BRCA1 germline mutations were found in a high proportion (14–34%) of patients with triple-negative breast cancer (TNBC). BRCA2 was either not analyzed or showed much lower mutation frequencies. Therefore, we screened a group of TNBC patients (n = 30) of white European descent for mutations in BRCA2 as well as in BRCA1. Cases were unselected for age of disease-onset , family history of cancer and BRCA1 and BRCA2 mutation status. Half of the patients (15/30) showed a family history of breast and/or ovarian cancer. A high frequency of deleterious germline mutations was observed in BRCA2 , and only one case showed a BRCA1 mutation (3.3%). Although the study group was small, these results point to BRCA2 mutations being important in TNBC.Recently,  BRCA1-associated breast cancers is either TNBC, basal-like breast cancer or both. TNBC patients have a relatively poor outcome and cannot be treated with endocrine therapy or therapies targeted to HER2 due to the lack of related receptors Recently, triple-negative breast cancer (TNBC) has been classified as a breast cancer subgroup that is negative for estrogen and progesterone receptors (ER/PR) and receptor 2 of human epidermal growth factor (HER2). TNBC accounts for approximately 15% of all breast cancer cases and seems to be closely related to basal-like breast cancer, which has an expression profile similar to that of normal basal-myoepithelial breast tissue. Approximately 80% of TNBC cases show a basal-like phenotype, and the majority of basal-like breast cancers can be classified as TNBC. Close to 75% of BRCA1 germline mutations in TNBC cases. In a Canadian TNBC cohort (n = 54) with an age of onset before 41 years and no familial breast cancer aggregation, five cases with mutations of BRCA1 (9%) and only one case with a mutation of BRCA2 (2%) were detected. The entire coding sequence of BRCA1 and the large exons 10 and 11 of BRCA2 were analyzed BRCA1 mutations and none had BRCA2 mutations when fully screened in both genes. Forty-three of these cases were from patients younger than 41 years of age at diagnosis and with no familial aggregation of breast cancer. An additional 30 cases were unselected for family history of breast cancer because they were younger than 31 years old at onset of the disease BRCA1 (n = 12) and 3.9% for BRCA2 (n = 3). The entire coding sequence and the exon-intron boundaries were screened in both genes BRCA1 and BRCA2. In all Ashkenazi Jewish breast cancer cases, approximately 10% of patients carry one of three different founder mutations in BRCA1 and BRCA2. In the investigated Ashkenazi Jewish TNBC cohort (n = 64), 19 BRCA1 (29.7%) and 6 BRCA2 (9.4%) mutations could be detected when screening for the three founder mutations BRCA2 mutations BRCA2 mutation screening approaches BRCA2 mutation frequency in a TNBC cohort of a Southern German population unselected for age of onset and familial aggregation of cancer. If there was a relevant mutation rate in such cases, TNBC would meet the clinical criteria for mutation screening in BRCA2 and should be added to the current guidelines.Recent reports have focussed on All selected patients underwent genetic counselling and gave their written informed consent for genetic testing. The study protocol was approved by the Ethics Committee of the State of Salzburg (Austria).1 mouse antibodies for estrogen (clone 6F11) and for progesterone receptors . For HER2-IHC we used polyclonal rabbit antibodies from clone A0485 .In a monocentric approach executed in Munich, Germany, newly diagnosed cases of individuals with TNBC diagnosed between 2005 and 2010 were selected from the Pathology Unit. Histological samples were classified as TNBC when the following criteria were met: less than 1% of cells demonstrated nuclear staining for estrogen and progesterone receptors, and immunohistochemical staining for HER2 showing a 0, 1-fold, or a 2-fold positive score and a FISH ratio  of less than 1.8 according to the relevant ASCO and CAP guidelines BRCA1 and BRCA2, we used primer pairs and polymerase chain reaction (PCR) protocols published elsewhere DNA extraction from whole blood samples (EDTA) was performed according to standard protocols. To amplify exons and exon-intron boundaries of BRCA1 and BRCA2. The following clinical criteria were evaluated in approximately 1,000 unrelated cases in a German-wide study:In Germany, patients with a 10% mutation probability are eligible for mutation screening in Two women with breast cancer, with one of them ≤50 years, or three women with breast cancer, independent of age.One woman with breast and one woman with ovarian cancer, independent of age, or one woman with breast and ovarian cancer, independent of age, or two women with ovarian cancers, independent of age.One male breast cancer and one woman with breast or ovarian cancer.One woman with breast cancer ≤35 years.One woman with bilateral breast cancer or two primary breast cancers, with first tumour diagnosed before ≤50 years BRCA1 and five  in BRCA2. We detected four known deleterious frame-shift mutations (BRCA1: c.5266dupC (old nomenclature is “5382insC”) which is one of the most common deleterious BRCA1 mutations BRCA2: c.1029delA http://research.nhgri.nih.gov]) and one unknown splice-site mutation (BRCA2: c.476-1G>A) with a clear predictive deleterious effect. One missense mutation in BRCA2 (p.W2626C) was classified as “predictive deleterious” in prior literature Characteristics of investigated patients are presented in BRCA1 leading to an exchange of methionine for isoleucine at amino acid position 1,652 of the protein . Variant c.5744C>T causes a point mutation of threonine to methionine at amino acid position 1,915 of BRCA2 and was detected in four cases . Within mutation carriers, two out of six cases (33.3%) did not meet the German clinical criteria for BRCA1 and BRCA2 testing. From all TNBC cases, 15 out of 30 (50%) showed an early age of onset or a familial aggregation which are German clinical criteria for mutational screening of BRCA1 and BRCA2. The median age at diagnosis of the total study group was 58 years (n = 30), for mutations carriers 50 years (n = 6), and for the remaining patients (n = 24) not showing deleterious mutations 61 years.In four cases, we detected c.4956G>A in BRCA2 germline mutations in triple-negative breast cancer (TNBC). We detected 6 deleterious BRCA1 and BRCA2 mutations in 30 TNBC patients (20%). The total mutation frequency for both genes is comparable with those described in recent studies BRCA2 germline mutations in our TNBC study group: four cases with previously published deleterious variants, and one splice-site mutation which was not published before (16.7%). The low rate of BRCA2 mutations found in a Canadian sample (1/54 = 2%) was probably the result of assay design given that only exons 10 and 11 of BRCA2 were tested. Additionally, only TNBC cases with an age of disease onset of less than 41 years were selected for the study BRCA2 mutations. No BRCA2 mutations were detected in 73 TNBC cases from the UK. BRCA1 and BRCA2 genes were fully screened, but patients were diagnosed with TNBC before age 41 (n = 43) and before age 30 (n = 30). Cases were selected for the UK study only if no familial aggregation was reported BRCA2 mutation frequencies. Interestingly, a similar study of 77 TNBC cases from the United States where cases were unselected for age of onset (average was 51 years) revealed a much lower BRCA2 mutation rate (3.9% vs. 16.7% in our study) The aim of this study was to investigate the role of BRCA1 and BRCA2 mutation carriers  showed that the proportion of TNBC increased with age at diagnosis in BRCA2 mutation carriers, while it decreased in BRCA1 mutation carriers BRCA2 and higher BRCA1 mutations rates in TNBC Recently, data of a multi-national approach containing approximately 7,000 BRCA1 . Analysis with protein prediction software showed some minor effect BRCA2 (rs4987117) was less frequent  in estrogen receptor-positive patients of a study cohort of 117 sporadic breast cancer cases from Poland BRCA1 and c.5744C>T in BRCA2) could therefore be investigated in much larger association studies to evaluate their impact in TNBC tumorigenesis.In four cases, we detected c.4956G>A in BRCA2 mutations in two patients not meeting the current German clinical criteria for BRCA1 and BRCA2 mutation screening. In our cohort of TNBC patients unselected for age at diagnosis and cancer family history, 15 cases  did not meet those criteria. The mutation rate in this subgroup was 13% (2/15). The main requirement for the German clinical criteria for BRCA1 and BRCA2 mutation testing was defined as being at least a 10% a priori probability to carry either a BRCA1 or BRCA2 mutation BRCA1 and BRCA2 mutation screening, and additional cases may be assessed not only if they present with early-onset of the disease or have a familial aggregation of breast and/or ovarian cancer. BRCA1 and BRCA2 mutation carriers are frequently diagnosed with triple-negative breast cancer BRCA1 and BRCA2 mutation screening in TNBC. In contrast, recent guidelines published by the National Comprehensive Cancer Network in the United States are less stringent. For instance, TNBC diagnosed before age 60, and breast cancer cases with a negative family history for cancer younger than 45 years should be tested according to these guidelines. In comparison, the German guidelines recommend testing single cases only if the diagnosis was made before age 35 We identified deleterious BRCA1 and BRCA2 genes in patients diagnosed with TNBC from Southern Germany and unselected for age of disease onset and familial aggregation of this malignant disease. In contrast to recent reports, an unexpected and high mutation frequency was seen in BRCA2  suggesting that this gene may play an important role in the development of TNBC when mutated. Our data suggest that Southern German patients diagnosed with TNBC may qualify for BRCA1 and BRCA2 mutation screening. Larger patient cohorts have to be investigated to validate our findings.In summary, we found a 20% germline mutation rate (6/30 cases) in


FoxP2 is widely involved in a number of important physiological and developmental processes. We systematically studied the evolutionary history and functional adaptations of FoxP2 in teleosts. The duplicated FoxP2 genes (FoxP2a and FoxP2b), which were identified in teleosts using synteny and paralogon analysis on genome databases of eight organisms, were probably generated in the teleost-specific whole genome duplication event. A credible classification with FoxP2, FoxP2a and FoxP2b in phylogenetic reconstructions confirmed the teleost-specific FoxP2 duplication. The unavailability of FoxP2b in Danio rerio suggests that the gene was deleted through nonfunctionalization of the redundant copy after the Otocephala-Euteleostei split. Heterogeneity in evolutionary rates among clusters consisting of FoxP2 in Sarcopterygii (Cluster 1), FoxP2a in Teleostei (Cluster 2) and FoxP2b in Teleostei (Cluster 3), particularly between Clusters 2 and 3, reveals asymmetric functional divergence after the gene duplication. Hierarchical cluster analyses of hydrophobicity profiles demonstrated significant structural divergence among the three clusters with verification of subsequent stepwise discriminant analysis, in which FoxP2 of Leucoraja erinacea and Lepisosteus oculatus were classified into Cluster 1, whereas FoxP2b of Salmo salar was grouped into Cluster 2 rather than Cluster 3. The simulated thermodynamic stability variations of the forkhead box domain (monomer and homodimer) showed remarkable divergence in FoxP2, FoxP2a and FoxP2b clusters. Relaxed purifying selection and positive Darwinian selection probably were complementary driving forces for the accelerated evolution of FoxP2 in ray-finned fishes, especially for the adaptive evolution of FoxP2a and FoxP2b in teleosts subsequent to the teleost-specific gene duplication.As one of the most conserved genes in vertebrates,  FoxP2 is a key transcription factor gene in the FoxP subfamily  distribution at a significance level of 0.05. LR represents the ratio of the maximum likelihood that θλ > 0 to that of θλ = 0. In addition, the critical amino acids that are responsible for the type I functional divergence can be identified using a reasonable cutoff value (> 1) for the posterior probability ratio based on the hidden Markov model. The coefficient of functional divergence (θlication . Type I on 1.04) . A statiFoxP2 of Sarcopterygii), Cluster 2 (FoxP2a of Teleostei) and Cluster 3 (FoxP2b of Teleostei) based on the teleost lineage specific FoxP2 duplication in the ancestral 3R-WGD event  show greater divergence than these functional domains, we also tested the functional divergence between Cluster 1 and Cluster 2 in Data set 2 to determine if the two clusters exhibit homogeneous evolutionary rates in an extensive region  were used to discriminate these sequences. With Data set 2, we investigated whether FoxP2 of Leucoraja erinacea and Lepisosteus oculatus were grouped together with Cluster 1 or with Cluster 2. In order to study whether the classification was a remaining trace after a long-term evolutionary history, we also used the three clusters to discriminate hydrophobicity profiles of ancestral sequences from Furthermore, we performed a stepwise discriminant analysis using SPSS software version 20) to evaluate the consistency of the classification in the four cluster trees constructed with each data set. The variable selection method used in the discriminant analysis was Mahalanobis distance. The prior probability for groups was computed according to size. We kept the F values for entry or removal of variables and the covariance matrix for classification as defaults. Coefficients for both the canonical discriminant function and Fisher’s linear discriminant function were computed. Finally we evaluated the classification results by using the percentage of original grouped cases and cross-validated grouped cases correctly classified. In Data set 1, we focused on the rationalization of groupings for FoxP2 of 0 to evalThe three-dimensional (3D) structure of the forkhead domain of human FOXP2 (PDB: 2A07) provides a model to investigate changes in the stability of the local 3D structure in different evolutionary scenarios. We applied FoldX (version 3.0 b5.1), a fast and effective computer algorithm for estimating the effect of mutations on stability , in ordeFoxP2a and FoxP2b in teleosts experienced dramatic evolutionary changes after gene duplication. Therefore, we tested natural selection pressures with Data set 1 based on the corresponding phylogenetic topology tree (l (twice the log likelihood difference) between a model and its nested model was compared to the value of the χ2 distribution with degrees of freedom equal to the numerical difference between the free parameters of the two models. The nested model was rejected at a significance level of P < 0.05.The analyses of functional and structural divergence suggest that ogy tree  using thogy tree . Codon sFirst, three pairs of site models were tested on the data sets and compared based on the LRT . Comparip (one tail probability for χ2 distribution) value was not the statistically significant (P < 0.05), we neglected Test 2 and positively selected sites.We used branch specific models to detect whether Cluster 1, Cluster 2 and Cluster 3 experienced different natural selection pressures . Due to FoxP2 shared a highly conserved synteny in four tetrapod species: Homo sapiens, Taeniopygia guttata, Anolis carolinensis and Xenopus tropicalis  could be pinpointed in the chromosomal segments in which FoxP2a and particularly FoxP2b were both greatly divergent from their FoxP2 orthologs. FoxP2a in teleosts possessed a 16-exon composition resembling that in human FOXP2  between Cluster 2 and Cluster 3 in Data set 1 was significantly greater than 0 (λ between Clusters 1 and 2 (or 3), the θλ between Clusters 1 and 3 was greater than that between Clusters 1 and 2. Moreover, a significant functional divergence between Clusters 1 and 2 was detected in Data set 2 , yielded highly consistent inconsistency coefficients and clusters . All copSalmo salar and Gadus morhua were singly classified in these dendrograms. FoxP2 of Leucoraja erinacea and Lepisosteus oculatus were grouped into Cluster 1, however FoxP2 of Rana daunchina was misclassified into Cluster 2 in the trees based on Correlation, Cosine and Euclidean pairwise distances. In addition, some links connecting dissimilar members (i.e. with inconsistency coefficients > 0.8) existed in these three clusters, indicating possible structural heterogeneity in the clusters. Notably, all dendrograms revealed that FoxP2a of Acanthopterygii were dissimilar to FoxP2a of Salmo salar, Gadus morhua and Otocephala. FoxP2a of Takifugu rubripes and Tetraodon nigroviridis showed significant dissimilarity from FoxP2a of other seven acanthopterygian fishes. Amphibian FoxP2 were possibly divergent from other tetrapod FoxP2. Subsequent stepwise discriminant analysis, which correctly classified high percentages of the original grouped cases (100%) and cross-validated grouped cases (97.2%), strongly demonstrated that FoxP2 of Leucoraja erinacea, Lepisosteus oculatus and Rana daunchina belonged to Cluster 1. Additionally, FoxP2b of Salmo salar and Gadus morhua were classified into Cluster 2 and Cluster 3, respectively. Moreover, FoxP2 in the ancestor of Osteichthyes and the ancestor of Actinopterygii, as well as all the ancestral FoxP2 in Sarcopterygii, were grouped into Cluster 1. FoxP2 in the ancestor of Teleostei, FoxP2b in the ancestor of Euteleostei and all the ancestral FoxP2a in Teleostei were classified as members of Cluster 2. All ancestral FoxP2b of Neoteleostei were classified into Cluster 3.According to the cutoff value (0.8) for inconsistency coefficients, the three clusters  were consistently separated from one another in the four hierarchical dendrograms from Data set 1 , althougLeucoraja erinacea and Lepisosteus oculatus were grouped into Cluster 1. The existence of a substantial inconsistency coefficient (> 0.8) in Cluster 2 indicated significant divergence between FoxP2a of Acanthopterygii and that of Salmo salar, Gadus morhua and Otocephala. FoxP2 of Leucoraja erinacea and Lepisosteus oculatus were confirmed to be members of Cluster 1 in subsequent stepwise discriminant analysis. In addition, FoxP2 in the ancestor of Osteichthyes and the ancestor of Actinopterygii were grouped together with all ancestral FoxP2 in Sarcopterygii into Cluster 1. All ancestral FoxP2a in Teleostei were classified into Cluster 2. Furthermore, 100% of the original grouped cases and cross-validated grouped cases were correctly classified.Similarly, analysis of structural divergence on Data set 2 produced consistent results , classifThe simulated ADTE variations of monomers and homodimers in different evolutionary scenarios showed that such ADTE variations were not always in unison . In 53 oThe comparison of Model 3 to Model 0 in both data sets revealed variable natural selection pressures across sites . NotwithSubsequent analysis of branch specific models in Data set 1 demonstrated that Cluster 3 experienced divergent natural selection pressures against both Cluster 1 and Cluster 2 . SpecifiFoxP2a in ancestor of Tetraodontiformes, FoxP2b in ancestor Neoteleostei and FoxP2b in the ancestor of Tetraodontiformes) were found to have experienced accelerated evolution using Test 1 for branch-site models A in Data set 1  likely experienced accelerated evolution . The test of selection pressures on 27 selected sequences in Data set 2, containing exons 6 and 16, produced similar results (not shown). These two exons with teleost-specific inserts were also demonstrated targets of positive natural selection and relaxed purifying selection.Likewise, five ancestral branches in Data set 2  in these chromosomal segments after the tetrapod-teleost split. Although FoxP2a and FoxP2b show remarkable divergence in teleosts, the phylogenetic trees reconstructed with four member genes  of the FoxP subfamily clearly demonstrate that FoxP2a and FoxP2b in teleosts are members of FoxP2 lineage (not shown). In comparison with FoxP2, greater divergence (e.g. exon-composition) of FoxP2b than FoxP2a suggests that FoxP2b evolves faster than FoxP2a after the gene duplication. Although dimerization of FoxP1, FoxP2 and FoxP4 has been demonstrated, which is important for their DNA binding and transcriptional activity [In this report, we identify the teleost-specific activity , it is nFoxP2b in Danio rerio probably results from pseudogenization [FoxP2a of Danio rerio, FoxP2a of two other species in Otocephala (Ctenopharyngodon idella and Pygocentrus nattereri) are phylogenetically determined. However, deletion of FoxP2b in Ctenopharyngodon idella and Pygocentrus nattereri is uncertain because their genomes are not available. The retention of FoxP2b in Euteleostei implies that the gene may conserve some functions of the parental FoxP2 through subfunctionalization or may gain novel functions through neofunctionalization. The linked paralogon shared by FoxP2a and FoxP2b in teleosts indicates that these two duplicated genes were generated in a large-scale genome duplication event, i.e. 3R-WGD event [FoxP2 may occur in polyploid vertebrates, such as Xenopus laevis and Salmo salar [The absence of nization  of the rGD event ,36  rather than the functional domain itself in FoxP2a and FoxP2 are targets of divergent evolutionary rates. Therefore, the functions of FoxP2a may be more conservative than those of FoxP2b in teleosts.The significant functional divergence between FoxP2a and FoxP2b in Data set 1 implies that FoxP2a and FoxP2b probably execute divergent functions in teleosts. The θSalmo salar groups together with FoxP2a, as well as FoxP2 in the ancestor of Teleostei and FoxP2b in the ancestor of Euteleostei together with all ancestral FoxP2a in Teleostei, implying that structural divergence between FoxP2a and FoxP2b was probably very weak at the early stage of post-duplication. Perhaps the weak structural divergence of FoxP2a and FoxP2b generated functional redundancy of FoxP2b in Otocephala, further leading to the lineage-specific loss of FoxP2b. The significant structural divergence between FoxP2a and FoxP2b seems to coincide with the split of Neoteleostei from Euteleostei, implying more divergent functions between the genes of Neoteleostei than Salmoniformes. Additionally, some teleost lineages show significant FoxP2a structural divergence, likely indicating divergent functional adaptations in the lineages.Hierarchical cluster analysis and stepwise discriminant analysis of hydrophobicity profiles in CC10 [The significantly divergent variations of thermodynamic stability among FoxP2a, FoxP2b and FoxP2 clusters imply divergent functional adaptations of the forkhead domains in FoxP2a, FoxP2b and FoxP2. Intriguingly, the significant destabilizing effects of mutations (E519D and R523M) on both monomers and homodimers suggest that functional divergence of FoxP2 in teleosts possibly began in the pre-duplication phase. It has been demonstrated that the two mutations, as well as 521S, are responsible for the weak repressive activity of the medaka FoxP2a on the lung-specific gene of CC10 . The theFoxP2b lineage has undergone a generally relaxed purifying selection pressure, which is significantly greater than that of the FoxP2a and FoxP2 lineages. The regions flanking the functional domain of the forkhead box, rather than the forkhead domain per se, in the FoxP2a lineage has experienced a milder purifying selection pressure than those in the FoxP2 lineage. Thus the asymmetrical natural selection pressures acting on the FoxP2a, FoxP2b and FoxP2 clusters, particularly between FoxP2a and FoxP2b, likely led to asymmetrical functional divergence among these clusters. Furthermore, natural selection pressures on branches and sites in each cluster show significant heterogeneity, especially positive natural selection acting on several ancestral branches. Notably, positive selection pressure mainly acts on regions flanking functional domains. Therefore, positive selection pressure possibly was the driving force in forming the teleost-specific inserts in FoxP2a and the truncated regions in FoxP2b. It is also notable that FoxP2 in the ancestor of Actinopterygii probably underwent positive selection, suggesting that positive selection played a role in variation during the pre-duplication phase [FoxP2b in Otocephala possibly resulted from excessively relaxed purifying selection on the redundant copy in the fixation phase [FoxP2a and FoxP2b after Neoteleostei branched off from Euteleostei, implying adaptive evolution of these two genes coincided with diversification of Neoteleostei. Our tests of natural selection pressures based on branch specific models strongly suggest the on phase . The poton phase ,58. PosiMus musculus for lung and esophageal development, especially for postnatal lung alveolarization [FoxP2 is critically involved in the development of foregut derived pulmonary organ systems through regulating lung-specific genes, e.g. CC10 and surfactant protein C (SPC).Studies of FoxP2 in both animal models and humans have shown that the pleiotropic gene is widely involved in a series of important physiological activities and developmental processes ,4,17. Inrization ,22,59. TFoxP2a in teleosts have provided only limited information concerning its functions for organ development [FoxP2a in development of CNS with other vertebrates. No FoxP2a expression has been detected in the swim bladder of adult Danio rerio [FoxP2a expression in early (7 days post-fertilization) embryonic development in Maylandia zebra [FoxP2a decreases to a light but visible level in the swim bladder of late larval (20 days post-fertilization) embryos of Mchenga conophorus [Danio rerio [The teleost swim bladder has been considered homologous to the lung on the basis of consistency of developmental blastema and molecular pathways ,29. It helopment ,19,21. Fia zebra . ExpressFoxP2 in foregut derived air-breathing organ, rapid evolution of FoxP2 (FoxP2a and FoxP2b) in teleosts is possibly correlated with adaptive evolution of the swim bladder [Lepisosteus oculatus retains a swim bladder to breathe air and possesses FoxP2 resembling that of tetrapods. The three species of Otocephala  and one species of Protacanthopterygii  are physostomous fishes with a pneumatic duct connecting the swim bladder to the gastrointestine. It has been suggested that the Otocephala-specific loss of FoxP2b possibly results from excessively relaxed purifying selection on the functionally redundant gene. In spite of retention of FoxP2b in Salmo salar, the divergence of FoxP2a and FoxP2b is noticeably weak. Positive natural selection probably acts on FoxP2a and FoxP2b to favor the divergence of the genes in Neoteleostei, which are physoclistous fishes (i.e. swim bladders of these fishes have lost a pneumatic duct). Taken together, the present study provides a useful introduction to studying the functional involvement of FoxP2a and FoxP2b in teleosts.Given the essential functions of  bladder . NotablyInformation S1Species coverage, PCR primers and predicted FoxP2 sequences.(PDF)Click here for additional data file.Information S2FoxP2, FoxP2a or FoxP2b in eight species.Protein-coding genes around  Twenty upstream (with negative order number) and 20 downstream (with positive order number) protein-coding genes.(PDF)Click here for additional data file.Information S3Amino acid or fragment inserts in FoxP2 and FoxP2a of ray-finned fishes.(PDF)Click here for additional data file.information S4Phylogenetic reconstructions based on Data sets 1 and 2.(PDF)Click here for additional data file.Information S5Posterior probability profiles of site-specific rate difference among clusters in Data sets 1 and 2.(PDF)Click here for additional data file.Information S6Hierarchical dendrograms of genes in Data sets 1 and 2.(PDF)Click here for additional data file.Information S7Test of natural selection pressures on Data sets 1 and 2.(PDF)Click here for additional data file.


The aim of the study was determination of serum Se concentration and identification of genetic variations in genes related to metabolism of selenium as markers of cancer risks for carriers of BRCA1 gene mutation and individuals with susceptibility to unselected breast cancer.Eight genotypes of 4 most common SNPs localised in GPX1, GPX4, TXNRD2 and SEP15 were selected. Genotyping was performed in 27 triplets matched BRCA1 carriers (cases and controls were matched 1:2) as well as on pairs matched 1:1 consisting of 220 unselected breast cancer (BRCA1 excluded). Cases and controls were matched for year of birth (+/-3 years), number and location of cancer among Iº relatives , smoking - the number of pack years (+/- 10%) and adnexectomy.For group of 27 triplets matched BRCA1 carriers, all patients were carriers of one of three Polish founder BRCA1 mutation  and serum was collected 1 – 2 years before tumor diagnosis. Whereas for 220 pairs of unselected breast cancer, serum was collected before treatment but during diagnosis.The following techniques for laboratory analyses have been applied: a) sequencing on ABI310, b) TaqMan analysis (a melting-curve genotyping with fluorescence-labeled probes based on the LightCycler 480 System (Roche Applied Science), c) determination of selenium concentration in serum using ICP MS – inductively coupled plasma mass spectrometry with ± 5% accuracy (Perkin Elmer).In both studied groups there was observed that the optimal concentration of selenium was in range 100 – 120µg/l of serum independently on variants in selenoprotein coding genes.If selenium level data were combined with some selenoprotein genotypes a tendency was found that the optimal selenium level for carriers of GPX1 nCC genotype  with genotype nCC in GPX1 gene the optimal concentration of selenium is around 75µg/l of serum.• The results of association studies require confirmation by a prospective observation of large groups of patients.• Investigation on larger number of patients are needed especially valuable may be observations of women with increased risk of cancers.


HTLV-1 is the ethiologic agent of adult T-cell leukemia (ATL). The viral regulatory protein Tax1 plays a pivotal role in T-cell transformation by deregulating several signalling pathways including CREB, NF-κB and AP-1 leading to the abnormal expression of several cellular proteins involved in cell survival and proliferation. Previous studies in the lab, using the yeast-2-hybrid approach to screen a T-cell library for Tax1 interacting partners identified the cellular Four and a Half Lim domain protein 3 (FHL3) as a possible Tax1 interacting candidate. FHL3 can regulate the actin-cytoskeleton and functions as a transcriptional activator. The aim of this study is to investigate the physical and functional interaction between Tax1 and FHL3. We have found that Tax1 and FHL3 interact both in vitro and in vivo and deletion analysis of FHL3 revealed that each LIM domain of FHL3 can interact with Tax1. Deletion analysis of Tax1 determined that the interaction with FHL3 occurs at both the N and C terminus, namely amino acids 1-50 and a fragment which encompasses the predicted LIM binding domain 207-353aa. We have demonstrated that FHL3 enhances Tax1 mediated LTR transcriptional activation without affecting basal activity. In contrast to this we show that FHL3 down regulates NF-κB activation by Tax1. Using confocal microscopy, we show that FHL3 co localizes with Tax in the nucleus and at the periphery of co transfected cells. In addition we demonstrate that FHL3 induces nanotubules in Cos7 cells and may be capable of transporting Tax1 from one cell to another. Overall our results so far suggest that the interaction between Tax1 and FHL3 alters both the transactivating activity and the sub cellular localization of Tax1 and provide new insights into molecular mechanisms that underlie the oncogenic nature of this HTLV-1 protein.


Atoh1, which is necessary for differentiation of hair cells, but not for their specification. Loss of several genes will disrupt ear morphogenesis or arrest of neurosensory epithelia development. We previously showed in null mutants that the loss of the transcription factor, Gata3, results specifically in the loss of all cochlear neurosensory development. Temporal expression of Gata3 is broad from the otic placode stage through the postnatal ear. It therefore remains unclear at which stage in development Gata3 exerts its effect. To better understand the stage specific effects of Gata3, we investigated the role of Gata3 in cochlear neurosensory specification and differentiation utilizing a LoxP targeted Gata3 line and two Cre lines. Cre∶Gata3f/fFoxg1 mice show recombination of Gata3 around E8.5 but continue to develop a cochlear duct without differentiated hair cells and spiral ganglion neurons. qRT-PCR data show that Atoh1 was down-regulated but not absent in the duct whereas other hair cell specific genes such as Pou4f3 were completely absent. In addition, while Sox2 levels were lower in the Cre:Gata3f/fFoxg1 cochlea, Eya1 levels remained normal. We conclude that Eya1 is unable to fully upregulate Atoh1 or Pou4f3, and drive differentiation of hair cells without Gata3. f/fPax2-Cre∶Gata3 mice show a delayed recombination of Gata3 in the ear relative to Cre:Gata3f/fFoxg1. These mice exhibited a cochlear duct containing patches of partially differentiated hair cells and developed only few and incorrectly projecting spiral ganglion neurons. Our conditional deletion studies reveal a major role of Gata3 in the signaling of prosensory genes and in the differentiation of cochlear neurosenory cells. We suggest that Gata3 may act in combination with Eya1, Six1, and Sox2 in cochlear prosensory gene signaling.Hair cells of the developing mammalian inner ear are progressively defined through cell fate restriction. This process culminates in the expression of the bHLH transcription factor  Jagged1 (Jag1), Lunatic fringe (Lfng), Bone morphogenetic protein  SRY-box containing gene (Sox2), Paired box gene 2 (Pax2) and several Fibroblast growth factors  in the prosensory areas of the mouse at E9.5–10.5 suggests that prosensory areas are molecularly defined by these factors at or before this time point Atoh1 expression Atoh1 is not necessary for specification of postmitotic precursors Atoh1 in the cochlea results in transformation of some cells into hair cell-like cells, but these hair cells are unstable and are morphologically and physiologically like vestibular hair cells Atoh1 transfection alone is inadequate for restoration of lost hair cells in the mammalian cochlea suggesting that Atoh1 action depends on the proper molecular context to develop organ of Corti-like hair cells as previously suggested for placodal development The mammalian inner ear develops through a cascade of diffusible factors and transcription factors to generate a complex labyrinth of ducts and recesses out of a flat epithelium, the otic placode Gata3 is particularly high in the ventral otocyst, the area of the cochlear anlage Gata3 expression continues in the organ of Corti and spiral ganglion neurons from specification through late postnatal stages The zinc finger transcription factor Gata3 is expressed throughout the early otic placode. Later Gata3 becomes restricted to future proneurosensory regions (except that of the saccule) concomitant with their segregation from non-sensory domains Gata3 null mutant. Moreover, what specific function the later expression of Gata3 has in these processes remains unclear as no inner ear-specific delayed deletion data exist for Gata3.Haploinsufficiency of GATA3 causes human hypoparathyroidism, sensorineural deafness, and renal dysplasia (HDR) syndrome Gata3 deletion mouse models. We utilized both CreFoxg1 and Pax2-Cre to recombine floxed Gata3 in the ear. CreFoxg1 is a knockin Cre that recapitulates the Foxg1 expression from the E8–8.5 otic placode Pax2-Cre is a BAC-transgene that does not fully conform to early Pax2 expression We investigated the function of Gata3 at later embryonic stages utilizing two conditional Cre:Gata3f/fFoxg1 mice have an earlier and more profound loss of Gata3 expression and exhibit a cochlear duct completely devoid of neurosensory cells. Atoh1 shows limited expression levels in the cochlea of these mice, suggesting that a specific Atoh1 level is critical for hair cell differentiation. Some prosensory genes such as Sox2 are expressed at lower levels compared to normal, while Eya1 and others are not altered in their expression levels. This suggests that Gata3 is an essential co-factor for neurosensory development. Utilizing f/fPax2-Cre: Gata3 mice resulted in a delayed deletion of Gata3 before differentiation of the organ of Corti. Only patches of the organ of Corti with abnormal hair cells formed in the cochlear duct along with improperly projecting spiral ganglion neurons. This indicates sustained expression of Gata3 is required to fully differentiate hair cells and spiral ganglion neurons of the organ of Corti.Our results show that All animal care and procedures were approved by the University of Iowa IACUC following the guidelines for use of laboratory animals (ACURF 0804066).Gata3 conditional knockout mice, we crossed CreFoxg1 mice Pax2-CreGata3 floxed mice Foxg1 locus as CreFoxg1 and Pax2 promoter with the Cre recombinase inserted as a BAC transgene as Pax2-Cre. Breedings were always conducted with a male expressing Cre and heterozygous for the Gata3 floxed allele and the female being homozygous for the floxed allele and no Cre. A few of the matings included a Pax2-Cre male with a Gata3 LacZ null allele instead of a Gata3 floxed allele LacZ/fPax2-Cre:Gata3 and f/fPax2-Cre:Gata3 ears. Genotyping by PCR analysis of tail DNA was accomplished under standard conditions with Gata3 specific primers . Embryos were collected from pregnant dams with noon on the day of vaginal plug considered embryonic day (E) 0.5. To collect embryos, pregnant dams were anesthetized with 1.25% Avertin at a dose of 0.025 ml/g by intraperitoneal injection. For non-qRT-PCR experiments, embryos were dissected from the uterus and fixed with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) transcardially using a peristaltic pump. The following genotypes were utilized as controls: Pax2-Cre:Gata3wt/wt; CreFoxg1:Gata3wt/wt; Gata3f/wt; Gata3f/f. All images are representative of assessments of at least 3 biological controls which were identical unless noted in the text.To generate in situ hybridization experiments were conducted as described in Whole-mount Whole-mount Immunochemistry was performed as described in Inner ear innervation was labeled with lipophylic (NeuroVue) dyes . Dyes were placed into the cerebellum and caudal hindbrain for selective labeling of vestibulo-cerebellar and vestibulo-spinal nerves as described Primers were created using the Roche Universal Probe library assay center which utilizes Advance Primer3 to calculate optimal size, melting point, and complementarity of primer sets. A list of primers used can be found in The process of staining, imaging, and reconstruction of inner ears has been previously described The mice for scanning electron microscopy were perfused with 4% PFA after sedating with Avertin. Ears were then dissected and fixed in a 2.5% glutaraldehyde, 4% PFA solution. Ears of postnatal mice were decalcified with EDTA. Following osmication in 2% osmium tetroxide in 0.1 Monophosphate buffer (pH 7.4) for 1 hour, the ears were dissected and prepared including removal of the tectorial membrane. The samples were then rinsed with distilled water to remove ions and dehydrated in a graded ethanol series, critical-point dried, mounted on stubs with carbon tape and coated with gold/palladium. Specimens were then viewed with a Hitachi S-4800 Scanning Electron Microscope with 3MeV acceleration. False coloring was accomplished with Adobe Photoshop CS6.Gata3 null ears fail to develop all inner ear neurosensory epithelia except a few saccular hair cells and neurons Gata3 mouse line CreGata3 line has LoxP sites flanking exon4 which contains the primary DNA binding domain. CreFoxg1 is expressed early in ear development Gata3 is proposed to lead to early embryonic lethality. To assess recombination of the floxed allele by Foxg1Cre we used qRT-PCR to assess Gata3 mRNA levels over time. We designed specific Gata3 exon4 primers to detect recombination. Gata3 exon4 mRNA expression was significantly reduced in mutant ears by embryonic day (E) 8.5 compared to control, and continued to decrease with age  were not detectable by qRT-PCR. Eya1 and Notch1, factors that are necessary for prosensory specification and differentiation in the inner ear Gata3 conditional mutant mice. Pax2, known to be necessary for normal cochlear development Gata2 in the absence of Gata3. This upregulation of Pax2 and Gata2 is obviously unable to compensate for the lack of Gata3. Tecta is a gene known to be an integral player in tectorial membrane development Tecta levels is consistent with the lack of tectorial membrane in these mutants. Axin2 is known to be expressed in the cochlear duct but not in the sensory epithelia and its expression remains unaltered We used qRT-PCR to identify potential down-stream genes for cochleae . Care waAtoh1 expression led us to speculate that Gata3 not only plays a role in specification but possibly also in the differentiation of neurosensory cells of the cochlea. This is consistent with differentiating spiral ganglion neurons and organ of Corti hair cells expressing Gata3 during differentiation. To analyze the timing of recombination of Gata3 for specification versus differentiation, we utilized another Cre strain (Pax2-Cre) which like CreFoxg1 is expressed in the otic placode, but somewhat later in development Pax2-Cre may be slightly less efficient than CreFoxg1, possibly because of a relatively delayed or more inefficient recombination The loss of Atoh1 downstream gene expression in hair cells without the complete elimination of Pax2-Cre to recombine the Gata3 floxed allele over time. The level of the Gata3 exon4 was not significantly altered from control ears until E10.5  in the cochlear duct. These spaces were absent in all mutants analyzed. We also analyzed morphology and neurosensory formation using LacZGata3 expression. The heterozygous wt/LacZGata3 ear contains no obvious morphological or embryonic histologic defects Pax2-Cre mutant mouse shows a high level of Gata3-LacZ expression in the cochlea as in the control (CreFoxg1:f/fGata3 mutant mice there was frequently a short curvature at the end of the somewhat longer cochlear duct (N = 11/14). One canal crista was present in the anterior part of the ear and a transient formation of a posterior canal crista occasionally occurred (N = 2/7). Unlike the null and CreFoxg1 mutants there was some formation of spiral ganglion neurons. These few remaining neurons were coalesced into a single location near the base of the shortened cochlear duct, and were not present as a “spiral” ganglion along the length of the cochlea as in the control ear , or Gata3 is necessary for Atoh1 function. This is consistent with some bHLH genes in Drosophila requiring the Gata3 homolog, pannier, for both upregulation of these factors and their DNA binding on downstream genes Our results indicate that in the absence or reduced presence of Gata3, some prosensory cells remain, but are not fully specified and lack full upregulation of Gata3 recombination (Pax2-Cre) we were able to partially assess hair cell and spiral ganglion differentiation in the absence of Gata3. While spiral ganglion neurons are completely absent in the CreFoxg1 ear few spiral ganglion neurons form in the Pax2-Cre ear. However, these spiral ganglion neurons do not differentiate properly as is evident by the aberrantly projecting to the sensory patches in the cochlear duct and the cochlear nucleus. There is a curious resemblance between this mutant and the conditional knockout of Neurod1 utilizing the same Cre line Neurod1 and Gata3 mutants have reduced numbers of spiral ganglion neurons, spiral ganglia cell bodies incorrectly migrate away from the ear towards the hindbrain, and have incorrect patterning within the cochlear nucleus. Interestingly both of these mutants have spiral ganglia that project to the correct regions (organ of Corti and cochlear nucleus), but, once there, do not correctly target appropriate cell types. In addition, timing of Gata3 expression has indicated a role in regulating the Insulin-like Growth Factor (IGF) pathway Pax2-Cre). This is interesting since hair cells in the cochlea do not exit the cell cycle before E11.5 With a slight delay in Atoh1 has been shown to be an essential factor for hair cell development in the ear Atoh1 are essential for hair cell development and viability Atoh1 does not result in long lasting and physiologically functional auditory hair cells Sox2 and Eya1/Six1 have been shown to be necessary for bHLH gene upregulation in the ear, how many other factors are needed in addition remains unclear. Until now no factor has been shown to be exclusively necessary for cochlear and not vestibular hair cells, with the former being known to be more susceptible to a number of genetic and pharmacological insults. We provide here evidence that Gata3 may enable Atoh1 mediated hair cell differentiation in particular in the cochlea. This conclusion is consistent with recent data showing that Gata3 enhances the ability of Atoh1 to upregulate downstream genes Eya1, Pax2, Jag1), which remain expressed in the Cre:Gata3f/fFoxg1 cochlea, are unable to drive Atoh1 expression or neurosensory differentiation in the cochlea in the absence of Gata3. Our data therefore establish Gata3 as an essential player in the emerging molecular network needed for neurosensory development.f/fGata3 cochlea suggests that sustained and profound expression of Gata3 is needed for normal development of these structures. The down-regulation of Tecta in the CreFoxg1 mutant mouse nicely fits into this phenotype as Tecta is necessary for tectorial membrane formation Cre) or severely reduced (Pax2-Cre) in Gata3 mutants. The mal-development of hair cell microvilli in the Pax2-Cre:f/fGata3 cochlea suggests that Gata3 is playing a role not only in hair cell specification but also in hair cell stereocilia differentiation. An assessment of genes selectively down-regulated in these hair cells would be beneficial in elucidating which genes are deregulated in the absence of Gata3 during differentiation. Among those genes could be many microRNA regulated genes as the overall development of hair cells is reminiscent of Dicer1 null mice cochlear hair cells The lack of a spiral limbus, the formation of an incomplete tectorial membrane and formation of only microvilli on hair cells in the Pax2-Cre: Gata3 expression early in ear development in Foxg1Cre: f/fGata3 mice abolishes cochlear neurosensory formation. However, the saccule, utricle and a single anterior canal crista form hair cells and receive afferent and efferent innervation, indicating that Gata3 is less critical in vestibular neurosensory development compared to the cochlea. These ears develop a very short cochlear duct, saccular and utricular recesses, but lack semicircular canals. The lack of canal formation could be caused by lack of canal crista known to be necessary for canal growth f/fGata3 mutant where a single toral structure forms despite the presence of up to three cristae. Formation of a short cochlear duct is consistent with findings in other vertebrates that form outgrowth of such ducts without neurosensory formation Knocking out Cre mutant. We find efferent fibers to vestibular epithelia and presumably some may be olivocochlear efferents which may also project with the facial nerve as previously reported Gata3CreFoxg1 and Pax2-Cre are not expressed in these populations, indicating that this is not a cell autonomous effect. Thus with the loss of their original target vestibular and cochlear efferents are able to segregate, and cochlear efferents are able to survive at least a short time without proper targets.Interestingly, we were able to label the olivo-cochlear efferents in the hindbrain in both mutant lines which migrate their soma into the correct periolivary location Gata3 expression in the forming semicircular canals plays an essential role in translating canal crista signals into proper canal growth. Sustained Gata3 expression is not only needed to initiate development of neurosensory cells but its reduction affects the normal differentiation leading to unusual hair cell formation and aberrant spiral ganglion cell projections. Cochlear development thus depends at multiple levels on the continued presence of Gata3 and future work is now needed to elucidate how Gata3 co-operates with an emerging network of other factors to achieve these multiple functions Click here for additional data file.


To the Editor: Human astroviruses (HAstVs) are a common cause of gastroenteritis in children, the elderly, and immunocompromised persons (––www.megasoftware.net) by using the neighbor-joining method and a p-distance algorithm. Bootstrap resampling was performed by using 2,000 replicates to demonstrate robustness of grouping. The nucleotide sequences determined in this study were assigned GenBank accession nos. HQ674630–HQ674650.Fecal samples were collected from a cohort of 364 symptomatic children <5 years of age who had diarrhea and were seeking medical care at Abu Homos Hospital in the Nile Delta of Egypt from September 2006 through September 2007. RNA was extracted from 10% fecal suspensions and reverse transcription PCR for astrovirus was performed as previously described (Consensus astrovirus reverse transcription PCR results were positive in 23 (6.3%) of 364 fecal samples. Five common serotypes of classical HAstV were identified constituting 16 (70%) of 23 positive samples; HAstV type I was most prevalent (n = 9). Alignment of the partial amino acid sequences of the ORF2 capsid region indicates that Egyptian HAstV type I strains share 99%–100% identity , panel AFive of the 7 remaining positive samples were most closely related to MLB1 on the basis of the partial ORF2 sequence analysis. Egypt MLB1 samples shared ≈99%-nt identity with each other, and all grouped in 1 phylogenetic cluster , panel BThe 2 remaining HAstV-positive samples were phylogenetically most similar to astrovirus VA2 (VA2) , panel BOur study describes the detection of the recently identified viruses MLB1 and VA2 in a cohort of symptomatic children with diarrhea residing in Egypt. This study expands the geographic range of these viruses to include northern Africa. The consensus primers used in our study were able to detect a higher percentage of positive HAstV serotypes I–VIII than the Mon340/Astman-2 primers (


Bupivacaine is an amide type long acting local anesthetic used for epidural anesthesia and nerve blockade in patients. Use of bupivacaine is associated with severe cytotoxicity and apoptosis along with inhibition of cell growth and proliferation. Although inhibition of Erk, Akt, and AMPK seemingly appears to mediate some of the bupivacaine effects, potential downstream targets that mediate its effect remain unknown. S6 kinase 1 is a common downstream effector of several growth regulatory pathways involved in cell growth and proliferation known to be affected by bupivacaine. We have accordingly attempted to relate the growth inhibitory effects of bupivacaine with the status of S6K1 activity and we present evidence that decrease in cell growth and proliferation by bupivacaine is mediated through inactivation of S6 kinase 1 in a concentration and time dependent manner. We also show that ectopic expression of constitutively active S6 kinase 1 imparts substantial protection from bupivacaine induced cytotoxicity. Inactivation of S6K1 though associated with loss of putative mTOR mediated phosphorylation did not correspond with loss of similar phosphorylations in 4EBP1 indicating that S6K1 inhibition was not mediated through inactivation of mTORC1 signaling pathway or its down regulation. Bupivacaine is an amide type local anaesthetic used in clinical pain management . Althougβ/S6K1 axis /IC50), with Graph Pad Prism Software (Version 4.0).Cytotoxicity of bupivacaine was evaluated by its effect on cell viability. Accordingly cell lines NIH-3T3, C6, and C2C12 myoblasts were allowed to grow in the absence (control) or presence of bupivacaine at various concentrations over a period of 24 hrs. While the control cells exhibited normal growth characteristics, significant dose-dependent decline in cellular density was observed in bupivacaine treated cells resulting in substantial cell death . The magGrowth inhibition and apoptosis have quite often been associated with dysregulation of signaling pathways with potential to influence S6K1 activity directly or indirectly. We therefore, sought to investigate any such possibility by analyzing activity status of S6K1 in the presence or the absence of the drug. Endogenous S6K1 was immunoprecipitated from NIH-3T3 cells grown in presence or absence of different bupivacaine concentrations for its ability to phosphorylate GST-S6. As seen in Catalytic and linker domain phosphorylations at the activation loop (AL) and hydrophobic motifs (HM) are established determinants of S6K1 enzyme activity. Accordingly their loss is a hallmark of S6K1 inhibition. We therefore sought to ascertain whether inhibition of S6K1 by bupivacaine did indeed correspond with loss of these phosphorylations. As seen in In an attempt to establish that S6K1 inactivation did indeed mediate the growth inhibitory effects associated with bupivacaine, cells transfected with HA-tagged S6K1 wild type and a constitutively active variant T412E-D3E were examined for their ability to resist bupivacaine induced toxicity. mTORC1 is considered to be a major input acting upstream of S6K1 and implicated in T412 phosphorylation of the enzyme and parallel phosphorylations of its other substrate, eukaryotic translation initiation factor 4E binding protein 1 (4EBP1). Possible inactivation of mTOR pathway suggested by inhibition of S6K1 should therefore be corroborated by its inability to cause phosphorylations in 4EBP1 (Threonine 70). Phosphorylation of 4EBP1 at Threonine 70 site  remainedn = 3) indicated that the extent of Erk inhibition coincided with the inhibition of S6K1 as much as it did with the kinetics of cell proliferation, to suggest that the effect of bupivacaine may be mediated through inactivation of Erk and S6K1.In the absence of mTOR as the mediator of S6K1 inhibition by bupivacaine, it was imperative to examine other signaling pathways that may directly or indirectly influence S6K1 activity in the cell. Since extracellular signal-related kinase/s (Erk) are believed to participate in the events leading to cytotoxicity by anesthetic drugs like bupivacaine, we sought to investigate the activation status of Erks to possibly relate it with observed inhibition of cellular proliferation and S6K1 activity. The activation of Erks was evaluated by immunoblotting with a phospho specific antibody. As seen in The use of local anesthetics like bupivacaine is associated with chondrotoxicity, myotoxicity, and neurotoxicity at variable propensities , 9, 32. We chose NIH-3T3 cells for their restrained response to bupivacaine induced cytotoxicity compared to C2C12 and C6 cell lines, to help us register the dynamics of signaling events responsible for preventing cell growth and proliferation. We demonstrate that the inhibition of cell proliferation by bupivacaine strictly correlated with the inactivation of ribosomal protein S6 kinase 1 and its relief by the use of constitutively active enzyme to suggest a possible dysregulation of events that control cell division, at least in part via protein translation. With long standing implications that bupivacaine may influence protein synthesis by interfering with amino acylation of tRNAs , the relMAPK signaling has a central role in regulating cell growth and proliferation . AccordiOur data suggests that bupivacaine induced inhibition of cell proliferation is mediated at least in part through inactivation of S6 kinase 1. We also demonstrate redundant role of mTORC1 signaling in mediating this effect to suggest the possible involvement of Erk pathway.


The input nucleus of the basal ganglia, the striatum, processes cortical inputs in two output streams, called the direct and indirect pathways. Medium Spiny Neurons (MSNs) that express dopamine D1 receptors constitute the direct pathway (D1 MSNs) and MSNs that express dopamine D2 receptors constitute the indirect pathway (D2 MSNs). The two populations of MSNs differ in their intrinsic excitability with D2 MSNs more responsive to somatic current injection  to produ


Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion antigen family are major contributors to the pathogenesis of P. falciparum malaria infections. The PfEMP1-encoding var genes are among the most diverse sequences in nature, but three genes, var1, var2csa and var3 are found conserved in most parasite genomes. The most severe forms of malaria disease are caused by parasites expressing a subset of antigenically conserved PfEMP1 variants. Thus the ubiquitous and conserved VAR3 PfEMP1 is of particular interest to the research field. Evidence of VAR3 expression on the infected erythrocyte surface has never been presented, and var3 genes have been proposed to be transcribed and expressed differently from the rest of the var gene family members.Members of the var transcript and PfEMP1 expression profiles of the generated parasites were investigated. The IgG reactivity by plasma from children living in malaria-endemic Tanzania was tested to parasites and recombinant VAR3 protein. Parasites from hospitalized children were isolated and the transcript level of var3 was investigated.In this study, parasites expressing VAR3 PfEMP1 were generated using anti-VAR3 antibodies and the Var3 is transcribed and its protein product expressed on the surface of infected erythrocytes. The VAR3-expressing parasites were better recognized by children´s IgG than a parasite line expressing a Group B var gene. Two in 130 children showed increased recognition of parasites expressing VAR3 and to the recombinant VAR3 protein after a malaria episode and the isolated parasites showed high levels of var3 transcripts.var3 is transcribed and its protein product expressed on the surface of infected erythrocytes in the same manner as seen for other var genes both in vitro and in vivo. Only very few children exhibit seroconversion to VAR3 following a malaria episode requiring hospitalization, supporting the previous conclusion drawn from var3 transcript analysis of parasites collected from children hospitalized with malaria, that VAR3 is not associated with severe anaemia or cerebral malaria syndromes in children.Collectively, the presented data suggest that  Plasmodium falciparum is a major cause of mortality and disease in sub-Saharan Africa. Immunity to malaria in areas with intense transmission is acquired during childhood as a broad repertoire of specific protective antibodies to parasite-derived polymorphic variant antigens present on the infected erythrocyte surface, develops [Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is the best characterized variant surface antigen [var genes per parasite genome [var genes, var1 (UPSA), var2csa (USPE), and var3 (aka Type 3) (UPSA) are conserved in their full length in the global parasite population [vars encoding more diverse domain cassettes not spanning the full length genes [Var3 genes have arisen from a recombination between a DBLζ-DBLε encoding sequence only found in the 3´end of var exon1 and an N-terminal DBLα1 sequence. Sequence analysis has shown that only the DBLζ-DBLε part of VAR3, which is 99% identical between VAR3 sequences, is unique to the protein sub-family [develops -3. Plasm antigen -6. A sin antigen -11 as th antigen . Members antigen ,14. The e genome , which ce genome ,17. Threpulation -22. Aparth genes . PfEMP1sth genes  and is tth genes . Var3 geb-family .var types expressed in severe malaria [var mRNA species by PCR amplification and sequencing of a short 350 bp DBLα tag present in all var genes except var2csa and var3. As this approach does not capture var3 sequences, quantitative PCR has been deployed to investigate var3 transcript levels in patient samples [var genes are associated with severe malaria in children.VAR2CSA binds chondroitin sulphate A (CSA) in the placenta and facilitates the parasite sequestration causing pregnancy-associated malaria. Similarly, organ specific PfEMP1 mediated sequestration has been linked to severe malaria in children , and evi malaria ,30,33-36 samples ,37. The var3 transcript levels in malaria patient samples have shown that var3 transcripts was not associated to any particular syndrome of severe malaria [P. falciparum-infected erythrocytes has never been achieved and evidence presented by Epp et al.[var3 transcription may be regulated differently from the rest of the var genes and expressed in a non-mutually exclusive manner due to an atypical intron activity.Because VAR3 is conserved, belongs to group A PfEMP1 and has an unusual domain structure, this protein could play a particular important role for the parasite and in development of malaria disease. Malaria-endemic populations has been shown to have acquired antibodies that react with recombinant VAR3 protein , suggest malaria . A formapp et al. suggestsvar3 mRNA and VAR3 protein is expressed similar to other PfEMP1s. Furthermore, in this study, evidence for in vivo expression of VAR3 in malaria-sick children is presented.In an effort to unravel some of the mystery surrounding this PfEMP1, parasites of different genetic background were successfully manipulated to express VAR3 on the surface of the infected erythrocytes. Analysis of these parasites suggests that both Plasmodium falciparum clones 3D7 and IT/FCR3 parasites were cultured in O Rh + erythrocytes in RPMI1640 supplemented with Albumax II, as previously described [escribed  by repeaet al.[P. falciparum negative controls, human DNA from Danish donors and blank samples, were included in the setup and the amplified PCR products were analysed by agarose gel electrophoresis.Isolate integrity of the cultures was tested using the method previously described by Snounou et al. with theseryl-tRNA synthetase gene. RNA was reverse transcribed from random hexamers, using Superscript II (Invitrogen), according to the manufacturer’s instructions (Invitrogen).RNA was extracted using Trizol (Invitrogen) according to manufacturer’s instructions. Samples were treated with DNase I (Sigma) to digest any genomic DNA and tested in quantitative PCR for contamination, using a primer set, p90, for the var gene of the P. falciparum clone 3D7 and to the endogenous control genes seryl-tRNA synthetase and fructose-bisphosphate aldolase, have been described previously [var genes extracted from the IT/FCR3 genome available at The Wellcome Trust Sanger Institute (UK) [PFI1820w/PFF0020c/IT4var3 (var3Xi) and PFA0015c (PFA0015cXi) transcripts, respectively, were designed as well   -∆CtQuantitative PCR was performed on a Rotorgene RG-3000 thermal cycler (Corbett Research), applying QuantiTect SYBR Green PCR Master Mix (Qiagen) with primers at 20 μM, and internal control genes + erythrocytes and incubating 24 h at 4°C. Besides the full length VAR3 protein, two full length and four multi-domain PfEMP1 proteins were included in the Luminex assay  and DAPI and laser scanning confocal microscopy performed using a Nikon TE 2000-E confocal microscope with 60x oil immersion objective lens (DIC) as previously described [Indirect immuno-fluorescence assays were performed on live 3D7VAR3/3D7 unselected and FCR3VAR3/IT/FCR3 unselected in order to observe the staining of individual infected erythrocytes with VAR3-antibodies. The staining was done using Alexaescribed .Levels of plasma PfEMP1 domain-specific IgG were analysed as described previously . In briein vitro model parasite lines expressing VAR3, parasites of two laboratory cultured parasite clones, 3D7 and IT/FCR3, were selected with rabbit antiserum against a 3D7 VAR3 (PFI1820w) recombinant protein. The expression of VAR3 on the surface of 3D7VAR3- and FCR3VAR3-infected erythrocytes, respectively, was investigated by flow cytometry and confocal microscopy using rabbit IgG specific for the clone 3D7 VAR3 PFI1820w which they had been selected with. The selected lines, 3D7VAR3 and FCR3VAR3, were recognized by the rabbit serum and stained the infected erythrocytes in a punctuate pattern typical for PfEMP1 [To generate r PfEMP1 ,51 wherevar transcript profiles of the selected 3D7VAR3 and FCR3VAR3 and unselected 3D7 and IT/FCR3 parasite lines were investigated using var gene specific primers in quantitative PCR. The transcript levels of all var genes relative to internal control genes are shown in Figure var3 transcripts, PFI1820w and IT4var3, than the unselected lines and the var3 transcript levels were similar to those observed for var genes in vivo[in-vitro parasite cultures with verified erythrocyte surface expression of PfEMP1 [var3 genes, resulted in selection of parasites expressing two of these genes (PFI1820w and PFF0020c) but not parasites expressing the third (PFA0015c) , with a peak in transcript level at 19 h PM corresponding to mid ring stages  Figure . A speciite line  and fourite line .var gene, IT4var60, thought to mediate rosetting [var gene, IT4var59  of the acute patient plasma samples. Only two boys, patient A and B, appeared to acquire IgG to VAR3 but not to VAR2CSA expressing parasites after being sick and hospitalized with malaria Figure . An incr primers  . In prechildren -32. The isolates ,37 does  general ,28-30. Ovar3 and the presence in almost every parasite genome, it is striking that only a fifth of 1,342 individuals from malaria-endemic areas in Tanzania showed acquisition of antibodies to this PfEMP1 [The pattern of Tanzanian children´s acquisition of antibodies to live VAR3-expressing parasites compared to UPSA and UPSB type PfEMP1-expressing parasites indicate a virulence of VAR3 similar to UPSA type PfEMP1. Yet, in spite of the conserved nature of s PfEMP1 . This mas PfEMP1 ,37 and tvar3 transcribing parasites and VAR3 antibodies in malaria patients can be challenged if var3 genes are regulated and expressed abnormally as appears to be the case for var1. Studies of laboratory parasite lines show that var1 is constitutively transcribed throughout the blood stages cycle, while the transcription of other var genes is silenced when parasites develop into schizonts [var1 often lacks or has a truncated exon2 which may cause a corrupted transcription regulation resulting in protein synthesis and host antibody development upon parasite destruction. It remains unclear if VAR1 serves a particular function for the parasite and why var1 is maintained in the population. Indeed, Epp and colleagues [var intron of the PFF0020c var3 gene did not show any transcriptional activity as opposed to other non-VAR3 var introns. Non-coding RNA is transcribed from var introns and is physically associated with chromatin and believed to be involved in the transcriptional regulation of var genes [var3 genes in general could be regulated differently from other var genes. From the present data it does not seem that var3 genes are transcribed and expressed differently from other var genes. In both 3D7VAR3 and FCR3VAR3 var genes other than var3 were silenced and the var3 transcript profile during the intra-erythrocytic life cycle was similar to that of other var genes in other parasite lines [These interpretations of chizonts . In spitchizonts , VAR1 erlleagues  found thar genes . Thus, ite lines ,52 and pAlso the VAR3 expression on the erythrocyte surface appeared similar to that of other PfEMP1. Antibodies raised towards a recombinant 3D7 VAR3 variant successfully selected VAR3 erythrocyte surface-expressing parasites from both 3D7 and IT/FCR3 parasite populations. The surface expression of VAR3 was confirmed by antibody staining in both flow cytometry and confocal microscopy with expression levels and a punctuate pattern characteristic for PfEMP1 expression in knobs. While the VAR3 in the IT/FCR3 line most likely is the only PfEMP1 on the infected erythrocyte surface, it is possible that both PFI1820w and PFF0020c VAR3 PfEMP1s are expressed by the same parasites, as previously seen in 3D7 parasites .var3 is transcribed and the encoded protein expressed on the surface of infected erythrocytes in the same manner as seen for other viable var genes. VAR3-expressing parasites sustain P. falciparum infections in children living in malaria-endemic areas although only found in a fraction of malaria hospitalizations and not associated with severe anaemia or cerebral malaria syndromes. Whether this structurally unique and conserved PfEMP1 confer parasites expressing it with a rare functional specialization remains unclear, but further studies of the cyto-adherence phenotype of the established VAR3-expressing parasite lines may elucidate this.In conclusion, The authors declare that they have no competing interests.CWW carried out molecular biology studies, analysed data and wrote the paper. TL and TGT participated in the design, coordination and analysis of the study and helped to draft the manuscript. LT designed and carried out molecular biology studies, analysed data and helped to draft the manuscript. DCB, SBS, AMM and MA carried out molecular biology studies and revised the manuscript. PAM and JPL participated in the coordination of the study and revised the manuscript. All authors read and approved the final manuscript.Specificvargene primers used for Q-RT PCR.Click here for filePrimers used forPlasmodium falciparumerythrocyte protein 1 full length (FL) and multi-domain expression.Click here for fileVargene transcript levels ofPlasmodium falciparumIT4var59 parasites.Var gene transcript levels of IT4var59 parasites relative to the average of two internal control genes. The average of control gene transcript levels is set to 100. The distribution of var transcripts is also shown (circular diagram). Transcript levels were measured by quantitative PCR using a set of primers that amplify 56 var genes from clone IT/FCR3. The grouping of var genes are as previously described [escribed ,17,23.Click here for fileTranscript levels ofvar3in patient parasite isolates. Transcript levels of var3-DBLε8 in P. falciparum parasites isolated from 130 hospitalized children  and the var subtype transcript levels in patient parasite isolates, A and B. The levels are relative to the average of two internal control genes, which are normalized to 100. Transcript levels were measured by quantitative PCR using a set of 42 subtyping quantitative primers [ primers .Click here for file


Epigenetic regulation of gene expression is commonly altered in human cancer. We have observed alterations of DNA methylation and microRNA expression that reflect the biology of bladder cancer. This common disease arises by distinct pathways with low and high-grade differentiation. We hypothesized that epigenetic gene regulation reflects an interaction between histone and DNA modifications, and differences between normal and malignant urothelial cells represent carcinogenic events within bladder cancer. To test this we profiled two repressive histone modifications (H3K9m3 and H3K27m3) using ChIP-Seq, cytosine methylation using MeDIP and mRNA expression in normal and malignant urothelial cell lines. In genes with low expression we identified H3K27m3 and DNA methylation each in 20–30% of genes and both marks in 5% of genes. H3K9m3 was detected in 5–10% of genes but was not associated with overall expression. DNA methylation was more closely related to gene expression in malignant than normal cells. H3K27m3 was the epigenetic mark most specifically correlated to gene silencing. Our data suggest that urothelial carcinogenesis is accompanied by a loss of control of both DNA methylation and H3k27 methylation. From our observations we identified a panel of genes with cancer specific-epigenetic mediated aberrant expression including those with reported carcinogenic functions and members potentially mediating a positive epigenetic feedback loop. Pathway enrichment analysis revealed genes marked by H3K9m3 were involved with cell homeostasis, those marked by H3K27m3 mediated pro-carcinogenic processes and those marked with cytosine methylation were mixed in function. In 150 normal and malignant urothelial samples, our gene panel correctly estimated expression in 65% of its members. Hierarchical clustering revealed that this gene panel stratified samples according to the presence and phenotype of bladder cancer. Bladder cancer is the fifth commonest malignancy in the United States with 70, 530 new cases and 14,680 deaths in 2010 Molecular changes in cancer arise from either genetic or epigenetic events. The latter is defined as stable heritable changes in a chromosome without alterations in the DNA sequence We have previously observed changes in DNA methylation and microRNA expression that reflect the molecular biology of UCC and are associated with the clinical phenotype of tumors We performed massively parallel sequencing to determine the distribution of DNA adjacent to H3K9m3 and H3K27m3 in urothelial cells. Each experiment yielded between 7.7 M and 18.9 M reads . The nat4 bases distant. For H3K27m3, the relationship was bimodal with peak enrichment occurring at both 104 and 107 base distances.Recent data suggest that many tissue and cancer-specific differentially DNA methylated regions (T and C-DMRs) occur within 2 kb around CpG islands , filtered for those with differences between the malignant and normal cells  and selected probes with differential hypermethylation in the cancerous lines ). We mapped these probes to their nearest neighbors to identify regions with adjacent (those <200 bp apart) enriched probes. Depending upon stringency, we found between 1,513 (5/5 adjacent probes) and 106 (11/11 adjacent probes) CpG islands around 711 and 40 protein-coding genes, respectively. Of these, 20% and 26% were located within a promoter and the remainder within (66% and 8%) or downstream from the nearest gene. We annotated the 5 mC profile in each cell line for these 68,292 probes using mRNA expression and obtained data for 10,568 genes . When measured an inverse correlation was seen between mRNA expression and 5 mC enrichment for these probes . In general, DNA and histone enrichment were negatively correlated to mRNA expression . This reTo determine specific loci important for urothelial carcinogenesis we identified those with cancer-specific silencing and associated epigenetic modifications. Specifically, we selected genes with one or more repressive epigenetic mark (defined as within the highest 20% of enrichment) that was shared in the two cancer lines and absent in NHU. From these, we chose genes with reduced expression in EJ and/or RT112, and preserved expression in NHU. This strategy identified 239 and 317 genes in EJ and RT112, respectively . Of thesTo evaluate epigenetically mediated gene upregulation in cancer we used the combined dataset  to identify those with DNA hypomethylation and/or loss of enrichment for either histone modification in the cancer cell lines, when compared to NHU. From these we selected genes with cancer specific expression  Here we have produced integrated epigenomic maps for two UCC cell lines and non-transformed normal urothelial cells. The latter are cultured expansions taken from disease free patients that grow as sheets of histologically normal appearing urothelial cells for 7–8 passages before senescence. Our experimental data reveal insights into the epigenetic control of gene expression in the biology of UCC and have specifically identified genes potentially involved in urothelial carcinogenesis.At the global level several observations are apparent. Firstly, H3K9m3 and H3K27m3 are located mostly around gene transcription start sites Our data detail the associations between H3K9m3, H3K27m3, CpG islands and DMRs , one would suspect that the pro-carcinogenic pathways associated with 5 mC are those contributing to malignant transformation.To assess the validity of the genes identified using cell lines, we examined their expression in 150 normal and malignant urothelial samples. Our epigenetic panel correctly estimated the expression of 65% of genes and stratified the tissues according to the presence and phenotype of cancer. This is important as previous epigenetic studies using candidate tumor suppressor genes reveal that low-grade non-invasive cancers differ to high grade and invasive UCC One of the most interesting candidates within our panel is the Cellular Apoptosis Susceptibility/CSE1 chromosome segregation 1-like gene (hCAS/CSE1L). This component of the nuclear import pathway is frequently upregulated in cancer and located at 20q13 within a region amplified in malignancy In conclusion, we have mapped repressive epigenetic events in malignant and normal urothelial cells. In genes with low expression we identified associated H3K27m3 and DNA methylation each in 20–30% of genes and both marks in 5% of genes. When all genes were analyzed H3K9m3 did not appear to be associated with expression. DNA methylation was more closely related to gene expression in cancerous than normal cells. H3K27m3 was the epigenetic mark most specifically correlated to gene silencing. We identified a panel of genes with cancer specific epigenetic mediated aberrant expression including those with known carcinogenic functions and members potentially mediating a positive epigenetic feedback loop.We analyzed bladder cancer cell lines representing non-invasive and invasive disease  grown in Dulbecco's medium with 10% fetal calf serum, and normal human urothelial (NHU) cells maintained in keratinocyte serum-free medium containing bovine pituitary extract, epidermal growth factor  and cholera toxin Malignant and normal human urothelial cells were cross-linked with 1% formaldehyde in PBS for 10 min at room temperature. Glycine was added to the final concentration of 0.125 M and the mixture incubated for 5 min to quench unreacted formaldehyde. Cross-linked cells were washed with PBS, lysed in 250 µl SDS lysis buffer and sonicated . Samples were pre-cleared with 15 µl of protein G dynabeads (Invitrogen) and 50 µl supernatant saved as total input control. Rotating samples were immunoprecipitated overnight at 4°C with antibodies against H3K9m3 , H3K27m3 (Millipore) and rabbit IgG as a negative control . The chromatin:antibody complex was incubated with 20 µl dynabeads protein G for 1 h at 4°C, before washing and DNA elution. Cross-links were reverted by overnight incubation with RNAse A at 65°C before DNA purification using columns . Immunoprecipitated DNA ends were repaired using the PNK and Klenow enzyme, adenosine was added to the 3′ end of the fragments and the DNA was ligated with the adapters. Following ligation, the ChIP DNA was amplified for 17 cycles and fragments around 200–300 bp isolated using electrophoresis in an agarose gel. The purified DNA was used for cluster generation and sequencing analysis using the Solexa 1 G Genome Analyzer according to manufacturer protocols  as detailed elsewhere Methylated DNA immunoprecipitation and tiling CpG island microarrays  (MeDIP-CHIP) were used to determine genome wide distribution of 5 mC Whole genome mRNA expression was determined by microarray  as detailed elsewhere Our analyses identified a panel of genes that appeared to be important in urothelial carcinogenesis. To evaluate their role in human UCC, we extracted expression data from Array Express . all genes and (b). for genes around CpG islands. As can been seen, H3K9m3 appeared more specific to CpG islands than H3K27m3.(PDF)Click here for additional data file.Figure S2Gene expression and histone enrichment. Gene expression was an average of more than 10 fold lower in TSS with H3K27m3 enrichment, compared to those without enrichment Click here for additional data file.Figure S3Epigenetic gene silencing in bladder cancer. Venn diagrams represent number of down regulated genes in EJ and RT112 bladder cancer cell lines, when compared to NHU, associated with each epigenetic mark.(PDF)Click here for additional data file.Figure S4Epigenetic gene upregulation in bladder cancer. Venn diagrams represent number of upregulated genes in EJ and RT112 bladder cancer cell lines, when compared to NHU, associated with each epigenetic mark.(PDF)Click here for additional data file.Figure S5Functional annotation clustering of genes with epigenetic marks in EJ cells. Using gene enrichment pathway analysis we determined clusters of genes for each set marked by an epigenetic modification. The number of gene clusters within each part of the diagram is indicated in these area proportional Venn diagrams.(PDF)Click here for additional data file.Figure S6The expression of genes within our combined epigenetic panel in an external dataset . The median expression of 124 genes is shown when stratified according to the epigenetic traits found in EJ, RT112 and NHU. As shown, genes with predicted epigenetic upregulation had higher expression than those with predicted silencing.(PDF)Click here for additional data file.Figure S7Unsupervised hierarchical clustering stratified malignant and normal urothelial samples according to phenotype. Previously reported microarray data (PDF)Click here for additional data file.Table S1Reads per experiment obtained from massively parallel sequencing.(PDF)Click here for additional data file.Table S2Details of the combined epigenetic gene panel stratified for associated events and mRNA expression. Below each column is the proportion of total events (as a percentage).(PDF)Click here for additional data file.Table S3Gene ontology analysis for each epigenetic mark. The table reveals the GO details for all identified pathways associated with each event.(PDF)Click here for additional data file.Table S4Phenotype specific gene expression in the epigenetically selected panel as determined using gene expression data .(PDF)Click here for additional data file.