development of human lung, breast, kidney, cervical and ovarian carcinomas. © 2001 Cancer Research Campaign http://www.bjcancer.comβ-catenin forms complexes with Tcf and Lef-1 and functions as a transcriptional activator in the Wnt signalling pathway. Although recent investigations have been focused on the role of the adenomatous polyposis coli (APC)/ β-catenin/Tcf pathway in human tumorigenesis, there have been very few reports on mutations of the β-catenin gene in a variety of tumour types. Using PCR and single-strand conformational polymorphism analysis, we examined 93 lung, 9 breast, 6 kidney, 19 cervical and 7 ovarian carcinoma cell lines for mutations in exon 3 of the β-catenin gene. In addition, we tested these same samples for mutations in the NH