Gallbladder cancer is an infrequent neoplasia with noticeable geographical variations in its incidence around the world. In Chile, it is the main cause of death owing to cancer in women over 40 years old, with mortality rates up to 16.5 per 100,000 cases. The prognosis is poor with few therapeutic options; in advanced cases there is only a 10% survival at 5 years.Several studies mention the possible role of DNA methylation in gallbladder carcinogenesis. This epigenetic modification affects tumor suppressor genes involved in regulation pathways, cell cycle control, cell adhesion and extracellular matrix degradation, in a sequential and cumulative way. Determining DNA methylation patterns would allow them to be used as biomarkers for the early detection, diagnosis, prognosis and/or therapeutic selection in gallbladder cancer. Gallbladder cancer (GBC) is the most frequent malignant tumor of the biliary tract and the fifth most common cancer of the digestive tract. The presenting symptoms are vague, so diagnosis commonly occurs at an advanced stage. This late diagnosis combined with the fact that the gallbladder lacks a serosa result in a rather dismal prognosis -3. The hSeveral factors have been associated with the risk of developing GBC. Lithiasis is one of the main risk factors, presenting in 65% to 90% of cases of GBC ,3,6,7; tin situ and subsequently becomes invasive  through the covalent bond of a methyl group ,19. Non-Genes that show oncogenic properties, that is, that exhibit a methylated promoter region in normal cells, can become reactivated in cancer cells by the loss of this methylation, resulting in hypomethylation . In geneet al..-38.31-38et al. reported), RUNX3 %, APC , CDH13 %, P15INKpatterns . We alsoPSCA is downregulated in non-neoplastic gallbladder lesions such as cholesterolosis, cholecystolithiasis and cholecystitis . However, it was disclosed that the expression was decreased in more than 90% of cancers (40 out of 44) [PSCA enhancer [PSCA was recently reported to be upregulated [Recently it has been reported that egulated , which sAlterations in DNA methylation patterns are commonly found in all cancers, often with concomitant changes in gene expression. In GBC, molecular information is reduced; however, a high rate of methylation of some genes in GBC has been reported and associated with carcinogenesis of other tissues of the human digestive tract.CDH1), located on chromosome 16q22.1, is one of the most important tumor suppressor genes [CDH1 encodes for a transmembrane glycoprotein of 120 kDa that intervenes in cell adhesion mediated by calcium. It is a component of the E-cadherin/beta-catenin complex, which is important for cellular polarity, normal tissue morphology and cellular differentiation [CDH1 belongs to a family of genes directly related to the processes of tumor invasion and cytoskeleton destabilization. CDH1 expression, it has been reported in less differentiated tumors, that generally have an unfavorable prognosis [CDH1 (77.8%) [CDH1 has been described to be hypermethylated in GBC  . CDH1 haCDH1 presented a high methylation frequency in poorly differentiated tumors and in cases with three or more positive nodes [CDH1 expression has been recorded as the lesion progresses, which may be due to methylation [In relation to the degree of tumor differentiation, it was observed that ve nodes . A signihylation . As far hylation ,39.CDH1 and the metastatic phenotype in GBC [Therefore, there is a significant correlation between the methylation of e in GBC , which he in GBC .CDH1 has been reported in two cell lines of biliary tract cancer (SNU-478 and SNU-1079), with silenced mRNA expression. After treatment with a demethylating agent (5-aza-2′-deoxycytidine), CDH1 was successfully re-expressed [Hypermethylation of xpressed .CDKN2A-p16 is a tumor suppressor gene that encodes the protein p16, which is an inhibitor of cyclin-dependent kinase, involved in cell cycle regulation at Checkpoint G1. The loss of p16 expression is usually connected to homozygote deletion, loss of heterozygosity, mutations and methylation. A lower expression of p16 has been reported to increase the activity of D-type cyclin-dependent kinase activity, which translates into aberrant phosphorylation of the retinoblastoma gene, producing uncontrolled cell proliferation [p16 is frequently inactivated by methylation of the promoter and rarely by deletion or mutation [. This is a tendency that is also observed in GBC with a loss of expression of up to 62.5% [et al.[feration ,46. Lossferation . In hepamutation . Inactivmutation -69. Thisto 62.5% ,50. Taka% [et al. reported% [et al..et al. published the methylation status of five genes in advanced carcinoma of the gallbladder and their correlation with the immunohistochemical expression, demonstrating that CDH1, APC, FHIT and CDKN2A might be important in the carcinogenesis of gallbladder [CDKN2A is also one of the main mechanisms that induce the loss of p16 expression [et al. studied different alterations of p16 in 68 tumors in Chinese patients, finding that only 14.8% presented aberrant hypermethylation [In 2006, Roa lbladder . Hypermepression . Furtherhylation . Other ihylation . When thhylation .p16 and p14, it was noted that they presented a high methylation rate (between 40% and 60%) for carcinoma of the biliary duct, GBC and for primary sclerosing cholangitis. In normal tissue and choledocolithiasis, either no or an extremely low methylation rate was observed in the two genes [In the analysis of wo genes .p16  in cases of GBC means a better overall survival rate, and it is thus considered a significant prognostic factor [p16 was related to poorer survival [The absence of alterations in c factor . A similsurvival . This wasurvival .p53, which is commonly altered in numerous human cancers. Reprimo is a highly glycosylated protein located in the cytoplasm that induces cell cycle arrest at the G2 phase, inhibiting the activity of Cdc2 and cyclin B1 [Reprimo by methylation was initially confirmed together with other genes in pancreatic cancer [This is a candidate tumor suppressor gene regulating yclin B1 ,72. The c cancer . As a rec cancer .Hypermethylation of this gene is infrequent in normal tissue; however, methylation percentages of 32% and 27% have been described in the gastric epithelium and colorectal polyps, respectively . A low mUCHL1  is the only gene with a potential oncogenic role that has found to be hypomethylated in the promoter region in GBC [. The product of the gene is a peptide responsible for eliminating Ub from proteins that have it, and to thereby avoid its degradation by the proteasome. Proteins degraded by this mechanism actively participate in cell cycle control, for example, p53 and a variety of cyclins [In vivo analysis of UCHL1-deficient mice suggested that it functions as a regulator of apoptosis in neurons [n in GBC . Is locan in GBC . The pro cyclins . Ub, a p cyclins . In vivo neurons  and also neurons .PGP9.5 promoter is a reliable marker in GBC and that DNA hypomethylation might play a significant role in the re-expression of the gene in GBC [UCHL1 had previously been found in its promoter region in colorectal cancer, and lymph node metastasis was significantly associated with a lower frequency of methylation [The accumulation of Ub has been documented in several types of primary cancers . During e in GBC . In addihylation , poorer hylation .p16CDH1REPRIMODAPK-1 and SEMA3B, is high in GBC [DNA methylation is the epigenetic alteration most studied in the cancer cell. The number of genes with aberrant methylation in the human cancer cell is not known, but it is estimated that around 1% or 250 genes) of the human genome can be aberrantly methylated in a tumor cell . The useh in GBC ,50,51,88 genes ofp16 is frequently inactivated in a wide variety of human cancers by at least three distinct mechanisms: point mutation, small deletions of both p16 alleles, and methylation of CpG islands [p16 promoter in two of eight (25%) cholangiocarcinomas, and four of seven (57%) tumors analyzed by immunohistochemistry, demonstrated an absence of p16 nuclear staining in primary sclerosing cholangitis-associated cholangiocarcinoma [CDH1 are rare events [et al.[et al.[RUNX3 was more frequent in elderly patients [Helicobacter pylori infection [E-cadherin methylation is an early event in gastric carcinogenesis, and is initiated by H. pylori infection [et al.[et al.[et al.[The tumor suppressor gene  islands . Other sarcinoma . Also, aarcinoma . The exparcinoma  and genoarcinoma . In chole events . Downrege events ,92,93, we events ,43. Thiss [et al. found a l.[et al. a frequepatients  and Helinfection  can accenfection . Anothern [et al. and Kogal.[et al. utilizedl.[et al.. All resl.[et al., who useMSP in its classical format is non-quantitative and cannot distinguish between low and high levels of a methylated target sequence. By contrast, combining real-time PCR probes with MSP, as in MethyLight assay, can achieve a quantitative assessment of the level of DNA methylation of a targeted sequence . The effUCHL1 has been reported to be repressed by methylation in other cancers, such as primary head and neck squamous cell carcinoma [UCHL1 is a controversial molecule from an oncologic point of view. Hypomethylation of its promoter has been identified in a subset of human cancers, including GBC, presumably due to its intrinsic oncogenic properties or as a result of transformation. However, arcinoma  and coloarcinoma  and pancarcinoma . Particuarcinoma  and it iChile is considered a very high-risk area for GBC, and its mortality rate reached 16.5 out 100,000 women in 2007 . It is iTherefore, the study of gene methylation has at least five potential clinical applications: reactivating genes inactivated by methylation using demethylating drugs; identifying tumor cells in biological samples, allowing an early diagnosis since the change in methylation frequently precedes the appearance of advanced tumors; determining the methylation of individual genes or methylation profiles for groups of specific genes; being used as response markers to chemo- or hormone therapy; and allowing the transition between neoplastic and normal tissue to be determined in the surgical section margins according to the gene methylation profile ,100,101.Finally, the reversible nature of the epigenetic changes that occur in cancer must be mentioned as these have made epigenetic therapy possible as a treatment option. This therapy is based on reversing the epigenetic modifications that occur in tumor cells and re-establishing a normal epigenome. The possibility of reversing DNA methylation and reactivating the affected genes is an attractive option for a new therapeutic target in the treatment of cancer or preneoplastic lesions. However, the main problem with demethylating drugs is their poor specificity and they therefore cannot be used in the treatment of previously selected genes . NeverthThe authors declare that they have no competing of interests.PL designed the article and participated in writing all sections of the manuscript. PB revised the manuscript and contributed to content relating to methylation studies on the gallbladder cancer. OT contributed to content relating to conclusions and provided guidance for the structure. JCR provided guidance for the overall structure and content of the manuscript. All authors read and approved the final manuscript.


The recently identified class of microRNAs (miRs) provided a new insight into cancer research, since abnormalities of members of microRNAs family have been found in various types of cancer. However, the relationship between five miRNAs  and colorectal cancer remains unclear. In the present study, we examined expression of these miRNAs in 25 pair-matched colon cancer tissues and normal colon mucosa. The expression levels of miR146a, miR155, miR21, miR135a, and miR147b were quantified by real-time PCR. We found that miR21, miR146a, and miR135a were all expressed at higher levels in colon tumors. On the other hand, miR146a and miR147b expressions are significantly higher in left colon compared to right colon. These two miRs, especially miR146a, seemed to be markers for the left colon tumors. Moreover, significant proportional and inverse correlations were found between miR expressions in tumor and healthy tissue, and the correlations profiles were different depending on cancer localization. Taken together, these results lead us to suggest the presence of different mechanisms regulating miRs expression and consequently their target genes in left and right colon. So the pathway of colorectal carcinogenesis would be different according to the site of the tumor. Colorectal cancer (CRC) is the third most common malignancy in the world, accounting for more than 1.2 million cases and 608,700 deaths per year  such as A large number of recent studies have shown the utility of microRNAs as cancer-related biomarkers, supported by the finding that some microRNAs display altered expression profiles in cancers compared to normal tissues .MicroRNAs (miRNAs) are evolutionarily conserved, endogenous, small, and noncoding RNAs of 19–25 nucleotides in length that repress protein translation through binding to target messenger RNAs . SubsequSeveral miRNAs have been identified in humans, but much remains to be understood about their precise cellular function and role in the development of diseases . MicroRNMicroRNA155 (miR155) gene located on chromosome 21q21 is involved in the process of carcinogenesis . Indeed MicroRNA21 (miR21) gene located on chromosome 17q23.2 is involved in inflammation, at least in part, by modulating cytokines responses . miR21 iMicroRNA146a (miR146a) gene is located on chromosome 5q34. A recent study provided evidence that miR146a acts as a modulator of the innate immune response and of the adaptive immune response as well. Additional studies showed that miR146a expression is upregulated in thyroid cancer and breast cancer in contrast to pancreatic cancer and gastric cancer in which the expression of this miR is downregulated . It is i stabilin 1 (STAB1) gene on 3p21 and in intron 5 of rhabdomyosarcoma 2-associated transcript (RMST) gene on 12q23. Some studies demonstrated elevated levels of miR135a in colorectal tumor cells [ adenomatous polyposis coli (APC) with a potent effect on Wnt pathway activity. These results suggest that upregulation of miR135a might be involved in CRC pathogenesis [MicroRNA135a (miR135a) is encoded in the human genome by two copies, which are located in the first intron of theor cells  and otheMicroRNA147b gene, which is located on chromosome 15q21.1, is involved in posttranscriptional regulation of gene expression by affecting both stability and translation of target mRNAs. Studies demonstrated the participation of mir147 in a negative feedback loop that is able to inhibit the proinflammatory response of macrophages to multiple TLR ligands . Few stuIn the current study, we aimed to investigate and compare the differential expression of the five miRNAs  in colon cancer (CC) tissue and normal colon mucosa and their relationships to the clinicopathological factors in Tunisian patients and this is in order to assess their involvement in colon carcinogenesis.Paired samples of colon cancer and adjacent normal mucosa in paraffin-embedded blocks came from 25 patients recruited from the Charles Nicolle Hospital of Tunis. They were classified on the bases of their histopathological profiles.Total RNA was extracted from tumor and corresponding normal tissue in paraffin-embedded blocks using the miRNeasy FFPE tissue kit (Qiagen) following the manufacturer's instructions. Total RNA was eluted in RNase-free water and stored at −20°C. The concentration and the quality of RNA were detected by spectrophotometer and by bioanalyser Agilent.−ΔΔCT method.Reverse transcription was performed using the miScript II reverse transcription Kit (Qiagen). Expression of miRNAs was measured using the miScript SYBR Green PCR Kit and miScript Primer Assays (Qiagen) according to the manufacturer's protocol. RNU6B-6 miScript Primer Assay was used as an endogenous control. For each sample, the standard control used is RNU6B-6 as endogenous control. RNU6B-6 corresponds to a small nucleolar RNA that was selected by a previous study from the best performing controls for miR quantification assays. Indeed, based upon its expression level, its relative abundance, and its stability, as determined by several tissues and cell lines analysis, RNU6B-6 is used as endogenous control snRNA chosen as candidate reference in several papers published in the literature on miR quantification , 24. miRt-test and by explorer test. All P values lower than 0.05 were considered statistically significant. Coefficient correlations were calculated using Spearman's rho test. Changes in miR expression between patient subgroups were determined using one-way ANOVA. Cases values with P < 0.05 were considered significantly different. Receiver operating characteristics (ROC) analysis was generated to tag expression of miR146a and 147b in colon tissue. The sensitivity and specificity of the optimum cut-off point were defined as those values that maximized the area under the ROC curve (AUC). All statistical tests were analyzed using SPSS software (version 20.0).For miR quantification performed by real-time PCR, we express our results relatively to endogenous RNA RNU6B-6; thus, it is possible to compare between samples. From this, we have compared the relative expression of each miR between groups that have been stratified according to epidemiological or clinical features. The different tests used for comparison are standard tests displayed by SPSS software and significance of the results is given by this software. The correlation between miR expression and colon cancer was assessed by paired samples The expression levels of five different miRNAs that were known to be deregulated in various cancers were quantified by real-time PCR analysis. We further established the CT values for these genes of interest, in colon tumor and normal mucosa.R which is presented as the n-fold change in gene expression allowed comparing the overall level of each miRNA. The value of R > 1 represented miRNAs overexpression in colon cancer relative to paired normal mucosa. The result reveals that miR21, miR135a, and miR146a are upregulated in colon cancer tissue compared to normal tissue (P = 0.011) and miR135a shows 8-fold overexpression in tumor tissue as compared to control tissue (P = 0.002), while miR146a overexpression is less high (4.02-fold) (P = 0.032). The relative expression ratio for miR155 suggests that it is also overexpressed in tumor tissue (2.84-fold) but statistical analysis did not carry out a significant difference in its expression between compared tissues (P = 0.087). The level of miR147b in colon cancer tissue compared to the healthy tissue tends towards downregulation (0.87-fold) (P = 0.793).In a first step, we analyzed the expression levels of miRNAs in tumor compared to normal mucosa . The rell tissue . Indeed,87-fold) . HoweverTo investigate the clinical significance of miR21, miR146a, miR135a, miR147b, and miR155 expression, the relationships between this expression and clinicopathological parameters of colon cancer patients were assessed. As shown in P = 0.012)  .Considering these results we have then evaluated the correlation between the expression of these miRs according to the tumor localization . As showIn these profiles the correlation between miR21 and miR155 is a constant feature. One has to notice that in healthy tissue correlation profiles in right and left colon are different indicating that regulation of gene expression is normally different according to left or right localization.In both healthy tissues, miR147b does not correlate with the other miRs studied.P = 0.008) and the dissociation of miR146a expression from the other miRs in left colon   .P = 0.003), miR155 (P = 0.04), miR135a (P = 0.02), and miR21 (P = 0.02) in normal tissue, indicating a significative change of miR146a expression in right colon tumor  and seemed to be in part determined in left tumor by that of miR135a in normal left colon (P = 0.03)  .According to this work, we considered miR135a, miR146a, miR21, and miR147b as potential markers in colon cancer and performed ROC curves in order to evaluate the pertinence of their use. We have scored the expression of miR135a, miR146a, and miR21 by curve ROC to confirm their association with colorectal cancer by measuring their degree of sensibility and specificity with respect to this type of cancer . MiR135aMoreover, it appears that miR146a could be considered as a marker of the left colon from a cut-off value of 14.76 with a sensitivity of 78.6% and a specificity of 73% and an AUC value of 0.79 .P = 0.04)   from a c = 0.04) .β-catenin pathways that are involved in pathogenesis and signaling pathway of CRC. Hence, miRs expression profile analysis enables us to predict prognosis, support diagnosis, and improve therapy responses in cancer [MicroRNAs are considered as potential molecular biomarkers for human malignancies. Indeed, they have an important role in differentiation, proliferation, and apoptosis. Both up- and downregulation of specific miRs have been described in CRC carcinogenesis . Connectn cancer , 25. Indn cancer . TherefoP = 0.011; P = 0.032; and P = 0.002, resp.). This result is in agreement with previous studies that have shown that miR21 is an oncogenic microRNA implicated as an inflammatory mediator and may promote inflammation-associated colon carcinogenesis [P > 0.05). Contrary to our results, miR155 is overexpressed in colorectal cancer and in various tumors, including breast, lung, and pancreatic cancers [In our study, we investigated expression profiles of five microRNAs  in tumor colon tissue compared to healthy adjacent tissue. We showed that miR21, miR146a, and miR135a are overexpressed in tumor tissue comparatively to control tissue  andinterferon regulatory factor 3 (IRF3). Induction of miR147 expression by TLR is capable of downregulating excessive inflammatory responses [On the other hand, we showed that miR147b is not significantly downregulated in tumor colon tissue compared to healthy tissue. On the contrary, some studies have reported significant downregulation of miR147b , 20. Othesponses .Taken together these data indicate that miR135a, miR146a, and miR21 could be considered as a marker of CRC tumor with good specificity and sensitivity.Further, we explored the relationship between expression of these five miRs and colon cancer stratified by tumor aspect, tumor differentiation, tumor histology, TNM stage, lymph node, and metastasis. We found no significant association or any significant differential expression of each miR according to clinical data. In fact, contrarily to our finding, other studies considered miR21 as biomarker for advanced stage of CRC, since its expression level was significantly higher in the late stages of the disease . In addiThe differences observed between this study and previous works could be related to environmental conditions as well as to factors involved in miRs expression and their dysregulation in carcinogenesis and CRC progression. These factors, such as way of life and intestine microorganisms, dramatically vary among populations.P = 0.012 and P = 0.04, resp.). This suggests that miR146a and at a lesser extent miR147b may be biomarkers for a subtype of colon cancer tissue. The other miRs are expressed in the same way in the right or left colon (P > 0.05). In fact, we showed a great difference between profiles of miRs expression correlation. The difference between expression levels of each miR and the correlation coefficient between miR expressions could be due to the mechanisms of regulation related either to genetic factors or to epigenetic factors or both. One has to notice that in normal right and left colon, miRs expression profile is different leading to the suggestion that cells at the two locations do not have the same gene expression profile. Moreover, in the past two decades, a growing amount of data has been reported suggesting that carcinomas of the right and left colon should be considered as different tumor entities. In fact, right- and left-sided lesions exhibit different genetic, biological, and demographical characteristics and risk factors, suggesting that the carcinogenetic mechanism and progression of colon cancer lesions may differ with tumor location [According to tumor location, we found that miR146a and miR147b are both more expressed in left colon tumors than in right colon tumors (location , 38. Thelocation –41. On tlocation . A high location . In the location . At the location . Hence, location , 50. Allβ1/Smad signal transduction pathway [On the other hand, from miRs dysregulation, it can be predicted which targets genes are involved in the carcinogenesis pathway. Few studies have focused on correlation between microRNA and targets. Zhong et al. confirmed gene Smad4 as predictive target of miR146a and showed downregulation of miR146a associated with upregulation of Smad4 at protein level. These data suggested that miR146a might influence proliferation through TGF- pathway . Shibuya pathway .Our study shows upregulation of miR21, miR135a, and miR146a in colon cancer tissue compared to healthy colon tissue. We showed also differences between correlations of miR expression in right- and left-sided colon cancers suggesting that they are differently regulated and that there are different mechanisms of carcinogenesis between the two sites of the colon. Particularly interesting is the miR146a; its expression and role are unclear in several cancers and especially in colon cancer. In addition, overexpression of this miR appears to be specific to the side of colon tissue.Further studies will be necessary to understand the biological role of these miRNAs in colon cancer in order to use them as potential targets for cancer prognosis and therapy.


We report a study that examined the existence of a cognitive developmentalparadox in the counterfactual evaluation of decision-making outcomes. Accordingto this paradox adolescents and young adults could be able to applycounterfactual reasoning and, yet, their counterfactual evaluation of outcomescould be biased in a salient socio-emotional context. To this aim, we analyzedthe impact of health and social feedback on the counterfactual evaluation ofoutcomes in a laboratory decision-making task involving short narratives withthe presence of peers. Forty risky , fortyneutral decisions , and emotions feltfollowing positive or negative outcomes were examined in 256 early, mid- andlate adolescents, and young adults, evenly distributed. Results showed thatemotional ratings to negative outcomes (regret and disappointment) but not topositive outcomes (relief and elation) were attenuated when feedback wasprovided. Evidence of development of cognitive decision-making capacities didalso exist, as the capacity to perform faster emotional ratings and todifferentially allocate more resources to the elaboration of emotional ratingswhen no feedback information was available increased with age. Overall, weinterpret these findings as challenging the traditional cognitive developmentalassumption that development necessarily proceeds from lesser to greatercapacities, reflecting the impact of socio-emotional processes that could biasthe counterfactual evaluation of social decision-making outcomes.  There is now a broad acknowledgement that counterfactual evaluation of outcomes playsa role in everyday decision making. Upon making the decision and observing theoutcomes, people are able to process not only what actually occurred but also analternative course of events that might have occurred if a different option had beenchosen. This complex evaluation requires the cognitive capacity to engage incounterfactual thinking, which is usually accompanied by emotions . Some stOther studies have also characterized regret and disappointment by differentialagency attribution: personal/controlled agency for regret and relief,external/uncontrolled agency for disappointment and elation . AccordiThe present study takes this second approach to the study of counterfactual emotionsin risk decision-making situations. We examined the adolescents’ and youngadults’ decisions involving controlled and uncontrolled events and theircounterfactual evaluation of their negative and positive outcomes to induce therespective emotions of regret, relief, disappointment, and elation. Adolescence is aperiod of increasing risk-taking behavior, including practicing dangerous sports,drinking alcohol, engaging in unsafe experimentation with addictive substances,among others . HoweverThe findings of this study may help to demonstrate the possible existence of acognitive developmental paradox not only in the decision-making process but also inthe realm of the counterfactual evaluation of outcomes. Traditional developmentaltheory presupposes that with age cognitive development proceeds from lesser togreater sophistication, and that increased cognitive skill should decrease thelikelihood of participation in risks . The cogWhat happens with regard to the counterfactual evaluation of outcomes in riskdecisions? Is there a cognitive developmental paradox, too? The studies on thedevelopmental progression of counterfactual reasoning from childhood to adulthoodhave shown that although five- to seven-year-old children are able to experienceregret and relief , 2012, tThere are reasons to suspect, however, that a cognitive developmental paradox couldalso exist in the counterfactual evaluation of outcomes. According to thedual-processes account , adolescThe existence of a cognitive developmental paradox and its possible explanationaccording to the dual-process proposal has not been tested in the realm ofcounterfactual evaluations of decision-making outcomes in social situations. Theexception is one recent study examining the ability to experience regret and reliefin children, adolescents, and young adults who performed a gambling task in asocio-emotional context of competition, in which they were informed that theiroutcome would be compared with that of a competitor . ResultsThe present study uses analogues to real-life social decision-making by means of thesocial context decision-making task SCDT, . This taBoth the risky and the neutral trials involved the same sequence of eventspresented on the screen as illustrated in 1) A second-person scenario describing “you” as accompanied by aclose friend;2) the two alternative options for the decision-making task in thatscenario;3) the outcome of the choice selected, either positive or negative;extremely bad) to +5 (extremely good), placedat the bottom of the screen; and4) the emotional rating scale, where participants had to indicate “how doyou feel about what just happened?” using a linear scale from -5(5) the feedback information , to inform participants about thegains and losses in health and peer popularity depending on the choice made and thenegative or positive consequence received.The outcomes presented were pre-set by the experimenter following a table ofcontingencies . First, Our main goal was to investigate whether providing feedback on health status and peerpopularity modulates participants’ performance on the counterfactualevaluation of the outcomes . We expected lower emotional ratings when this feedback isprovided, compared to the no feedback condition. When presenting feedback, thenegative peers’ reactions (decrease in popularity) were highlighted, whichmade more salient the socio-emotional context leading to bias in the evaluation ofoutcomes. Specifically, the feeling of regret resulting from risky choices andnegative outcomes would be lessened in the feedback condition. The reason is that inour task risky choices with negative outcomes, though involving health dangers, wereassociated to gains in peer popularity. This would not be the case for the feelingof relief resulting from risky choices with positive outcomes both in health andpopularity. To support our expectation, the feeling of regret was also attenuated ina gambling task by providing a socio-emotional context of competition with peers. We alsoPrevious studies on gender effects reported that adolescent women are more prone thanmen to perceive situations as risky . In factAltogether, the results of the present study could help to demonstrate the existenceof a cognitive developmental paradox in the counterfactual evaluation ofdecision-making outcomes to the extent that the presence of feedback with health andsocially relevant information could affect this evaluation process, as well as thepossible impact of age, and gender effects.MAge = 13.5 years, SD = 0.5; 64mid-adolescents (MA), aged 15-16, 32 female and 32 male,MAge = 15.6 years, SD = 0.5; 64late adolescents (LA), aged 17-18 years, 32 female and 32 male,MAge = 17.50 years, SD = .50, fromone public high school and 64 young adults (YA), aged 19-20, 32 female and 32 male,MAge = 19.5 years, SD = 0.5, from apublic university and one public technical school. After explaining the aim of thestudy to the teaching staff of each academic center and receiving the permissionfrom the officials, students volunteered to participate. Written parental consentwas obtained for children and adolescents prior to the assessment session. Writtenconsent was also obtained for adult participants, who also volunteered afterreceiving information about the research. The procedure was approved by theCommittee for Research Ethics and Animal Welfare at the University of La Laguna.A total of 256 volunteers participated in the study, belonging to four age groups: 64early adolescents (EA), aged 13-14, 32 female and 32 male,very negative)to +5 (very positive). The length of the sentences in the scenarioswas matched in the number of words and unfamiliar words were avoided in all thescenarios.The study used a social context decision-making task (SCDT) involving two types ofverbal materials: forty risk situations and 40 neutral situations . Pilot sExample of a risk situation:“You are in a disco with your close friend. In the toilet you and your friendmeet a guy who offers you cocaine”.Decision: 1) “You buy it” (risky choice), 2) “You tell him thatyou are not interested” (safe choice).Outcomes (risky choice): 1) Negative: “You got very sick and had to go tothe hospital”, or 2) Positive: “You had a big ’high’ andfelt great”Outcomes (safe choice): Positive: “You enjoyed dancing with yourfriends”.Example of a neutral situation:“You are in a restaurant with your friend checking the menu forlunch”.Decision: 1) “You decided to get a hamburger”, 2) “You decidedto get a hotdog”.Outcomes: 1) Negative: “The mayonnaise was spoiled and you got sick and had togo to the hospital”; 2) Positive: “You enjoyed the meal as it was verygood”.Participants received the scenarios of the risk and neutral situations auditorily andthe choices and outcomes in written format. The presentation of each piece ofinformation was self-paced, allowing for the recording of chronometric data inaddition to the behavioral data. The 80 trials were separated by an inter-trial interval of 5 s, and preceded by a 5-trial practicephase. The stimulus presentation was controlled by means of Cogent 2000, a MATLABToolbox for presenting stimuli and recording responses with precise timing.The task was administered individually to the participants in a quiet room at theirSecondary School. They were asked to imagine themselves (“imagingyou”) as vividly as possible in each situation accompanied with a closefriend and choose between the two alternative actions. They were informed that theirdecisions would have positive or negative outcomes with more or less impact on theirhealth status and their popularity among friends. Half of the participants, randomlyselected from the total sample, were submitted to the feedback condition beinginformed at the end of every trial (by means of bars diagrams) of the specific gainsand losses obtained in peer popularity and health see . Every 1With respect to the procedure, once participants entered a quiet room at the schoolhalf of them completed the battery of self-report assessment measures first  and then, individually, the SCDT inanother room; the other half followed a reversed order (first SCDT and thenquestionnaires).p2 )were calculated . T-test and post hocBonferroni corrected comparisons were used when necessary. Data points lying > 3SD from the grand mean of the dependent variables involvingtime measures in each analysis were considered outliers and were excluded from thatanalysis (2% of the data as average).A mixed factorial design was used with age (four groups) or gender (two groups) andfeedback (present/absent) as between-participant factors, and type of choices and outcome valence (negative and positive) as within-participantfactors. The dependent variables were the emotional rating, rating time, andobservation time of feedback (just in the feedback condition). Rating time is anindex of the cognitive cost allocated to the performing of the emotional rating.Observation time is the time spent watching the feedback information, being anindication of the cognitive effort required to process this information. Fouremotional conditions were analyzed resulting from the combination of type of choicesand valence of outcomes: (a) risky choice and negative outcomes (regret); (b) riskychoice and positive outcomes (relief); (c) neutral choice and negative outcomes(disappointment); (d) neutral choice and positive outcomes (elation). The emotionalcondition resulting from choosing the safe option was not included in the analysesas it always involved positive consequences and could not be crossed factoriallywith the other emotional conditions. In all the cases analyses of variance (ANOVAs)were used and effect sizes , which would suggest that participants experienced regret and disappointmentfor negative emotion scores and relief or elation for positive emotion scores.Means and standard deviations of emotional ratings after reading the consequences areshown in F = 40.4, p = .001,ηp2 = .164 (on average 2.0 and 2.9 respectively).There was a main effect of type of choice, F = 69.85,p = .001, ηp2 = .255, showing thatthe emotional ratings were higher in neutral choices than in risky choices. Therewas also a main effect of outcome valence on emotional ratings,F = 1191, p = .001,ηp2 = .854), showing higher emotional ratings fornegative outcomes (regret and disappointment) than for positive outcomes (relief andelation). However, there was a significant interaction of feedback by outcomevalence, F = 45.88, p = .001,ηp2 =.187  and no feedback conditions . By contrast, emotional ratings for negative outcomes (regret anddisappointment) were significantly lower under feedback conditions than under no feedbackconditions . Significant ageeffects were not observed.Overall, emotional ratings were lower in the feedback condition than in the nofeedback condition, p2 =.187 . Thus, eF = 5.26, p = .023,ηp2 = .025. Women reported higher emotional ratingsthan men when they were told the negative outcome of their decision (regret anddisappointment), whereas gender differences were not significant for positiveoutcomes (relief and elation).There was a significant gender effect according to the outcome valence on emotionalratings, F = 7.49, p = .050,ηp2 = .020. There was a main effect of outcomevalence, F = 19.1, p = .001,ηp2 = .087, indicating that emotional ratings fornegative outcomes took more time than those for positive outcomes. No interactioneffects with feedback were observed.Means and standard deviations of emotional rating times after reading theconsequences are shown in F = 3.52, p = .016,ηp2 = .087 , with only the difference between earlyadolescents and young adults being reliable, p < .05, all othercomparisons p > .10 . However, sensitivity ofrating times to feedback conditions also changed with age, F = 3.27, p = .032, ηp2 = .097,(p < .001) and young adults (p <.05).Age and gender differences were observed in rating times according to feedbackconditions. Overall, rating times significantly decreased with age, = .097,. OverallF = 4.37, p = .038,ηp2 = .021. Simple effects showed thatwomen’s rating times in risky choices (regret and relief) were significantlyshorter in the feedback condition than in the no feedback conditions(p < .01). Men’s rating times in neutral choices(disappointment and elation) were significantly shorter in the feedback conditionthan in the no feedback condition (p < .05).There was an interaction of gender by feedback and type of choice,F = 81.67, p = .001,ηp2 = .415. and after receiving negative outcomes as compared to positive outcomes , F =4.97, p = .029, ηp2 = .028. Botheffects were qualified by a type of choice × outcome valance interaction,F = 13.24, p =.001,ηp2 = .103, showing that the above difference wassignificant in the risky choices  butnot in the neutral choices .The last set of analyses was performed in the feedback condition only, since thedependent variable was the observation time of feedback information see . ParticiF = 3.09,p = .030, ηp2 = .075, with thedifference being reliable between early adolescence and mid-adolescence(p = .024), late adolescence (p = .007) andyoung adults (p = .024), .Overall, the observation times decreased with age, This study examined the existence of a cognitive paradox in the counterfactualevaluation of risk decision making by means of exploring the impact of gains andlosses in health status and peer popularity, as well as age and gender effects. Asexpected, the presence of feedback in risk situations determines an attenuation ofthe participants’ emotional experience derived from outcomes as compared tothe no feedback conditions, as shown by the lower emotional ratings, and shorterrating times in the former condition. No effect was obtained from the manipulationof feedback in the neutral situations, as expected. Moreover, what seems to bespecifically affected in risk situations are the emotions of regret anddisappointment which were attenuated in the feedback condition as compared to the nofeedback condition, whereas emotions linked to positive outcomes (relief andelation) were not affected by the presence or absence of feedback. This isremarkable, since overall the processing of negative outcomes demands more effortand provokes higher emotional arousal than the processing of positive outcomes, assuggested by higher emotion ratings, and longer rating times. The impact of feedbackon risky choices is not likely to be due to the instructions received at thebeginning of the task, since in both the feedback and no feedback versionsparticipants were informed that their decisions would have positive or negativeconsequences with more or less impact on their health status and their popularityamong friends. Also it is not likely to be due to a game-like effect according towhich participants tend to disregard the task information waiting for the finalfeedback, since the emotional attenuation is confined to the negative consequencesbut not to the positive ones.As expected, participants evaluating the outcomes in the feedback condition were lesssensitive to regret feelings as compared to relief feelings , both deUnexpectedly, participants were also less sensitive to disappointment feelings underfeedback conditions, as compared to elation, which suggests that the surprise effectprovoked by non-controlled negative circumstances  is also Emotional ratings to outcome information did not vary across age groups, in line withprevious results indicating that the children’s ability to reasoncounterfactually is already in place after 12 years of age . In factGender effects on emotional ratings showed that women experienced more emotionalintensity than men when confronted with negative outcomes (regret anddisappointment) but not when facing positive outcomes, with no feedback effects.That means that overall women become more emotionally activated than men not onlywhen faced with the prospect of receiving negative results but also when actuallyexperiencing harmful consequences . HoweverIn keeping with the ecological validity of the task we are aware of two possiblelimitations of the study. First, feedback information differs under risk  and neutral  situations.Introduction of an arbitrary weight towards gains and losses in peer popularitydepending on the neutral choices, instead of a “zero” score, couldhave affected results in unknown ways. However, we managed to perform the analysesand to draw the main conclusions from the comparisons performed within each type ofchoice . Second, it would be interesting to have included a measureof executive functioning to support our interpretation of decreasing times with agein the counterfactual evaluation of outcomes. Third, arguably using real-lifescenarios would have entailed some misunderstandings in the interpretation of riskand neutral situations. To keep this possibility at minimum, we have performed pilotstudies to elaborate verbal material that could be comparable across ages andgenders. We think that simulation of real-life situations is worthwhile to increasethe participants’ chances of visualizing the course of actions and foreseeingtheir consequences, and to facilitate the participants’ actual engagement incounterfactual-related emotional states.In conclusion, this study demonstrated that the cognitive developmental paradox alsoexisted when considering the counterfactual evaluation of outcomes in a salientsocio-emotional context. Evidence of development of cognitive decision-makingcapacities does exist, as young adults were more capable of performing fasteremotional ratings than early adolescents. Moreover, late adolescents and youngadults were able to differentially allocate more cognitive resources to thecounterfactual evaluation than younger participants in situations when no feedbackinformation is available. However, the paradox exists since across the age groupsenhancing the health and the socially relevant consequences of the choices by meansof feedback negatively affected the counterfactual evaluation in risk situations byattenuating the emotional sensitivity to the outcomes of the choices, especially thenegative ones. This could be potentially damaging since there would be less chancesto anticipate regret or to avoid the previously chosen option in the future, twocognitive capacities that are already present in older children . Altoget


The p53-related gene p63 is required for epithelial cell establishment and its expression is often altered in tumor cells. Great strides have been made in understanding the pathways and mechanisms that regulate p63 levels, such as the Wnt, Hedgehog, Notch, and EGFR pathways. We discuss here the multiple signaling pathways that control p63 expression as well as transcription factors and post-transcriptional mechanisms that regulate p63 levels. While a unified picture has not emerged, it is clear that the fine-tuning of p63 has evolved to carefully control epithelial cell differentiation and fate. At first glance, the tumor suppressor p53 and its family member p63 seem quite similar in function and exhibit a high degree of evolutionary conservation. In particular, the DNA-binding domains are about 60% identical at the amino acid level; however, the adjacent domains and C-termini diverge drastically . While ip63 is also unlike its family member p53 in that it is rarely mutated in human cancers. Instead, mutations in p63 lead to disorders with ectodermal dysplasia such as ankyloblepharon-ectodermal dysplasia-clefting (AEC)/Hay–Wells syndrome, which can include symptoms like cleft lip/palate and skin erosions –9.Waf1/Cip1 signaling, indicating that these domains can modulate target gene specificity , and the other set lacking the N-terminal transactivation domain (ΔN). While the ΔN form can be dominant negative to the TA isoforms , the ΔNpcificity , 12.−/− mice were found to have severe limb and epithelial defects, including partial or missing epithelial stratification, and truncated forelimbs  , 31. IndSo how does p63 impact cancer formation? The last decade has seen a preponderance of direct targets unearthed, including adhesion-related β4 integrin, the tissue integrity factor Perp, the Notch ligands Jagged1 and Jagged2, keratins 5 and 14, and EGF receptor , 37–41. p63 is also known to regulate a diverse set of microRNAs. A prominent target is miR-205, a repressor of epithelial–mesenchymal transition (EMT) and metastasis in bladder and pancreatic cancers , 47, 48.A data mining approach also identified p63 and the p53-related p73 gene as key regulators of microRNAs differentially expressed in ovarian carcinomas, including miR-200a, miR-200b, and miR-429 . For morTaken together, p63, like p53 and p73, can regulate a host of processes, some of which are known regulators for or against tumor growth. As suggested by the opposite expression of p63 in different tumor types, the context of the cell type appears to be critical to which p63 targets have the dominant effects in each cell. Whether targets are differentially expressed or have different activities in different cell types needs to be investigated further.As ΔNp63 is required for the formation of stratified epithelial layers and is the primary isoform expressed in the basal layer of epithelial tissues, it is subject to multiple modes of tissue-specific regulation , 14. As activation of p63 in fibroblasts  are associated with craniofacial abnormalities like cleft lip and/or palate . Both IRR  can also lead to craniofacial disorders such as cleft lip and Crouzon’s syndrome , 88. TheInterestingly, FGFR2 can induce expression of the epithelial-specific transcription factor Elf5, and deletion of Elf5 causes altered expression of ΔNp63 in the luminal compartment of mouse mammary tissue –94. ThisThe tyrosine kinase receptor EGFR has also been found to induce ΔNp63 expression. In one case, this was through phosphatidylinositol-3-kinase (PI3K) signaling in keratinocytes , while iEpithelial cells can undergo an EMT during development and during carcinogenesis, progressing to a more invasive and metastatic phenotype. This differentiation is thought to allow the cancerous cells greater motility and increased metastatic potential [see reviews by Thiery  and KangOther transcription factors involved in control of EMT are Ovol1 and Ovol2 , 105. ThWhile we have mentioned a number of transcription factors as regulators of ΔNp63, a clear picture has yet to emerge on which factors are critical direct regulators of ΔNp63 and through which sequence elements they act near the ΔNp63 gene.It is possible that multiple pathways regulate p63 through the C/EBP family of transcription factors, as they have been repeatedly found to regulate p63. C/EBPδ was found to bind to multiple regions of the ΔNp63 gene in human keratinocytes , 107. AnOther transcription factors have also been found to regulate the p63 gene. An OCT4 binding site within the TAp63 promoter activates its expression, suggesting its involvement in stem cell regulation . Anotherp63 positively activates its own expression through binding to the C38 and C40 intronic enhancers as well as to its own proximal promoter , 113. OvInitially, p63 expression was found to be suppressed by stresses, such as UV irradiation, that stimulate p53 expression –116. Binp63 levels are also regulated by miRNA, ubiquitin-dependent proteasomal degradation, and protein phosphorylation. Notably, miR-203 can repress p63 expression in supra-basal epithelial cells, contributing to definition of the border between progenitor and differentiated epithelial cells , 119. InOther miRNAs have also been found to regulate p63 expression. miR-92 targets ΔNp63α and β in the HaCaT keratinocyte cell line and in myeloid cells, respectively, and miR-302 suppressed p63 expression in germ cells , 122. Thp63 protein stability is also regulated by the ubiquitin–proteasome system, adding another layer of regulation . One exaWhile p53 is stabilized by DNA damaging agents, such as UV irradiation, ΔNp63 is degraded , 132. TwNF-κB can also activate the TAp63 promoter, suggesting that a shift to the TAp63 form could also underlie the DNA damage response . Again, An alternative mechanism for ΔNp63 degradation as part of the DNA damage response is phosphorylation on threonine 397 by the protein kinase HIPK2 . HIPK2 hp63 has been termed as a master regulator of epithelial cells, and it is often suppressed in order for these cells to differentiate , 142. WeIt will also be important to understand how modulation of p63 levels affects cancer formation. The combination of heterozygous p63 and p53 genotypes in mice yielded conflicting results, giving either greater or reduced tumor burdens , 144. AdOther open questions concern p63 promoter usage and splicing – what factors determine the balance of usage of the TA and ΔN promoters, and what governs the presence of different 3′ splicing isoforms? How does the balance of these 3′ isoforms lead to differences in development or oncogenesis? Finally, can the signaling pathways that control p63 levels be controlled to provide a therapeutic benefit in specific cancers? We can hope that the following years will bring a greater understanding of this master regulator of epithelial biology.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The Guest Associate Editor Wen Zhou declares that, despite being affiliated to the same institution as authors Ron Prywes and Kathryn Yoh, the review process was handled objectively and no conflict of interest exists.


Spontaneous neural activity in the auditory nerve fibers and in auditory cortex in healthy animals is discussed with respect to the question: Is spontaneous activity noise or information carrier? The studies reviewed suggest strongly that spontaneous activity is a carrier of information. Subsequently, I review the numerous findings in the impaired auditory system, particularly with reference to noise trauma and tinnitus. Here the common assumption is that tinnitus reflects increased noise in the auditory system that among others affects temporal processing and interferes with the gap-startle reflex, which is frequently used as a behavioral assay for tinnitus. It is, however, more likely that the increased spontaneous activity in tinnitus, firing rate as well as neural synchrony, carries information that shapes the activity of downstream structures, including non-auditory ones, and leading to the tinnitus percept. The main drivers of that process are bursting and synchronous firing, which facilitates transfer of activity across synapses, and allows formation of auditory objects, such as tinnitus. Spontaneous neural activity is very often considered as neural noise that sets limits on sensory performance. This neural noise idea has been the basis for the optimal processor model in psychoacoustics that typically worked on activity in auditory nerve fibers Green,  and trieThe neural noise hypothesis for spontaneous activity in auditory nerve fibers was apparently boosted by Kiang et al.’s  extensivA decade later, Liberman  raised cJavel et al.  showed fn = 41) having SFR <1 sp/s but in a few others (n = 2) >35 sp/s, the remaining 60 cells had SFRs between 1–35 sp/s. Eggermont and colleagues performed studies on spontaneous activity in AI of ketamine-anesthetized cats. In a series of studies  or AI . Huang et al. .Anesthesia will affect SFRs in a way that depends on depth and type of anesthesia. Barbiturates and urethane have a strong effect, ketamine very little. In contrast to studies that measure anesthesia effects on stimulus-evoked activity, there are only a few studies that compared SFRs under anesthesia and awake. Torterolo et al.  reportedg et al.  comparedAnesthesia also changes the prevalence and properties of burst firing in neocortex. Ketamine anesthesia results in spindle-like LFP activity in the 5–15 Hz range in cat auditory cortex, depending on the depth of the anesthesia  on SFRs in auditory nerve fibers. Liberman and Kiang  “exposedSuperficial multi-unit recordings, likely from fusiform cells, in the dorsal cochlear nucleus (DCN) of hamsters with 10 kHz, 125 dB SPL, 4 h induced noise trauma  was significantly elevated (about a factor two) compared to sham controls (N = 143). This was more evident in primary-like and onset types of neurons. In addition, mechanical damage to the high frequency region of the cochlea (N = 258) showed similar results as noise exposure, suggesting that it is the hearing loss and not the induction method that causes the SFR changes.Vogler et al.  exposed Ma et al.  exposed Vogler et al. , using tAs in the DCN, acoustic trauma (10 kHz tone at 124 dB SPL for 1 h) in guinea pigs did not immediately resut in SFR changes in the ICC. Two weeks recovery after acoustic trauma resulted in more neurons with high SFR compared to control animals, and a significant increase in the average SFR from control (mean = 1.2 sp/s) values  were further assessed by recording, with two 8-microelectrode arrays, from the same sorted-unit clusters before and after the trauma  and Ab1 (P < 0.001) groups. However, when the three frequency bands were combined, unpaired t-tests revealed a significant increase in the percentage of time of burst-firing (P < 0.0001), number of spikes per burst (P < 0.0001), mean burst duration (P < 0.0001) and mean ISI within a burst (P ≤ 0.001), immediately after the trauma (“After1” condition). The burst properties returned to normal a few hours after the trauma (“After2” condition). However, as we have seen SFRs increased significantly  and 1.4 for Ab2-Ab2 groups (P < 0.0001). For the After2 condition (well after the trauma), one observes that ρ is significantly increased for the Be-Ab2 (P = 0.001), Ab1-Ab2 (P = 0.0004) and Ab2-Ab2 groups (P < 0.0001). Again, SFRs increased significantly in the After2 condition over a wider frequency range compared to the changes in ρ.Spontaneous neural synchrony between spike firing of two neurons is also affected by noise exposure  structure, whereas in auditory cortex there is a hyperexponential distribution  to in particular the ICC and MGB and then increases somewhat in some auditory cortex recordings, likely because they reflect multi-unit activity in contrast to single-unit activity at more peripheral levels Figure . Could oAfter noise trauma, it is often observed that the increase in SFR Figure , and alsThese two distinct views of spontaneous activity, either as unwanted noise or as information carrier, may thus determine how one views the neural mechanisms of tinnitus. If one considers spontaneous activity in the auditory system as unwanted noise, the favored concept about tinnitus is likely that it results from too much noise. The suggested neural substrate will then be increased SFR in the auditory system. On the other hand, if one considers spontaneous activity as the information carrier of the brain, sound modulates this firing rate and reorganizes it. In this model external sound can suppress tinnitus. Tinnitus in this model results from increased neural synchrony, i.e., the pathology also reorganizes the spontaneous firing times either in the form of serial correlations, i.e., burst firing, or as parallel correlations, i.e., as synchronous firing among neurons (Eggermont and Tass, Both serial and parallel synchrony will enable efficient synaptic transmission and may amplify each other. Eggermont et al.  phrased The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tumor suppressor molecules play a pivotal role in regulating DNA repair, cell proliferation, and cell death, which are also important processes in the pathogenesis of Alzheimer’s disease. Alzheimer’s disease is the most common neurodegenerative disorder, however, the precise molecular events that control the death of neuronal cells are unclear. Recently, a fundamental role for tumor suppressor molecules in regulating neurons in Alzheimer’s disease was highlighted. Generally, onset of neurodegenerative diseases including Alzheimer’s disease may be delayed with use of dietary neuro-protective agents against oxidative stresses. Studies suggest that dietary antioxidants are also beneficial for brain health in reducing disease-risk and in slowing down disease-progression. We summarize research advances in dietary regulation for the treatment of Alzheimer’s disease with a focus on its modulatory roles in BRCA1 and p53 tumor suppressor expression, in support of further therapeutic research in this field. BRCA1 and p53 tumor suppressor genes comprise a variety of cancers. In general, tumor suppressor molecules play a pivotal role in regulating both cell proliferation and cell death in many cell types. Those may also play an important role in progression of the AD lesion coinciding with changes in the cellular composition. For example, alteration of the tumor suppressor gene p53 function, essential in DNA repair and cell apoptosis, often exacerbates cognate behaviors [Alzheimer’s disease (AD) is a progressive degenerative disorder that commonly affects emotional stability and memory domains, and is the most predominant reason of dementia ,2. Pathoehaviors . Other tehaviors ,14 as weehaviors ,16. Suchehaviors ,18. Stanehaviors . The DNAehaviors ,21. ReceThe tumor suppressor BRCA1 implicated in breast and ovarian cancers exerts various properties on the DNA repair system ,22. ActuBRCA1 gene is generally expressed in dividing neuronal cells during development [The elopment , and encelopment   extract up-regulates expression of p21 WAF1 and p27 KIP1 [M. officinalis effectively prevents memory impairment in a transgenic mouse model of AD [M. officinalis, on beta-amyloid-induced neuronal toxicity have been reported [Ginsenoside, one of components in American ginseng herb, activates p53 , which cp27 KIP1 . In addiel of AD . Moreovereported . Baicalireported . The effreported .Gleditsia sinensis, have been shown to increase cell cycle arrest during the G2/M phase, and is associated with increased p53 protein level [Gleditsia sinensis thorns is also associated with up-regulation of p21 WAF1 [Saussurea involucrate induces p21 WAF1 and p27 KIP1 expression, independent of the p53 pathway [Several other promising herbs or life-style conditions involved in p53 activity are as follows. Treatment with thorns from a medicinal herb, in level . Treatmep21 WAF1 . In addip21 WAF1 . On the  pathway . Zinc is pathway . Signali pathway . Zinc th pathway ,85. Furt pathway . Hypoxia pathway . The DNATo maintain normal cellular function, neurons can struggle to avoid oxidative damage throughout aging processes. If the cells cannot keep the balance, the excess toxic stresses may cause neuronal cell death. Because AD is a disease that could last more than ten years, neurons may attempt to survive by attempting to control the damaged or deregulated cells rather than succumbing to apoptosis immediately. These speculations raise the possibility that BRCA1 and p53 accumulate in neurons early stages of AD. In addition, the association of BRCA1 and p53 with neurodegenerative pathology in AD may implicate a neuro-protective function in healthy neurons in AD. Genomic instability as a feature in the pathogenesis of neurodegeneration in AD may as well be a feature of the DNA repair activity in survival neurons. Alteration of BRCA1 and p53 simultaneously and complexly indicate oncogenic and/or neurodegenerative stimuli which are found in cancer, AD, and other neuropathology. There are several examples showing cognitive improvements in dementia patients undergoing chemotherapy against cancer. Notably, tumor suppressors are crucial molecules at this point. Neuroprotective factors have been suggested as a possible target for drug design efforts with the goal of delaying neurodegeneration from neuronal apoptosis. This may represent the rational basis for the development of dietary treatment of AD. Further mechanistic studies are required to understand the precise molecular mechanisms, and to determine whether an appropriate dietary intake is related to the improved brain function and the preservation of brain health. Long-term clinical studies are also necessary to clarify effects of treatment in the management of AD.The functions of BRCA1 and p53 are involved in the pathogenesis of AD as well as carcinogenesis. Some diets may contribute to neuro-protection, disease prevention, and possible better prognosis in AD via regulation of the expression of tumor suppressors.


AMTN was characterized in rodents only. In this study, we applied various approaches, including in silico screening of databases, PCRs and transcriptome sequencing to characterize AMTN sequences in sauropsids and amphibians, and compared them to available mammalian and coelacanth sequences.Amelotin (AMTN) is an ameloblast-secreted protein that belongs to the secretory calcium-binding phosphoprotein (SCPP) family, which originated in early vertebrates. In rodents, AMTN is expressed during the maturation stage of amelogenesis only. This expression pattern strongly differs from the spatiotemporal expression of other ameloblast-secreted SCPPs, such as the enamel matrix proteins (EMPs). Furthermore, AMTN structure changed during tetrapod evolution. To infer AMTN function, we studied spatiotemporal expression of AMTN during amelogenesis in a salamander and a lizard, and compared the results with available expression data from mouse. We found that AMTN is expressed throughout amelogenesis in non-mammalian tetrapods, in contrast to its expression limited to enamel maturation in rodents.We showed that (i) AMTN is tooth (enamel) specific and underwent pseudogenization in toothless turtles and birds, and (ii) the Taken together our findings suggest that AMTN was primarily an EMP. Its functions were conserved in amphibians and sauropsids while a change occurred early in the mammalian lineage, modifying its expression pattern during amelogenesis and its gene structure. These changes likely led to a partial loss of AMTN function and could have a link with the emergence of prismatic enamel in mammals.The online version of this article (doi:10.1186/s12862-015-0329-x) contains supplementary material, which is available to authorized users. AMTN, AMEL, ENAM and AMBN) in the coelacanth genome  was commissioned to GATC Biotech.Pleurodeles waltl , and two sauropsids, Caiman crocodilus  and Tarentola mauritanica  were assembled at ISEM-Montpellier 2 (France) and screened for AMTN transcripts with BLAST using the Montpellier Bioinformatics Biodiversity (MBB) platform [The sequenced jaw transcriptomes (see below “Jaw transcriptome sequencing”) of the caudate amphibian, platform .AMTN were translated into amino acid sequences and aligned to mammalian AMTN using Se-Al v2.0a11 [The protein-coding regions of  v2.0a11 . The put v2.0a11 ,39 and p v2.0a11 ,41.P. waltl, and several young adults A. carolinensis (3 month- to 2 year-old) were used. Immediately after dissection, the lower jaw quadrants were fixed in a mixture of glutaraldehyde (1.5%) and paraformaldehyde (1.5%) in PBS for 2 h, at room temperature. They were demineralized for three weeks, under gentle agitation at 4°C, in the same fixative to which 5% EDTA was added. The solution was changed every two days. After washing overnight in PBS, tissues were post-fixed for 2 h in 1% osmium tetroxide, rinsed in PBS, dehydrated through a graded series of ethanol then immersed in propylene oxide prior embedding in Epon 812 (EMS). 2 μm-thick sections were obtained using a Reichert OMU-3 ultramicrotome, deposited on a glass slide, stained with toluidine blue, and photographed with an Olympus BX61 microscope equipped with a QImaging camera using Image Pro Plus software .One adult AMTN transcript fragments were amplified from A. carolinensis  and P. waltl (703 bp) cDNA, using primers designed from the gDNA and transcriptome sequences, respectively , and transformed into competent E. coli TOP10F’ bacteria. Colonies containing the vector and the insert were selected for plasmid purification . Purified plasmids were linearized by PCR using M13 forward and reverse primers and the sense and antisense RNA probes were synthesized using SP6 and T7 RNA polymerases  in the presence of digoxigenin-UTP  and purified . waltl 70 bp cDNA,The jaws were dissected in quadrants and fixed overnight, at 4°C, in Formoy  solution. Tissues were demineralized in 10% acetic acid for 20 to 30 days at room temperature and under gentle agitation. The solution was changed every two days. Samples were then dehydrated through an increasing series of ethanol, shortly immersed in toluene and embedded in Paraplast . The sections (8 μm-thick) were obtained with a Leica RM2245 microtome and deposited on Superfrost PLUS slides .2). The digoxigenin-labeled probes were revealed at 37°C using NBT/BCIP (nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indodyl phosphate). The slides were mounted in Aquatex mounting medium , and photographed (Olympus BX61 microscope).The sections were dewaxed in toluene, rehydrated through a decreasing series of ethanol, then in PBS, treated with proteinase K (0.6 μg/ml) for 5 min at 37°C, rinsed in PBS, post-fixed for 30 min in 4% paraformaldehyde, rinsed again in PBS and then in 2× SSC. The slides were incubated overnight, at 65°C, with the digoxigenin-labeled antisense probe (0.25 ng/μl) in the hybridization buffer . The following day, the slides were washed three times, at 65°C, in the washing buffer , and rinsed, at room temperature, in the Maleic Acid Buffer Tween (MABT), pH 7.5. Non-specific binding sites were blocked for 2 h in a blocking solution . Then, the slides were incubated overnight with the anti-digoxigenin antibody coupled to alkaline phosphatase  in the blocking solution. After four baths of MABT, the slides were rinsed in NTM pH 9.5  and the French Rural Code .AMTN sequences of sarcopterygian species were obtained using in silico screening of public databases, RT-PCR on jaw cDNA, and screening of assembled jaw transcriptomes: six species representative of mammalian lineages , eight sauropsids , two amphibians , and one coelacanth . Genbank accession numbers of AMTN sequences obtained from cDNA are indicated in Additional file AMTN was detected in the PCR products obtained from the cDNA of all non-mammalian species used in this study. In the gDNA sequences analyzed in GenBank, AMTN was always found upstream AMBN, from 5 kb in X. tropicalis to 73 kb in B. taurus.A total of 17 AMTN sequences were inferred either from the comparison of cDNA and gDNA sequences obtained for the same species  or from sequence comparison of related species . The AMTN sequences in P. molurus and P. regius, on the one hand, and in A. mississippiensis and C. crocodilus, on the other hand, were nearly identical . For H. sapiens, M. musculus, B. taurus, L. africana, M. domestica and O. anatinus the exons were previously identified [P. waltl AMTN (cDNA data only) the exon-intron boundaries were estimated from the alignment of all AMTN sequences. However, the limit between exon 4 and exon 5 could not be clearly defined because of the high variability of this region when compared to the homologous region in other AMTNs. For L. chalumnae (gDNA data only), the exons were identified with UniDPlot then aligned to other AMTN sequences.The exon-intron boundaries of nine non-mammalian entified . For P. AMTN structure resulting from these comparisons is represented in Figure AMTN (exons 1–9), and three newly identified exons, i.e. two exons located between exons 2 and 3, and one exon inserted between exons 3 and 4. In order to conserve the current nomenclature of AMTN exons, we named 2b, 2c and 3b these three new exons, the former exons 2 and 3 becoming exons 2a and 3a , and the smallest was seven in X. tropicalis . It is worth noting that the opossum AMTN (gDNA data only) may have either the same structure as in other mammals, i.e. the short exon 8 plus the exon 9 as suggested by Gasse et al. [AMTN, i.e. only a large exon 8 as proposed by Kawasaki and Amemiya [AMTN sequences could be present in opossum as splice variants , most in exon 1 plus 15 nt located at the beginning of exon 2a. In non-mammals, the sequence ends with exon 8 that is larger than in placental mammals and comprises the stop codon followed by the 3′UTR. The latter is also composed of a variable number of nucleotides. In placental mammals, the sequence ends with exon 9 that includes the termination codon and the 3′UTR. In non-placental mammals, platypus and opossum, both structures could occur (see below).The 5′ UTR of X. tropicalis) amino acids (aa), and the first two residues of the mature protein are encoded by the last six nucleotides of exon 2a. Given the different AMTN structure in the various lineages, the length of the encoded protein varies from 252 aa in X. tropicalis to 355 aa in P. waltl.A single signal peptide (SP) encoded by exon 2a was detected in the N-terminal region of all AMTN obtained in this study  and a SxE (SeE: aa 82–84) phosphorylation motifs adjacent one another in the region encoded by exon 7. A conserved Arg-Gly-Asp (RGD) motif (putative integrin-binding site) was found in the C-ter region encoded by exon 8 in non-mammals but it is missing in mammals, with the possible exception of marsupials  previously identified in mammals in the region encoded by exon 7 is conserved in sarcopterygian AMTN (SseE)  while a conserved motif “PQQL” is encoded by exon 5. Also, exon 6 encodes 12 amino acids that are well-conserved in all species studied. No particular function is known for these conserved motifs.In addition to these features, in sauropsid AMTN exon 8 is in average 975 bp-long and encodes a variable region of the protein, the C-terminal region, which houses a well-conserved RGD motif . A ninth exon possessing eight nucleotides followed by a stop codon and the 3′UTR was then recruited. In platypus and opossum, however, screening of the gDNA sequence provided two putative sequences depending on whether the intraexonic splicing occurs in exon 8 or not  could be identified within the 15 kb genomic region upstream φ AMBN. They display many nucleotide substitutions and indels  were identified using UniDPlot within the 10 kb upstream φ AMBN. They display less variations compared to A. platyrhynchos AMTN  and of a monotreme (platypus: Ornithorhynchus anatinus) (not shown). This exon 2c (57 bp) possessed high nucleotide identity with sauropsidian exon 2c and displayed correct putative splice sites, which suggests that exon 2c is functional in marsupial and monotreme AMTN. Exon 2c was not found in placental mammals but weak nucleotide similarity in the homologous genomic region of various mammalian species (not shown) suggests that exon 2c was present in an ancestral placental AMTN, then invalidated early, before the diversification of current placental lineages. In contrast, neither functional nor pseudoexons 2b and 3b were found in any mammalian genomes studied to date, which suggests that these two exons were either not present in the AMTN sequence of the last common ancestral mammal or invalidated early in the mammalian lineage, and no longer recognizable in gDNA.In mammals, many onfirmed , but pos Figures  and 2, tAMTN is composed of 11 exons in squamates (no exon 9) and of 10 exons in crocodiles (lack of exons 3b and 9)  of sauropsids  using mammalian exon 9 sequence did not provide valuable hit. Exon 3b was identified in squamates only. We checked whether this exon was missed in A. mississipiensis gDNA, but no hit (even as pseudoexon) was obtained.In sauropsids,  Figures . The exoAMTN was found to be composed either of 10 exons (absence of exons 3b and 9) in P. waltl (caudates) or of seven exons  in X. tropicalis (anurans)  of X. tropicalis did not provide valuable hit. Similarly, exons 2b, 3b, 4 and 5 were not identified in the target genomic regions (introns 2 and 3) of X. tropicalis and no pseudoexons were found.In amphibians, AMTN .In the late cytodifferentiation stage, ameloblasts have differentiated from the inner dental epithelium of the enamel organ and they are facing mesenchymal cells of the dental papilla differentiated into odontoblasts. As soon as a thin layer of predentin was deposited between the ameloblasts and odontoblats, h Figure A, B. AMTd Figure C-F. In ts Figure G, H. AMTe Figure I, J. AftAMTN expression was monitored in larvae (from stage 34 onwards) and in adult specimens .s Figure . In larvx Figure A. In conx Figure F, G. Dure Figure B, H. Onct Figure C, I. Befl Figure D, J. AMTIn this study, we combined RNA-seq data (transcriptomics), genomic data screening, isolation, characterization and expression of the AMTN gene to investigate the evolution of this SCPP in tetrapods. Our results suggest that AMTN was a component of the enamel matrix in the last common ancestor of tetrapods and that its structure changed and the function most likely changed in mammals, probably as a consequence of drastic events that occurred in the coding and/or regulatory region of the gene in the last common ancestor of mammals.AMTN is located close to AMBN and ENAM in the same DNA region, and always upstream AMBN in the genome of sarcopterygian species annotated to date [AMTN and AMBN could be closely related SCPP genes, an hypothesis which is supported by the presence of remarkable motifs in the two proteins, in particular a SxxE motif encoded by a small exon in the middle region of the protein  [AMTN and AMBN (but not that of AMEL or ENAM) is modified by the FAM20C kinase in mice [e.g. polypterids and lepisosteids, and of chondrichthyans to better understand the evolutionary history of these EMPs in gnatosthomes.According to its genomic position, AMTN could be related to ENAM and AMBN because  to date ,18. This in mice . NeverthAMTN structure in sarcopterygian lineages suggests that the ancestral sequence was composed of either nine or 10 exons, depending on whether exon 5 was present in the ancestor and then lost in the coelacanth lineage (or not found in gDNA) or acquired in the lineage leading to tetrapods , nor in Dipnoi (lungfishes), nor in representatives of the basal actinopterygian lineages . Furthermore, when only gDNA is available coding exons could be missed, as illustrated by the discovery of the putative exon 2c in monotreme and marsupial AMTN, after this exon was identified in sauropsids. Indeed, this exon was not detected in opossum AMTN when using comparison with mammalian sequences that do not possess exon 2c [The comparison of s Figure . To date exon 2c  but was  exon 2c  when usiAMTN and in most of the tetrapods analyzed here derive from those of an ancestral SCPP, from which AMTN and EMPs originated after duplication. Indeed, AMTN exons 2a and 2b are homologous to exons 2 and 3 (exon 4 and 5 in ENAM) of all SCPPs, respectively. In addition, AMTN shares several other characteristics with all EMPs, i.e. the signal peptide encoded by exon 2a, the conserved Ser-X-Glu motif (putative phosphorylation site) encoded by exon 2b, and the presence of a variable region encoded by a large exon, as previously observed by Kawasaki and Weiss [Our results suggest that some coding exons present in the coelacanth nd Weiss .AMTN exon 2b in salamander cDNA and its absence in clawed frog, for which gDNA and cDNA sequences are available, indicates that this exon has been lost either early in the anuran lineage (no AMTN sequences available in other frog lineages), or later in the lineage leading to Pipidae  encoded by exon 2b, the presence of which characterizes most SCPPs, is not required for the correct AMTN function during amelogenesis in clawed frog and mammals. This leads to the question of whether or not the phosphorylation site (SxxE motif) encoded by exon 7 compensates, in mammals, for the lack of that encoded by exon 2b.The presence of e Figure . Exon 2bAMTN and in non-placental mammals. The presence of this exon in representative species of the four sarcopterygian lineages  indicates that its origin is to be found before the divergence of these lineages. On the other hand, its absence in placental mammals, suggests that exon 2c was probably lost in the common ancestor of placentals.Exon 2c was found in non-mammalian sarcopterygian AMTN only, a finding which suggests that this exon was acquired either in the lepidosaurian lineage after its divergence from other sauropsid lineages or soon after Squamata diverged from the sphenodontian lineage , a finding which indicates that, as proposed for exon 2b this exon was lost either early in the anuran lineage or later, in the lineage leading to Pipidae. We did not find this exon in platypus AMTN, indicating that exon 4 was secondarily lost in this lineage. However, in absence of AMTN cDNA in this lineage, the lack of exon 4 could also result from a genome assembly artefact.Exon 4 is absent in the clawed frog AMTN cDNA) and in platypus, although the lack in the latter could result from a badly assembled genomic sequence.Exon 5 has not been found in coelacanth, clawed frog and platypus genomes, and is not present in clawed frog cDNA either. As discussed above we cannot know whether this exon was present in the sarcopterygian ancestor or was recruited later in a tetrapod ancestor. Parsimonious analysis suggests that exon 5 was secondarily lost in clawed frog  the motif is functional (conserved during more than 430 Ma), and (iii) AMTN exon 7 is homologous to AMBN exon 12 and to ENAM exon 8, all of them preceding the large exons 8, 13 and 10, respectively. In clawed frog AMTN, the exon 7 not only encodes a SxxE/D motif, but also codes for a second phosphorylation site. Because the SxE motif encoded by exon 2b was lost in the pipid (or anuran) lineage, we suggest that the second SxE motif, encoded by exon 7, could compensate for the loss of the former. The presence of two phosphorylation sites, that could be located in different regions of the protein, was probably essential for AMTN to fulfill its function in non-mammalian sarcopterygians.Exon 7 encodes an SxxE motif (putative phosphorylation site) that is found in all AMTN sequences analyzed so far. When phosphorylated, this motif promotes hydroxyapatite precipitation in vitro . Such moin vitro . These fAMTN exon 8 is shorter than in the other lineages and has lost a large part of the 3′ region, including the region encoding the RGD motif in non-mammals [AMTN possesses an exon 9, which encodes the three last amino acids of the protein followed by the stop codon and the 3′UTR. Sequence comparison showed that a mutation in exon 8 generated an intraexonic splice site in an ancestral placentalia AMTN. This intraexonic splicing occurred within a codon, leading to the recruitment of exon 9 encoding an appropriate stop codon. It appears that encoding the stop codon could be the only role of exon 9 as several mammalian species lack this last exon because a mutation generated a stop codon at the extremity of exon 8 [Exon 8 experienced two different evolutionary histories in sarcopterygians. In non-mammals, exon 8 is large and encodes a variable region of the protein, the C-terminal region of which includes a well-conserved Arg-Gly-Asp (RGD) integrin-binding motif that likely plays a crucial role for the protein function. RGD motifs are recognized by integrins and mediate the attachment of the cell to the extracellular matrix . As for -mammals , and hen-mammals . In addif exon 8 .AMTNs displaying a similar environment favorable for intraexonic splicing as observed in the homologous region of placental mammals, and (ii) exon 9 sequence, similar to that in placental mammals, present in both gDNA. The second possibility, suggested by Kawasaki and Amemiya [AMTN exon 8 did not occur in an ancestral marsupial but took place later in an ancestral placental. This finding is supported by the conservation of an RGD motif in the extremity of opossum AMTN, as in non-mammalian species. Such a long lasting conservation of RGD motif, although the rest of the sequence is less conserved, could indicate that this region is functional. A third interpretation could be that both AMTN variants are present in marsupials, the one with a short exon 8 and exon 9 (no RGD encoded), the other with the large exon 8 only (encoding an RGD). In platypus, the interpretation could be the same. However, if the intraexonic splicing does not occur and a large exon 8 transcribed, the latter does not encode an RGD. This suggests a different evolution of AMTN in this lineage. We previously showed in mammals that AMTN is enamel specific [AMTN sequence, including mutations in the sequence encoding the RGD motif.In monotremes and marsupials, the question of the efficient presence of the intraexonic splicing is posed. Indeed, the unavailability of cDNA data in both lineages has led to various interpretations. The first possibility we already proposed  is that  Amemiya , is thatspecific . In platspecific . This coA RGD motif is present in all members of the acidic SCPPs (dentin and bone SCPPs) and is also found in the ENAM sequence of several mammalian species . This sui.e. from the maturation phase onwards, previously reported during murine amelogenesis [AMTN expression in two selected tetrapods has answered partially this question.Taken together our results suggest that in non-mammalian sarcopterygians AMTN may play an important role in ameloblast adhesion to the enamel matrix. This role in ameloblast adhesion was then probably lost, either in the last common mammalian ancestor or later in the last common ancestor of placentals, as a consequence of a mutation generating an intraexonic splice site in exon 8, suppressing the encoded RGD motif. Could the absence of this RGD motif explain the late expression of AMTN, ogenesis ,3,24? The.g. opossum [AMEL expression during amelogenesis was similar in all tetrapods [AMBN, ENAM, or MMP20 in non-mammalian tetrapods.In lizard and salamander, tooth formation is roughly similar to that described in other vertebrates, but unlike mammals, ameloblasts do not exhibit Tomes’ processes, rod and interrod enamel structures are absent, and hence enamel is not prismatic -53. Ther opossum . These cetrapods ,55. No dAMTN appear when comparing lizard or salamander amelogenesis to the one of rodents. In the incisors and molars of the latter, AMTN is predominantly expressed in maturation-stage ameloblasts [AMTN expression is detected in ameloblasts as soon as enamel matrix deposition has started, and is maintained from secretion to late maturation stage. AMTN is no longer expressed in the reduced ameloblasts after tooth eruption. In salamander larvae, AMTN expression is detected in early stages of amelogenesis while in adults, the onset of expression is delayed and appears slightly later than in the lizard, during enamel matrix deposition. Then, in both larvae and adult salamanders, AMTN expression is maintained in ameloblast until maturation stage. The disappearance of AMTN signal in ameloblasts occurs earlier than in lizard but it persists longer in the inner dental epithelium along the dentin cone. This late AMTN expression in the cells facing the region lacking enamel along the tooth base could be related to the formation of the amphibian pedicel [AMTN gene nor the protein was ever detected beyond the cemento-enamel junction in the epithelial cells along the tooth root. Nevertheless, AMTN was found in Hertwig’s epithelial root sheath cells entrapped in cementum [AMTN expression is detected earlier in secretory stage ameloblasts, and later in the cells of the inner dental epithelium along the tooth root. Immunolocalization of the protein should be performed to better understand the significance of the mRNA expression pattern.Differences in the spatial-temporal expression of loblasts . In liza pedicel ,57. Futu pedicel ,24, alth pedicel ,58. In rcementum . In lizaAMTN being expressed during the various steps of amelogenesis in lizard and salamander, we concluded that (i) AMTN must be considered an enamel matrix protein (EMP), as are AMBN, ENAM and AMEL, and (ii) this status was the ancestral condition in non-mammalian tetrapods. When comparing lizard and salamander to mammals, one could hypothesize that the differences observed both in the timing of AMTN expression and in the gene structure could be responsible for the differences in enamel microstructure  in these lineages. The absence of both Tomes’s process and of an organized rod/interrod enamel structure in lizard and salamander, on the one hand, and in mice overexpressing AMTN under AMEL promoter, on the other hand, support this hypothesis [pothesis .AMTN expression and the gene structure changed in mammals could be visualized as the succession of several events that occurred early in mammalian evolution. One event probably occurred at the promoter level  resulting in late AMTN expression during enamel mineralization. Two other, probably independent events resulted in the loss of exon 2b encoding the SxE motif and in the intraexonic splicing of exon 8 eliminating the RGD motif. The occurrence of these events (in any order) may have led to important changes in ameloblast structure (differentiation of Tomes’ processes) and in enamel organization (formation of prisms).The putative evolutionary scenario we propose to explain how the spatiotemporal A. platyrhynchos, we found that AMTN is also a pseudogene. As observed for EMP genes, AMTN underwent pseudogenization after the capability to develop teeth was lost in the common ancestor of modern birds, 80–100 Mya [φ AMTN sequence that is now hardly recognizable from the non-coding gDNA. In turtles, the capability to build teeth was lost in the last common ancestor of chelonians, 250 Mya [C. mydas, AMTN also underwent pseudogenization, producing a φ AMTN, in which a few exons could be identified. These findings indicate that in the ancestors of these two sauropsid species AMTN had no other functions than being involved in amelogenesis and is, therefore, an enamel specific protein in toothed sauropsids. In addition, it appears that, after the loss of function, mutations in this gene accumulated more rapidly in birds than in turtles although teeth were lost three folds earlier in turtle than in the bird ancestor [In birds, pseudogenization of EMP genes is related to tooth loss ,31,60. I–100 Mya ,61. Nume 250 Mya . In C. mancestor . This coancestor .AMTN pseudogenization in two xenarthran mammals, armadillo and sloth, in which teeth are devoid of enamel cover [In a previous study, we showed similar el cover . These rIn situ hybridization of AMTN during tooth formation in lizard and salamander revealed that AMTN is expressed in ameloblasts during all stages of amelogenesis (with some differences in both species). These findings suggest that AMTN is an essential EMP for amelogenesis in these two species. The differences in spatiotemporal expression of AMTN described in murine vs lizard and salamander teeth would then suggest that in sauropsids and amphibians AMTN could have different functions than in mammals.This is the first comparative study of AMTN sequence in representatives of various mammalian and non-mammalian vertebrates. AMTN was present in the last common sarcopterygian ancestor more than 420 Mya. Various loss or gain of coding exons were identified in coelacanth, amphibians, reptiles and non-placental mammals, indicating different evolutionary pathways in sarcopterygian lineages.


Bovine cutaneous fibropapillomas are benign hyperproliferative lesions induced by Bovine Papillomaviruses (BPVs). Bcl-2 is an important anti-apoptotic protein which is expressed in several cancer types. In contrary, p53 is a tumour suppressor protein that mediates cell cycle arrest, apoptosis and senescence in response to cellular stresses.Here, we investigated immunohistochemically and biochemically, the expression of bcl-2 and p53 in a subset of BPV positive fibropapillomas and bovine normal skin. Normal skin samples showed a weak signal for both proteins in the cytoplasm of the basal cells. Nine out of twelve (75%) tumour samples stained positive for bcl-2 throughout basal and parabasal layers, with most of cells showing strong cytoplasmic immunoreactivity. Nine out of twelve (75%) fibropapillomas were found to be positive for p53 expression, showing a strong cytoplasmic and perinuclear staining of p53 protein mainly in the basal and parabasal layers.Our data reveal an altered bcl-2 and p53 immunoreactivity in bovine cutaneous fibropapillomas, suggesting involvement of these two proteins in the cutaneous neoplastic transformation through an impaired apoptotic process.The online version of this article (doi:10.1186/1750-9378-10-2) contains supplementary material, which is available to authorized users. Bovine cutaneous fibropapillomas are benign hyperproliferative lesions induced by different BPVs types, infecting both the epithelial cells and the underlying dermis . These lBPV infection is supposed to be the most important causal factor in fibropapilloma development and our previous studies on these tumours have demonstrated alterations of GAP junction proteins and signaling transduction pathways . HoweverPapillomaviruses (PVs) are oncogenic viruses causing both benign and malignant tumours in humans as well as in several animal species. These viruses have developed numerous strategies in order to block host-mediated apoptosis, contributing to cancer development .in situ and mammary carcinoma [Programmed cell death, known as apoptosis, is a pivotal mechanism of cell death that plays an important role during diverse biological processes, including development, cell differentiation and proliferation . The regarcinoma .P53 is a tumour suppressor protein that mediates cell cycle arrest, DNA integrity and repairing, apoptosis and senescence in response to specific cellular stresses  . As a coP53 has a significant role in cell cycle control, therefore the loss of its suppressive function has been reported for different types of human neoplasia and animal tumours, such as BPV induced tumours –14. HoweIn this study, we investigated the expression of p53 and bcl-2 in bovine fibropapillomas according to the detailed “Materials and Methods” in the Additional file Nine out of the 12 (75%) bovine fibropapillomas were found to be positive for p53 expression. In most samples, a strong expression of p53 protein was detected in the basal layer and parabasal layer, where cells with a cytoplasmic and perinuclear staining were recorded  did not expressed neither bcl-2 nor p53 proteins. The lack of reactivity might be due to the loss of antigenicity.Bovine cutaneous fibropapillomas are benign lesions known to affect cattle worldwide. BPV are considered as causative agents of these tumours, inducing the proliferation of both epithelial and dermal cells . ApoptosIn our study, bcl-2 positive samples showed a reduced number of TUNEL positive cells when compared to the bcl-2 negative fibropapillomas. This is in accordance with human counterpart, where in head and neck squamous cell carcinomas the number of apoptotic cells was noted to be reduced in bcl-2 positive tumours when compared to bcl-2 negative tumours . This fiNormally, a very low expression of p53 protein is detected in cells; in response to cellular stresses, however, the protein is stabilized and cell division is inhibited in order to respond to such damage .Our results indicate that p53 is weakly expressed in basal epithelial cells of normal skin samples. Accordingly, a similar p53 expression pattern in the basal epithelial cells has been recorded in human normal skin, suggesting that it may exert its physiological function in bovine healthy epithelium , 20, 21.Fibropapilloma samples showed a strong cytoplasmic and perinuclear p53 expression in the basal and parabasal cell layers. Cytoplasmic accumulation of p53 has been proposed as a considerable mechanism to disrupt its function as a tumour suppressor and its expression has been reported in different human and animal tumours , 23. RecHowever, the higher expression of p53 in epithelial component of fibropapilloma may be due to different stresses followed by a temporary increase of the normal p53 protein or accumulation of mutated p53, resulting in a permanent abnormal form of p53 that is not functional and/or resistant to degradation .Our results indicate that bcl-2 expression may promote the survival of the neoplastic cells possibly depending on p53 impaired expression.Despite a number of studies have higlighted a role for BPV’s oncogenes in impairing some cellular pathways and leading to neoplastic transformation, nothing is known about a possible interplay among the viral oncoproteins and bcl-2 and p53 dysregulation. Thus, the unraveling of new pathways possibly involved in the pathogenesis of bovine cutaneous fibropapillomas is of a great importance to complete the molecular scenario triggering to the development of these tumours.in vivo carcinogenesis.In summary, the current study provides evidence for p53 and bcl-2 expression in a majority of BPV-induced bovine tumours, suggesting an involvement of these two proteins in bovine cutaneous fibropapilloma development. Further studies are needed to better understand bcl-2 and p53 function Materials and Methods.(DOCX 32 KB)Additional file 1: