CNGA3 and CNGB3, encoding two structurally related subunits of cone CNG channels, lead to achromatopsia (ACHM). ACHM is a congenital, autosomal recessive retinal disorder that manifests by cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color blindness and photophobia. Here, we report the first canine models for CNGA3-associated channelopathy caused by R424W or V644del mutations in the canine CNGA3 ortholog that accurately mimic the clinical and molecular features of human CNGA3-associated ACHM. These two spontaneous mutations exposed CNGA3 residues essential for the preservation of channel function and biogenesis. The CNGA3-R424W results in complete loss of cone function in vivo and channel activity confirmed by in vitro electrophysiology. Structural modeling and molecular dynamics (MD) simulations revealed R424-E306 salt bridge formation and its disruption with the R424W mutant. Reversal of charges in a CNGA3-R424E-E306R double mutant channel rescued cGMP-activated currents uncovering new insights into channel gating. The CNGA3-V644del affects the C-terminal leucine zipper (CLZ) domain destabilizing intersubunit interactions of the coiled-coil complex in the MD simulations; the in vitro experiments showed incompetent trimeric CNGA3 subunit assembly consistent with abnormal biogenesis of in vivo channels. These newly characterized large animal models not only provide a valuable system for studying cone-specific CNG channel function in health and disease, but also represent prime candidates for proof-of-concept studies of CNGA3 gene replacement therapy for ACHM patients.Cyclic nucleotide-gated (CNG) ion channels are key mediators underlying signal transduction in retinal and olfactory receptors. Genetic defects in  In the visual system, rod-mediated phototransduction utilizes outer segment membrane CNG channels assembled from three CNGA1 subunits and one CNGB1, whereas the cone-specific CNG channels are composed of CNGA3 and CNGB3 subunits, arranged in a proposed stoichiometric ratio of 3:1, respectively  for PDE6C; 4 SNP for GNAT2 ; 4 SNPs for PDE6H ; and 9 SNPs  and one STR (TCCCT)16 for CNGA3.Genomic DNA was isolated from whole blood according to the manufacturer’s instructions . The control panel consisted of DNA samples from 100 unrelated dogs from 18 different breeds. Cone phototransduction cascade genes http://genome.ucsc.edu) using Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/primer3/input.htm). See CNGA3 gene screening. All amplicons were purified using ExoSAP-IT (Affymetrix), subjected to direct sequencing  and analyzed using Sequencher 5.2.4 software.Gene-specific primers were designed based on Broad CanFam3.1 genome assembly (CNGA3 cDNA was inserted into the pEYFPN1 expression vector and site-specific mutations were introduced as described previously [A full-length canine eviously . All genhttp://www.graphpad.com).Inside-out patches were excised from fluorescent cells as previously described . The batmaplot™) . Subcellmaplot™) . Fluoresex vivo data, IHC staining was performed on 10 μm thick canine retinal cryosections that were incubated overnight at +4°C in the presence of rabbit polyclonal anti-canine CNGA3 antibody  at a concentration of 1:750. Primary antibody was visualized with Alexa Fluor 568nm goat anti-rabbit secondary antibody and cell nuclei were stained with DAPI. Slides were mounted  and examined by epifluorescence  or confocal  microscopy using previously published methods [For the  methods .http://blast.ncbi.nlm.nih.gov/Blast.cgi), the chimeric voltage-gated K+ channel Kv1.2/2.1 (PDB ID: 2R9R), a molecule of solved crystal structure [Amino acid sequences of human (GI: 4502917) and canine (GI: 345777245) CNGA3 were used as a template for transmembrane topology prediction and MD simulations. Using PSI-Blast search  bilayer in a 120 mM NaCl solution was considered for all MD simulations . The R42-1 = 0.5 mol/kcal, a typical value used in many previous reports (summarized in [http://www.grigoryanlab.org/drawcoil/).The CLZ domain from the human CNGA3, consisting of the complete trimeric coiled-coil region, , was identified as the closest homolog of known structure of the canine CNGA3 CLZ domain. We therefore used it as a backbone template for the wild-type and V630 deletion models. Before beginning homology modeling, the structures were relaxed using 200 steps of conjugate-gradient minimization with the generalized Born implicit solvent model implemented in the NAMD2.10 software package. This initial relaxation was used to remove any structural perturbation due to crystallization conditions and to enhance the designability of protein structures, particularly in the core positions of coiled-coils. A probabilistic design algorithm was then used to identify optimized (the most probable) side-chain rotameric states consistent with the relaxed backbone structures for both the wild-type and V630del sequences . For therized in ). Side-crized in . No symmAll MD simulations involving the CLZ domain were performed in an identical manner to that of the CNGA3 MD simulations with a few modifications listed below. All simulations were performed using the NAMD 2.10 (CVS) software package. Simulations were run at a temperature of 310 K fixed using Langevin dynamics and a pressure of 1.0 atm using the Nosé-Hoover Langevin piston pressure control. Typical system setup and equilibration involved solvating the coiled-coil complex with 1 nm of water homogeneously from the surface of the protein followed by neutralization by adding 150mM NaCl using the Solvate and Autoionize plugins, respectively, provided in the VMD software package. Initial velocities were sampled from a Maxwell-Boltzmann distribution with a target temperature of 310K. Initially, 5 kcal/mol/Å constraints were placed on all heavy protein atoms followed by slow removal of the constraints over a 20 ns period to encourage the formation of favorable atomic interactions and to reduce initial large perturbations leftover from the modeling process. Trajectories were then collected for 325 ns and 430 ns for the hCNGA3 CLZ wild-type and V630del mutants, respectively.S1 Fig). Scotopic electroretinogram (ERG) showed normal rod function, but no light-evoked cone responses under photopic conditions in affected dogs compared to the age-matched controls, indicating a complete loss of cone photoreceptor function . Pedigree analysis in the Labrador retriever line [Young dogs from German shepherd and Labrador retriever breeds developed complete loss of photopic function at 8–10 weeks of age, soon after canine retinal development is complete . Clinicaver line  along wiCNGB3 (OMIM# 605080), GNAT2 (OMIM# 139340), PDE6C (OMIM# 600827) and PDE6H (OMIM# 601190) loci from causative association in both breeds. Direct sequencing of all exons and flanking intronic sites of CNGA3 (OMIM# 600053) revealed a missense mutation in exon 7 (c.C1270T/p.R424W) in the German shepherd breed and a 3nt deletion in exon 7 (c.1931_1933delTGG/p.V644del) in Labrador retrievers . In the homozygous affected German shepherd, the C1270T transition results in substitution of an evolutionarily conserved basic arginine (Arg/R) at the amino acid position 424 for an aromatic, hydrophobic tryptophan (Trp/W) residue, the largest amino acid. The homozygous 1931_1933delTGG mutation spontaneously occurring in Labrador retrievers eliminates a hydrophobic valine residue (V644del) from the core of the CLZ domain of CNGA3. Both mutations showed perfect segregation with disease phenotype and pedigree and none of the identified alleles were present in 200 control chromosomes from 100 dogs of 18 breeds.Phenotype-based candidate gene analysis combined with homozygosity mapping excluded CNGA3 gene screening were submitted to NCBI dbSNP database (http://www.ncbi.nlm.nih.gov/SNP): both pathogenic variants (c.1931_1933delTGG SS#1067414233 and c.C1270T SS#1067414234) and two newly discovered polymorphisms (c.T1266A silent SS#1067414235 and CFA10:44249374 G>A intronic SS#1067414236).All sequence variants identified during CNGA3 gene product was found to be exclusively expressed in the outer segment of cone photoreceptors in the normal canine retina  and comparative sequence analysis confirmed a high level of sequence conservation among vertebrate genomes for the regions involved . Because the canine protein is 14 aa longer, canine R424 corresponds to human R410 residue, while the canine V644 to V630 of human CNGA3 . For full-length amino acid sequence alignment of human and canine CNGA3 see S3 Fig.The + channel, Kv1.2/2.1 [Fig 2A. This model suggested a salt bridge interaction between R424, the C-terminal residue of S6 and E306, the N-terminal residue of the S4-S5 linker. To investigate the prediction that R424 conserves a salt bridge among CNG channels, the S4-S5 linker region and S6 TM domain of CNG channel subunits were aligned with selected members of the Shaker K+ channel superfamily . The residue of interest, R424 is conserved in CNGA1, CNGA2, and CNGA3 subunits as well as in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. In Kv channels, this site favors an acidic residue (Glu/E). The predicted interacting partner of R424, CNGA3 residue E306, demonstrated a high degree of conservation among the CNG channel subunits and in HCN channels. The Kv channels share the pattern, but with reversal of charges at these positions i.e., the positively charged amino acid residue K in the S4-S5 linker and the negatively charged E at the C-terminus of S6. These observations are consistent with a conserved salt bridge interaction between the S4-S5 linker and S6 in both CNG and Kv channel families.To examine the consequences of the R424W mutation, structural modeling with molecular dynamics and electrophysiological studies were employed. The formerly constructed open state canine CNGA3 (cCNGA3) homology models were used in this study [v1.2/2.1  and scheFig 2C and 2D). First, the MD simulations were used to relax the structure of the wild-type CNGA3 homotetramer channel in its membrane/solution environment for ~130 ns until equilibrated. The simulation was initiated from a complex structure in which all subunits formed the R424-E306 salt bridge. During the entire simulation time, the salt bridge interaction was lost only in one of the subunits, but was maintained intact in the other three subunits . However, with the introduction of the R424W point mutation, all of the salt bridge interactions were lost. Due to the dual hydrophobic/polar nature of Trp (W), these mutant residues did not tend to extend into the solution, but rather stayed at the protein/solution interface, an environment where the hydrophobic moiety could be buried in the hydrophobic core of the protein, but where the polar NH group could stay in a favorable solvated environment . Interestingly, after ~120 ns of simulation, the R424W mutant pore began to close . Full pore closure was not observed over the course of these short simulations, yet our results indicate that the closed state of the channel may be favored in the R424W mutant model .In order to explore the molecular role of R424 and substantiate the premise of salt bridge formation, the R424 and E306 residues were carefully examined in the CNGA3 homology model relaxed with MD simulations . On the other hand, R424K and E306D, the side-chain charge-conserving mutant constructs, were thought to have the potential to preserve channel function albeit with possible alterations in the channel properties. Indeed, the E306D and R424K mutant channels resulted in either cGMP- and cAMP-activated currents with cAMP/cGMP efficacy values similar to those of CNGA3-WT, or diminished cGMP-activated currents with no cAMP-activated currents, respectively . Finally, the double charge-reversal mutant channel, E306R-R424E, was assessed for a potential rescue of the channel function. As expected, the E306R-R424E restored cGMP activation producing large currents similar to the CNGA3-WT channel; however this double mutant was unresponsive to the partial agonist cAMP with an efficacy in the WT channels of ~20% compared to cGMP activation . Dose-response relations of cGMP-activated currents for the CNGA3-WT and E306R-R424E double mutant channels are shown in Fig 3B and the average K0.5 values are summarized in S2 Table. The dose-response curves for cGMP activation showed an average K0.5 value of ~102 μM for E306R-R424E mutant, which is ~8 fold higher than that of the CNGA3-WT, 12.3 μM (S2 Table). The loss of cAMP-activated currents in all R424-mutant channels examined and the cGMP K0.5 shift in the E306R-R424E double charge-reversal mutant channel, suggest that changes in the S4-S5 linker to S6 salt bridge alter nucleotide recognition and gating. However, since the K0.5 values reflect both nucleotide binding and pore opening, deeper insights into the properties of this double mutant will need to be addressed in future studies.To gain more insights into the CNGA3-R424W mutant channel function, a series of R424- and E306-targeted site-directed mutant constructs were examined and assessed using patch clamp technique as previously described , 24. Basin vitro. Representative photomicrographs of CNGA3-WT and CNGA3-R424W channels are shown in Fig 3C. The wild-type channels showed specific expression and trafficking to the plasma membrane and Golgi-like organelles with few cytoplasmic aggregates. In contrast, cells transfected with CNGA3-R424W demonstrated a significant increase in intracellular aggregates and a reduction of Golgi-like expression suggesting ER retention and altered protein folding . The averaged subcellular fluorescence data for all R424- and E306-mutant channels are summarized in Fig 3D. No significant variation in abundance of membrane expression was observed among the mutant constructs; however, cells transfected with R424W, R424E and E306D revealed reduction of Golgi-specific expression, while the highest percentage of cells with intracellular aggregates was calculated for R424W and E306D mutant channels. Notably, the double charge-reversal mutant, CNGA3-E306R-R424E, exhibited a fluorescence pattern similar to CNGA3-WT with a significant reduction in intracellular aggregates .Since documented ion channelopathies are often associated with misfolding and/or mistrafficking of the affected membrane proteins, the subcellular localization patterns of YFP-tagged R424- and E306-mutant channels were studied Fig 4A).The second novel canine disease model of ACHM2 is causally associated with Val644 deletion, a residue located in a coiled-coil domain of the CLZ in CNGA3. This 47-amino acid CLZ sequence is found in all CNG A-type subunits, but not in the CNGB1 or CNGB3. CLZ mediates subunit interactions among the A-type subunits through a trimeric coiled-coil structure during channel assembly in the 3A3:1B3 stoichiometry in the cone outer segment membranes [Fig 4B, human CNGA3-V630 residue  is in an a heptad position in the second turn of the coiled-coil domain. The trimeric CLZ domain coiled-coil structures of CNGA3-WT and CNGA3-V630del were constructed using the relaxed backbone of the hCNGA3-CLZ domain and their stability was assessed qualitatively by MD simulations. The wild-type CLZ structure appeared structurally stable, at least on the 325 ns timescale examined here . The WT coiled-coil core remained intact and tightly compact with the exception of fluctuations of the last a-position valine, likely due to solvent exposure and typical end-effects. The V630del mutant, however, exhibited rapid loss of the coiled-coil and tertiary structure over the course of only 100 ns, with unfolding beginning at the C-terminus within 30 ns, followed by the N-terminus within 50 ns. From the model structures alone, it was clear that the CLZ-V630 deletion mutant could significantly destabilize the coiled-coil assembly, unfolding the helical turns near the mutation at best and potentially leading to complete unfolding of the CLZ domain at worst. The average backbone root-mean-square deviation (RMSD) calculated over the entire trajectory was 9.06Å, with a maximum of 12Å when compared to the deletion model’s initial backbone configuration, a clear sign of structural instability , further highlighted by the wild- type’s 1.54Å average backbone RMSD over a similar simulation timescale. For the full-length video recordings of MD trajectories of hCNGA3-WT and V630del mutant CLZ trimeric structures see S4and .A high-resolution X-ray crystal structure (PDB ID: 3SWY) of the human CNGA3 (hCNGA3) CLZ domain (sequence identity with canine CNGA3 CLZ: 83%) provided a template for exploring the V644del in the canine CLZ using MD simulations. As depicted in Fig 5A). A series of patch clamp recordings of CNGA3-V644del channels revealed that approximately 40% of the mutant patches were unresponsive with no cGMP-activated currents, in contrast to 0% unresponsive patches from the CNGA3-WT-transfected cells. This reduction in the number of responsive patches might indicate a failure of proper subunit assembly due to disruption of the coiled-coil structure as illustrated in the simulation studies. However, 60% of patches showed cyclic nucleotide-activated currents, and these responsive mutant patches exhibited currents indistinguishable from the wild-type channels . The mean cGMP-activated currents for CNGA3-WT were -375pA , while those for CNGA3-V644del were -417pA  with the cAMP efficacy of ~20% for both CNGA3-WT and the responsive subgroup of V644del mutant channels.In addition to MD simulations, the effects of V644del were examined with regards to its subcellular localization and electrophysiological properties. In the previous studies on CNGA3 S1-domain mutant models an increase of intracellular aggregates was observed in all ACHM2-associated mutant channels examined and this predominant fluorescence distribution colocalized with ER-specific marker, GRP94 . SimilarFig 5C demonstrates that increasing the V644del component in the WT:V644del mixture resulted in a dramatic reduction of fluorescence associated with Golgi-like structures and a gradual increase in intracellular aggregates. However, regardless of the WT:V644del ratio, the plasma membrane fluorescence was comparable in all cells examined. We questioned whether cells with cGMP-activated currents might retain membrane fluorescence by expressing “rogue” subunits improperly assembled into a channel. This assumption would be consistent with the 40% of V644del mutant patches exhibiting no channel function.To explore the potential effects of CNGA3-V644del on the wild-type subunit folding and assembly, CNGA3-WT and CNGA3-V644del mutant constructs were co-transfected at varying ratios. The histogram in Fig 6). Both structures exhibited enhanced stability when compared to their all-deletion counterparts but also did show some signs of unfolding during the evolution of the trajectory . Predictably, the 2:1 (WT:V630del) structure, which retained two thirds of the hydrophobic core packing and avoided the electrostatic repulsion introduced in the all-deletion mutant, was more stable than its 1:2 (WT:V630del) counterpart with an average backbone RMSD of 2.99 Å versus 4.05 Å .To further examine structural stability of CNGA3-WT:V644del at different stoichiometric ratios, intermediate CLZ trimer models were evaluated using fully atomistic classical MD simulations. The stability of two hybrid structures composed of helical subunits from the hCNGA3-WT and V630del was qualitatively assessed in comparison to the established wild-type structure and the inherently unstable mutant model . Based on our previous work suggesting involvement of the S4-S5 loop and S6 in voltage-activated gating , we examAdditional insights regarding the molecular defects of the R424W mutation were derived from cellular localization studies. Although cells transfected with R424W mutant construct showed an increase in intracellular aggregates (from ~10% in the wild-type to ~40% in the R424W mutant), the expression profile of plasma membrane fluorescence did not vary significantly. These results differ from a previous canine ACHM missense mutation, CNGB3-D262N, located in the S2 transmembrane segment of CNGB3 . In thesIn CNG channels, the S2, S3 and S4 TM segments bear a large number of positive and negative charges. In contrast to the Shaker channels, the positive charges are not confined to the S4 segment, but are dispersed throughout the VSD sequence. Recently, we proposed that the charges of both signs form an electrostatic zipper that in effect laces the charge interactions locking the VSD of CNG channels into an immobile state . The pre0.5 suggesting coupling differences between the ligand binding and channel gating between these two ligands. The nucleotide activation properties of the E306R-R424E double mutant provides clear evidence for differences in the communication of ligand binding information to regions of the channel involved in gating, possibly with the S4-S5 loop and S6 salt bridge. Since cAMP and cGMP differ only at the C2 and C6 positions on the purine, the double mutant current profiles eventually might contribute to a deeper understanding of how ligand recognition and contacts within the binding domain are coupled to conductance properties.Although the exact mechanism of CNG channel gating involving the intracellular nucleotide-binding domain (NBD) remains to be determined, structural studies revealed that the NBDs form a tetrameric ring below the transmembrane pore that interacts via the C-linker with the S4-S5 linker –55. Strun caused by winding the canonical alpha-helix from 3.6 to 3.5 residues per turn. Every seventh residue then lies on the same face of the coil’s helices, giving rise to the familiar periodicity from which the coil derives its structure and stability [a and d positions  are tantamount to the coiled-coil. The core residues are often characterized by their tight “knob-into-holes” or “zipper like” packing making van der Waals contacts with neighboring core side-chains, and often encode both the orientation and oligomerization state of the folded structure [Coiled-coil structures, including the CLZ domains, are one of the most widely observed helical motifs among proteins , 58. Thetability . In suchtructure . Typicalb and e heptad positions now populate the core of the coiled-coil and exhibit significant electrostatic repulsion. Larger residues are unable to pack in the core without expansion of the coil’s radius, while “small” residues , do not provide the packing and van der Waals contact necessary to stabilize the core. Therefore, we hypothesize that the CNGA3-V644del mutant subunits in vivo do not form proper CNGA3 subunit assemblies essential for stable channel biogenesis and, as discussed below, an altered N- to C-terminal interaction may also contribute to the ACHM phenotype segregating in the Labrador retriever breed.We used a robust statistical protein design algorithm to identify the most energetically favorable configurations of the side-chain rotamers for both the wild-type CNGA3 and V644del mutant models , 42, 61.It should be noted, however, that the MD simulations were not performed to examine how individual CLZ domains with the deletion would assemble or behave. Instead, we initiated the simulations with the assembled coiled-coil structure of mutant CLZ domains, which led to disruption of the assembly. Moreover, the simulations were performed only with the CLZ domain structure, missing the VSD, pore-forming region, C-linker, and cyclic NBD. Thus, it is still to be determined how misassembly of the CLZ domain coiled-coil structure caused by this mutation would affect the whole channel structure and function. Nonetheless, the simulations suggest that significant interruption of the N-terminal region of the CLZ domain is a direct result of the V644del mutation.CNGA3 models represent either null phenotype (CNGA3-/- mouse model) [CNGA3 mutation classes   or premae model) , 72, and classes  and thusin vitro [An R410W mutation analogous to the canine R424W was found to be causatively associated with loss of cone function in human ACHM2 . The autin vitro . More rein vitro .d heptad position, only one turn downstream from V630. Using an experimental Xenopus laevis oocyte model, Dai and Varnum [in vitro. Although not investigated here, future studies on the co-expression of the CNGA3-V644del and CNGB3 subunit will aim to determine if aberrant channel regulation and changes in the PIP feedback, similar to those seen with the human CNGA3-L633P mutation, contribute to the loss of cone function observed in V644del-affected dogs in addition to the structural consequences of this mutation presented here.A pathogenic hCNGA3 missense mutation affecting a conserved hydrophobic residue within the CLZ domain was described by Goto-Omoto and colleagues . The repd Varnum  demonstrd Varnum . Consistin vitro findings indicate that the CLZ domain is involved in long-range intersubunit interactions important for channel regulation in the cone outer segments, and the V644del described here, similar to the L633P reported in patients, may produce a complex change in channel activation properties. More importantly, such cryptic regulatory features are not readily revealed while examined in the in vitro system. In vivo studies utilizing the canine CNGA3-R424W and-V644del models for translational purposes are currently under development.Taken together, our S1 FigSubjective vision testing shows complete loss of vision during daylight hours, but normal visual performance under dark, twilight, artificial, and low-level fluorescent light conditions. Movie illustrates visual behavior in the R424W-affected dog that is similar to what was observed in dogs homozygous for V644del mutations.(MOV)Click here for additional data file.S2 FigS3 Table) for additional details.Schematic representation of human CNGA3 molecule deduced from multiple sequence alignments. Canine R424 residue affected in German shepherd with achromatopsia is located at the terminal cytoplasmic end of S6 (arrow) and corresponds to R410 of the human CNGA3. The V644 mutated in Labrador retriever is positioned within CLZ domain and corresponds to hCNGA3-V630 (arrow). All known human disease-associated mutations are denoted and color-coded. Mutations in homo-, hetero- as well as both, homo- and heterozygous states are scattered throughout all domains with mutational hotspot located in the C-terminal part of the protein. See also (TIFF)Click here for additional data file.S3 FigMutlalin v.5.4.1.Orthologs of human (NCBI# NP_001289.1) and canine (NCBI# NP_001288041.1) CNGA3 demonstrate >82% of amino acid sequence identity with the highest degree of conservation in the C-terminal half, a region affected by the two novel canine mutations (arrows). Evolutionarily conserved residues are highlighted in red. h = human, c = canine; Sequence alignment was prepared using (TIFF)Click here for additional data file.S4 Figa-position [All simulations were run with a temperature of 310 K fixed using Langevin dynamics and a pressure of 1.0 atmosphere using the Nose-Hoover Langevin piston pressure control. The wild-type CLZ structure appeared stable during the entire 325 ns trajectory leading to impressive long timescale stability and an average backbone root-mean-squared deviation (RMSD) of 1.54 Å. The WT coiled-coil core remained intact and tightly compact with the exception of fluctuations of the last position , likely (MOV)Click here for additional data file.S5 FigAll simulations were run with a temperature of 310 K fixed using Langevin dynamics and a pressure of 1.0 atmosphere using the Nose-Hoover Langevin piston pressure control. The V630del mutant exhibited rapid loss of the coiled-coil tertiary structure over the course of about 100 ns, with unfolding beginning at the C-terminus within 30 ns, followed by the N-terminus within 50 ns. After 100 ns, the tertiary structure is mostly lost, leaving an amalgam of helices and an average backbone RMSD of 9.09Å.(MOV)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


CTNNB1, APC or other pathway components are rare in this tumor type, suggesting alternative mechanisms for Wnt/β-catenin activation. Recent studies have implicated the (pro)renin receptor ((P)RR) is related to the Wnt/β-catenin signaling pathway. We therefore investigated the possible role of (P)RR in pancreatic carcinogenesis. Plasma s(P)RR levels were significantly (P < 0.0001) higher in patients with PDAC than in healthy matched controls. We also identified aberrant expression of (P)RR in premalignant PanIN and PDAC lesions and all the PDAC cell lines examined. Inhibiting (P)RR with an siRNA attenuated activation of Wnt/β-catenin signaling pathway and reduced the proliferative ability of PDAC cells in vitro and the growth of engrafted tumors in vivo. Loss of (P)RR induced apoptosis of human PDAC cells. This is the first demonstration that (P)RR may be profoundly involved in ductal tumorigenesis in the pancreas.Although Wnt/β-catenin signaling is known to be aberrantly activated in PDAC, mutations of  Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with few effective therapies and a very poor prognosis113CCND112In vitro studies also have shown that inhibition of Wnt/β-catenin signaling reduces cell proliferation and increases apoptosis in cultured PDAC cells1115161718The Wnt/β-catenin signaling pathway plays a critical role in multiple developmental events during embryogenesis, and is implicated in adult tissue homeostasis68910et al.ATP6ap2/(P)RR, which is located on the X chromosome (Xp11.4) in humans21+-proton adenosine triphosphatase (V-ATPase)23et al. also showed that (P)RR amplifies activation of the renin-angiotensin system targeted for renal and cardiovascular disorders. However, activation of (P)RR was shown to stimulate several intracellular signaling pathways independent of the renin-angiotensin system. Recent studies in XenopusDrosophila26The (pro)renin receptor ((P)RR) was discovered and successfully cloned by Nguyen In the present study, we aimed to elucidate the role of (P)RR in the pathogenesis of human PDAC focusing on PDAC-associated (P)RR expression and functions through the Wnt/β-catenin signaling pathway. We could demonstrate for the first time that (P)RR is profoundly involved in genesis of PDAC.P < 0.0001) higher in patients with PDAC (14.67 ± 3.64 ng/mL) than in healthy controls (8.45 ± 1.37 ng/mL).s(P)RR is generated intracellularly through a furin cleavage mechanism and secretedImmunohistochemical analysis showed significant (P)RR expression in the cytoplasm of neoplastic epithelial cells in 21 of 22 samples of PDAC tissues obtained from patients who underwent resection . SupplemFull-length (P)RR expression in cell lysates was higher in all six PDAC cell lines than in HPDE cells .The consWe aimed to investigate whether (P)RR is a key factor for the canonical (β-catenin-dependent) Wnt signaling pathway in human PDAC cells. We examined the effect of Wnt3a, which is essentially involved in cell survival2913Treatment with (P)RR siRNA decreased in (P)RR expression in PK-1 cells . Wnt3a-iImmunohistochemistry revealed that β-catenin was overexpressed in almost all human PDAC tissues . The immCell proliferative ability was evaluated by Water-soluble tetrazolium salt (WST-1) assays in three human PDAC cell lines . Treatmein vivo, we injected PANC-1 cells expressing a scrambled siRNA or (P)RR siRNA. The 5 × 105 cells were injected subcutaneously into the upper right flanks and tumor growth was measured with an electric caliper . Harvested xenografts were identical histologically to the PDAC  (To examine the effects of (P)RR silencing on pancreatic tumor growth the PDAC . The mea 0.0001) . We succ 0.0001) . These rXenopus embryosTo investigate whether inhibition of (P)RR reduces Wnt3a-induced PDAC cell proliferation through inactivation of β-catenin, we examined the effects of co-transfection of (P)RR siRNA and a plasmid harboring wild-type β-catenin or constitutive activation of mutant β-catenin on Wnt3a-induced cell proliferative ability. Consistent with the above-mentioned data , treatmeTo investigate the mechanism by which (P)RR siRNA suppresses PDAC cell proliferation, DNA content was measured by PI in PK-1 cells, with detection of cells undergoing late-stage apoptosisCell apoptosis was also confirmed by measuring caspase-3 activity. (P)RR siRNA treatment of cells followed by Wnt3a stimulation significantly increased caspase-3 activity in all three human PDAC cells . These dThe present study revealed new critical aspects that strongly indicate a potential role of (P)RR in Wnt/β-catenin signaling-dependent genesis of PDAC. To our knowledge, this is the first demonstration that (P)RR might be profoundly involved in PDAC development. First, aberrant expression of (P)RR in premalignant PanIN and PDAC lesions as well as elevated plasma levels of s(P)RR was observed in patients with PDAC. Second, cultured human PDAC cell lines revealed substantial increases in full-length (P)RR expression and secretion of s(P)RR. Third, (P)RR was found to be crucial for Wnt/β-catenin-dependent proliferation of PDAC cells. Finally, loss of (P)RR triggered apoptosis of human PDAC cells through the activation of caspase-3.CTNNB1, APC and LRP5, has been noted in several cancers1115161718SLIT2 and ROBO2, which enhance β-catenin complex formation, were present in only 5% of cases14in vivo. Furthermore, transfection of a plasmid causing constitutive activation of β-catenin significantly attenuated siRNA (P)RR-induced reduction of cell proliferative ability, although this proved to be temporary. These data indicate that one mechanism of activation of the Wnt/β-catenin signaling pathway in PDAC is by virtue of inappropriate activation of (P)RR in the absence of mutations in genes encoding regulatory proteins of the Wnt/β-catenin signaling cascade.Inappropriate activation of the Wnt/β-catenin-dependent signaling pathway due to mutations in genes encoding regulatory proteins of this cascade, including CCND1, which increases transition through G0/G1Accumulation of β-catenin in the nucleus promotes cell proliferation through activation of Wnt target genes, such as In the present study, transfection of (P)RR siRNA triggered apoptosis, resulting in decrease of the proliferative ability of human PDAC cells. Studies in tissue-specific (P)RR knock-out mice showed that specific deletion of (P)RR in cardiomyocytes or glomerular podocytes resulted in death a few weeks after birth2435KRAS mutations and differential expression of other genes15et al. reported aberrant nuclear localization of β-catenin expression in high-grade PanIN (PanIN-2 and -3) and PDAC lesionset al.DKK and SFRP genes, which encode inhibitors of the receptor-ligand binding at the top of the Wnt signaling cascade, was established as another mechanism known to driving Wnt signaling in cancers including PDACPanIN is a histologically well-defined precursor to invasive PDACThe present study provided another novel insight into PDAC. Although data supporting the essential role of the Wnt/β-catenin signaling pathway in pancreatic carcinogenesis are accumulating as stated above, specific biomarkers of Wnt/β-catenin activation are lacking. One of the major problems is that no components of the Wnt/β-catenin signaling pathway have been detected in serum or plasma to dateThe institutional committee for ethics of Kagawa University and the National Cancer Center approved our studies using human plasma and tissue samples and informed consent was obtained from all subjects before the study was started. Eligible subjects were defined in advance as 20 patients with PDAC who were admitted to the National Cancer Center Hospital, Tokyo, Japan, between July 2012 and August 2012. The plasma of healthy volunteer examinees was collected at the Research Center for Cancer Prevention and Screening, a branch of the National Cancer Center, during the same period (between July 2012 and August 2012). To prevent sampling biases, 20 frequency-matched volunteer examinees in eight age categories  and matched sex were chosen as potential healthy controls. No cancer was detected by cancer screening at the institution in any of these 20 volunteer examinees.Patients with PDAC and healthy volunteer examinees were scheduled for blood collection before any procedures on the first day of admission or screening, respectively. Fasting venous blood was drawn into a vacutainer tube with EDTA-2Na. All 20 patients with PDAC and all 20 healthy volunteer examinees had fasted. The blood samples were centrifuged to obtain blood plasma and these specimens were preserved at −80°C until analysis.Human s(P)RR assays were performed in duplicate with use of a commercial kit . Recombinant human (P)RR was employed as the standard. The human (P)RR standard  and plasma (1:9 dilution in ELISA buffer) samples (100 μL/well) were individually loaded into wells of 96-well plates, which were incubated at 4°C overnight. Then, the plates were washed a total of five times with washing buffer (PBS containing 0.05% Tween 20). After incubation with horseradish peroxidase-labeled anti-human renin receptor antibodies  at 4°C for 1 h, the plates were washed a total of five times with washing buffer. They were incubated with 3,3′,5,5′-tetramethylbenzidine solution (100 μL/well) under light-protected conditions at room temperature for 45 min. The reaction was stopped by the addition of sulfuric acid  and absorbance was measured at 450 nm with a plate reader.Immunohistochemistry of (P)RR was performed using surgical specimens of PDAC. Briefly, paraffin sections were deparaffinized and incubated with methanol containing 0.3% hydrogen peroxide for 15 min, then 10% normal goat serum  was added to the sections to block non-specific staining. The sections were incubated with an anti-human (P)RR rabbit polyclonal antibody402/95% air in a humidified incubator. Serum-free conditioned medium was obtained by starving each cell line for 24 h. Conditioned medium was concentrated with an Amicon Ultra Device . Cells were lysed with 100 μL of ice-cold lysis buffer, pH 7.4 , 1% Triton X-100, protease inhibitors , and phosphatase inhibitors  and the supernatant was obtained after centrifugation for 10 min at 12,500 rpm.Dr. T. Furukawa  kindly supplied BxPC-3, PCI-35 and PK-8 cells for us. PANC-1 and MIAPaCa-2 cells were purchased directly from the American Type Culture Collection . PK-1 cell was obtained from Tohoku University. BxPC-3, PCI-35, PK-8, PK-1, MIAPaCa-2 and PANC-1 were grown in RPMI-1640 media  supplemented with 10% fetal bovine serum , penicillin (50 U/mL) and streptomycin . HPDE cells were cultured in Hu-Media KG2 containing 10 mg/mL insulin, 0.1 μg/mL human EGF, 0.5 mg/mL hydrocortisone hemisuccinate, 50 mg/mL gentamycin, 50 μg/mL amphotericin B and 0.4% V/V BPE . According to the cell culture method recommended by ATCC, hTERT-HPNE (CRL-4023) were grown in one volume of Medium M3 Base  and three volumes of DMEM without glucose , 5% FBS, 5.5 mM D-glucose, 10 ng/mL human recombinant EGF, and 750 ng/mL puromycin. These cell lines were maintained at 37°C under 5% COATP6AP2) siRNA and a plasmid carrying constitutively active aa 1–90-deleted β-catenin tagged with Myc or a full-length β-catenin cDNA before stimulation by Wnt3a. Stealth Select RNAi , a predesigned (P)RR siRNA, was synthesized by Life Technologies. Plasmid carrying constitutively active β-catenin was obtained from Addgene, Cambridge, MA Cells were treated with 150 ng/mL recombinant human Wnt3a , and harvested for analyses. For transient gene transfection, BxPC-3, PANC-1 and PK-1 cells were serum starved for 24 h and then transfected with (P)RR RR siRNA), in 200 μL of PBS were injected subcutaneously into the upper right flanks using a 26G needle (n = 7 per group). Size of local tumors at the implanting site was measured with an electric caliper. Volumes of tumors were calculated as follows: tumor volume (mm3) = length × (width)2 × 0.5. The tumor tissue of each mouse was fixed, embedded, sectioned serially, stained with hematoxylin and eosin, and examined under a microscope.Experimental protocols and animal care were performed according to the guidelines for the care and use of animals established by Kagawa University. The Animal Experimentation Ethics Committee at Kagawa University approved the experiments. Five-week-old male BALB/c nude mice (nu+/nu+) purchased from CREA, Japan were housed under pathogen-free conditions. Mice were anesthetized with sevoflurane. PANC-1 cells, 5 × 104 cells were seeded per well of 6-well plates. After siRNA-transfected cells were stimulated with Wnt3a for 24 or 48 h, WST-1 assays were performed according to the manufacturer's protocol  to determine proliferative ability. One hundred microliters of WST-1 reagent was added to the cell culture medium in each well. After incubation for 2 h, the absorbency of the samples was measured with a microplate reader at a wavelength of 450 nm. Direct cell counting was performed to count the cells at 24, 48 and 72 h after Wnt3a stimulation. Then, the cells were treated with 0.25% Trypsin-EDTA , and then resuspended in PBS after centrifugation. The cell suspensions were stained with 0.5% trypan blue stain solution  to distinguish living cells from dead cells, and 10 μL of each cell suspension was loaded into the sample injection area within C-Chip . Living cell numbers per mL were calculated.Prior to transfection, 5 × 10After 48 h of Wnt3a stimulation, siRNA-transfected cells were resuspended in 0.5 mL of ice-cold 70% ethanol after centrifugation and kept at −20°C for 24 h. Following washing in PBS, cells were resuspended in 10 mg/mL RNase A  in PBS for 30 min at 37°C and stained with 1 mg/mL PI  for 30 min at 37°C. After washing with PBS, cells were resuspended in fresh PBS, and data were acquired using a DNA flow cytometer. After treating the siRNA-transfected cells with Wnt3a, caspase-3 assays were performed according to the manufacturer's protocol  to detect caspase-3 activity. Cell lysates, 2× reaction buffer and caspase-3 substrate were added to each well and incubated for 2 h at 37°C, then the absorbency of the samples was measured with a microplate reader at a wavelength of 405 nm. The data were calculated based on the standard curve and normalized to the total protein concentration of the cell extracts.t test for s(P)RR concentration in human and nude mice and tumor volume in vivo. We used one-way ANOVA with Scheffe's post hoc test for analyzing protein expression, Wnt3a-stimulated cell proliferative ability measured by WST-1 assays. We also performed two-way ANOVA to detect the effects of treatment by siRNA and stimulation period by Wnt3a in number of PK-1 cells. P < 0.05 was considered significant. All the statistical analyses were performed using IBM SPSS Statistics 20  software.Results are expressed as mean ± SEM, with the exception of s(P)RR concentrations (mean ± SD). Continuous variables were compared using Student's The author(s) have made the following declarations regarding their contributions: Y.Sh., G.N., S.Y. and A.N. designed this study. Y.Sh., T.F. and S.Y. acquired the data. Y.Sh., T.F., G.N., D.N., S.Y. and A.N. performed the analysis and interpretation of data. Y.Sh., G.N., S.Y. and A.N. wrote the paper. A.I. and H.K. revised the manuscript. T.H., K.T., K.K., M.K., T.M. and Y.Su. contributed reagents/materials/analytical tools.Supplementary information


Genomic features of whole genome sequences emerging from various sequencing and annotation projects are represented and stored in several formats. Amongst these formats, the GFF  has emerged as a widely accepted, portable and successfully used flat file format for genome annotation storage. With an increasing interest in genome annotation projects and secondary and meta-analysis, there is a need for efficient tools to extract sequences of interests from GFF files.We have developed GFF-Ex to automate feature-based extraction of sequences from a GFF file. In addition to automated sequence extraction of the features described within a feature file, GFF-Ex also assigns boundaries for the features , which are not explicitly specified in the GFF format, and exports the corresponding primary sequence information into predefined feature specific output files. GFF-Ex package consists of several UNIX Shell and PERL scripts.http://bioinfo.icgeb.res.in/gff.Compared to other available GFF parsers, GFF-Ex is a simpler tool, which permits sequence retrieval based on additional inferred features. GFF-Ex can also be integrated with any genome annotation or analysis pipeline. GFF-Ex is freely available at  GFF represents genome feature data in a tab-delimited table, single feature per line, making it ideal for use with various data analysis pipelines. Currently, the scientific communities are becoming more and more reliant on these information for secondary analysis and laboratory experiments too, which has resulted in a growing need for efficient tools to extract desirable sequences based on annotations[Technical advancements in the field of high-throughput DNA sequencing,2, the eotations. This mohttp://bioinfo.icgeb.res.in/gff/) is a collection of various modules developed using Shell and PERL string parsing scripts. The current version of GFF-Ex  equipped with an installation file, copies program scripts to the user specified installation directory. The package also includes few example files and a README file to help users install and execute GFF-Ex. GFF-Ex is robust and adaptable towards future integrations of the GFF-Ex plug-ins. The software enables GFF-Ex users to pass input arguments and parameters as a single command statement, along with invoking GFF-Ex. This helps GFF-Ex to switch to the specified modules/plug-ins without taking much of kernel time. For an input file size of about 500 MB, the CPU time between invocation and termination of GFF-Ex is 2 m2.305 s. The current GFF-Ex architecture is suitable for transparent integration with various sequencing data analysis pipelines, meeting the requirements of researchers handling large datasets. To evaluate the GFF-Ex performance with other publicly available stand-alone parsers, we have performed comparison with functions of few parsers performing similar tasks. We compared GFF-Ex with other publicly available stand-alone gff parsers (accessed on 27-02-2013), namely, Sanger Institute GFF Perl modules[GFF-Ex . “COLx” plugin exports feature based coordinates from specified columns and translates them into sequences using GFF-Ex. Another plug-in “FETx” (http://bioinfo.icgeb.res.in/gff/gffdownloads/FETx.tar.gz) is a plugin for feature extraction, which extracts sequences on the basis of user-specified custom features declared in the input GFF annotation file. Keeping in mind the growing rates of genomic data and GFF files, the next version of GFF-Ex (under development) shall be made compatible with high performance clustered nodes.GFF-Ex is freely available from With the rapid advancements and automation of sequencing projects, the interests of scientific groups involve studies and experimental designs that make use of sequencing and GFF annotations. Handling large datasets to parse desirable information, especially sequence files, have always been an important and complex startup step in such studies. GFF-Ex is a reliable GFF parsing tool, which integrates with various applications and pipelines requiring large volumes of sequence extraction from GFF files, based on customized or GFF defined annotations.Project Name: GFF-Ex (A genome feature extraction package)Project home page:http://bioinfo.icgeb.res.in/gff/Operating system(s): UNIXProgramming Language: PERL and UNIX ShellLicense: NAAny restrictions to use by non-academics: None.The authors declare that they have no competing interests.DG and AR conceptualized development of the package. DG guided AR to develop the package; AR and DG tested the package. DG and AR wrote and approve the manuscript.


Background. The safety of endoscopy after an acute coronary syndrome (ACS) is poorly characterized. We thus performed a systematic review assessing the safety of endoscopy following ACS. Methods. Searches in EMBASE, Medline, and Web of Science identified articles for inclusion. Data abstraction was completed by two independent reviewers. Results. Fourteen retrospective studies yielded 1178 patients  having suffered an ACS before endoscopy. Patients underwent 1188 endoscopies primarily to investigate suspected gastrointestinal bleeding (81.2%). Overall, 810 EGDs (68.2%), 191 colonoscopies (16.1%), 100 sigmoidoscopies (8.4%), 64 PEGs (5.4%), and 22 ERCPs (1.9%) were performed 9.0 ± 5.2 days after ACS, showing principally ulcer disease  and normal findings . Overall, 108 peri- and postprocedural complications occurred , with hypotension , arrhythmias , and repeat ACS  as the most frequent. All-cause mortality was 8.1% (95% CI 6.3–10.4%), with 4 deaths attributed to endoscopy . Conclusion. A significant proportion of possibly endoscopy-related negative outcomes occur following ACS. Further studies are required to better characterize indications, patient selection, and appropriate timing of endoscopy in this cohort.  Performing endoscopic procedures in the setting of an acute coronary syndrome (ACS) can prove challenging. These patients are at increased risk of arrhythmias, heart failure, further ischemic events, and death , 2. The As a result of these concerns, physicians may be hesitant to perform endoscopy following an ACS. Currently, there exists no consensus regarding the optimal timing of an urgent endoscopy following an ACS. We present a systematic review of the literature on the safety, efficacy, and complications of luminal endoscopy in this setting.A comprehensive computerized medical literature search was performed using MEDLINE, EMBASE, Cochrane library, and the ISI Web of Knowledge from 1990 to April 2014. A highly sensitive search strategy was used to identify all observational studies  with a combination of controlled vocabulary (MeSH) and text words related to (1) upper or lower gastrointestinal endoscopy or ERCP and (2) myocardial infarction or acute coronary syndrome (in the appendix).All adult human studies in English were included as well as published abstracts from scientific meetings only if the data were not duplicated in subsequent publications. Recursive searches and cross-referencing were also carried out using a “similar articles” function; hand searches of articles were identified after an initial search.Two authors independently reviewed all abstracts for potential inclusion. Relevant abstracts were then further reviewed based on manuscript content, with a third independent reviewer resolving any disagreements.Case reports were excluded, and case series with a sample size of <5 were also excluded. Papers published by the same author(s) were screened for duplication of results. In order to be included, publications had to have assessed patients undergoing endoscopy within 60 days of suffering an ACS. ACS was defined as unstable angina, non-ST elevation myocardial infarction (NSTEMI), ST elevation myocardial infarction (STEMI), or ACS not specified. All forms of endoscopic diagnostic or therapeutic procedures involving the gastrointestinal tract were considered for inclusion into the study.Information from all relevant papers, including demographic information, type of endoscopy, indications, complication rates, and ACS subtypes, was compiled. Complications were defined based on initial descriptions provided in the papers being reviewed. Complications were subsequently classified according to general categories. Two authors independently classified complication events as major or minor. For every study, we determined weighted data for timing of endoscopy and rates of endoscopic complications and all-cause mortality. Descriptive statistics included categorical variables expressed as proportions and 95% confidence intervals and continuous variables as means ± standard deviation or medians and ranges. All statistical analyses were performed using SAS 9.2, SAS Institute Inc., Cary, NC, USA.Initial search of the databases yielded 1343 citations. After review, 1329 were excluded for the following reasons: incorrect outcome, non-English language article, and incorrect population , 191 colonoscopies (16.1%), 100 sigmoidoscopies (8.4%), 64 PEGs (5.4%), and 22 ERCPs (1.9%). The primary indications for endoscopy were unspecified symptoms of gastrointestinal (GI) bleeding (28.8%), occult blood loss/anemia (20.3%), hematemesis (14.2%), melena (12.7%), hematochezia (5.3%), and other indications (18.8%) . The aveOutcomes following initial endoscopy included 10 patients requiring repeat endoscopy . Following endoscopy, 3.6% of patients (95% CI 2.2–4.8%) required gastrointestinal surgery or angiography for ongoing management. Including deaths attributable to endoscopy, 108 complications occurred across all endoscopic modalities . Of all complications, 72.4% were classified as major , accounting for a major complication rate of 6.4% (95% CI 5.2–8.0%). The complication rate for the different endoscopic modalities was 11.5% (95% CI 9.2–14.4%) for EGD, 9.0% (95% CI 4.8–16.2%) for colonoscopy, 2.5% (95% CI 0.7–8.7%) for flexible sigmoidoscopy, 10.3% (95% CI 3.6–26.4%) for PEG, and 14.3% (95% CI 5.0–34.6%) for ERCP . When thThere is a paucity of data surrounding the safety of endoscopy following ACS. This stems from the unpredictable nature, varying indication, and low incidence of endoscopy after ACS. This systematic review was performed to summarize existing information to guide clinicians in future management of their patients. The primary outcome of this study revealed an overall complication rate for all endoscopic procedures to be 9.1% (95% CI 7.6–10.9%), suggesting that this group of patients is prone to adverse events related to endoscopy. Even in the absence of recent ACS, endoscopy has been documented to provoke silent ischemic changes in patients with underlying coronary artery disease –19. WhenThe close proximity of endoscopy to initial ACS (mean 9.0 ± 5.2 days) raises concerns about increased cardiopulmonary complications. The important issue of elapsed time between the ACS and endoscopy with regard to incidence of complications was only assessed in one study. Indeed, Spier et al. noted an inverse relationship between endoscopic complications from timing of ACS; in 135 patients, the only 2 complications occurred with endoscopy performed the same day as ACS, in contrast to no complications in procedures performed 24 hours or more after an ACS .Sharma et al. recently looked at all GI endoscopies entered into the Clinical Outcomes Research Initiative database over a 5-year period to identify factors predictive of endoscopic complications . ConscioThe results presented here are in contrast to previous reports of complications encountered during elective endoscopy. Existing data have shown complications of upper GI endoscopy ranging from 1 in 200 to 1 in 10,000 –25, withn = 64, ERCP n = 22). Despite this, the high rate of complications seen with ERCP can be explained by its technical difficulty and the concomitant disease processes, largely cholangitis or choledocholithiasis  and 9.0% (95% CI 4.8–16.2%), for EGD and colonoscopy, respectively; because of the size of the sampling, these values were felt to be more generalizable than the observed complication rates for PEG  and ERCP  that were based on smaller number of procedures  of patients received some form of sedation for the procedure. In a prospective study including 17,999 endoscopic procedures, 96% of patients received propofol as the primary sedative with an observed sedation-related complication rate of 4.51% . This isIn this review, the majority of patients underwent endoscopy to evaluate clinical or biochemical signs of GI bleeding (81.2%). Patients receiving standard medical therapy for ACS, namely, dual antiplatelet therapy and therapeutic low molecular weight heparin, are at risk of clinically significant GI bleeding, with a previously reported incidence of 2.7% . Among aPreventative measures should be employed to decrease the need for endoscopy, including the appropriate use of gastroprotective agents for those on antiplatelet agents. The routine use proton pump inhibitors (PPI) are preferred over histamine-2 receptor antagonists (H2RA) . FurtherMethodological limitations include the poor quality of the data, precluding the ability to perform a formal meta-analysis. In addition, adjudication of outcomes is not feasible given the nature of the data, limiting attribution of specific complications directly to endoscopy. The data collected are heterogeneous; the changing definition of ACS over time and the variation in the definition of complication across the articles force one to interpret the data cautiously. This highlights, however, the need for prospective data on the safety of endoscopy after ACS. Currently, these data are the best that exist regarding this topic. Finally, one author was responsible for 7 of the 19 publications included in our review, raising the possibility of selection bias. As they were felt to be of higher quality, we kept these studies, but steps were taken to ensure that we excluded duplicate data among studies.This systematic review nonetheless clearly quantifies an increased rate of negative outcomes in ACS patients undergoing digestive endoscopy. The clinical management of ACS patients must therefore include minimizing risks such as GI bleeding. While not validated in this systematic review, the temporal relationship between the ACS event and the procedure may contribute to exacerbating cardiopulmonary complications. The risks and benefits must therefore be carefully considered on a case-by-case basis, limiting urgent endoscopy to clinical scenarios where a therapeutic benefit is likely to alter the patient course.


During isometric voluntary contractions of a healthy human muscle, motoneurons (MNs) fire usually with low mean rates, rarely exceeding 25/s  and fast  human muscle (Exp. 1). Within the motor unit potential trains, recorded in these experiments during steady-state voluntary isometric muscle contractions, we found also doublets and triplets  from another study, designed specifically for the investigation of double discharges  were calculated after grouping the MISIs in 10 ms bins with 5 ms overlap. After rejection of points calculated from insufficient number of CD values, the mean data were fit with two linear regression lines and the intersection of these lines used to determine transition ISI, being an estimate of AHP duration.It is commonly accepted that each motor unit discharge is equivalent to the discharge of its MN. Motoneuron AHP duration was estimated from single motor unit potential trains by the method based on the observation of Person and Kudina  that theThe AHP duration was estimated for 80 MNs from BB, 90 MNs from SOL, and 28 MNs from TB. From these, MNs firing doublets constituted 5% for BB (4 MNs), 9% for SOL (8 MNs), and 64% for TB (18 MNs). Two MNs recorded in Experiment 1, one from BB and one from SOL, fired also triplets that were much scarcer than doublets: for BB MN 11 triplets vs. 1592 doublets, for SOL MN 6 triplets vs. 342 doublets. Figure Figure At the first sight, TB data do not seem to confirm this observation. The difference in mean values of transition ISIs between MNs with and without doublets is negligible: 80.40 vs. 80.00 ms and many of MNs from the former group present transition ISIs longer that those from the latter. However, there are striking differences between TB and BB in doublet incidence and transition ISI ranges. Since both TB and BB are fast muscles, it could be presumed that the AHP duration ranges of their MNs should be comparable. Moreover, it should be noted that the contraction strengths in the Exp. 2 were lower than in the Exp. 1 . Thus, the MNs recorded from TB could be expected to have the lower thresholds than those from BB. Consequently, according to the commonly known rules of the orderly recruitment  are in favor of this presumption. Summing up, the results from all three muscles seem to be in line with the earlier observation of Kernell .Figure In contrast to the doublet ISI, the triplet ISI measured for SOL (blue filled square) was substantially longer than that measured for BB (red filled square). This observation may suggest that its firing might not be caused by DD, but by the later hump in the time course of AHP conductance described by Baldissera and colleagues  underlying triplet firing. Nevertheless the specificity of the opinion article encouraged us to formulate the above presented speculative hypothesis about the unusual, intriguing phenomenon of triplet firing.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.


CTNNB1 (encoding β-catenin protein) or other antagonists targeting Wnt/β-catenin signaling are low in HCC patients, suggesting that genetic mutations are not the major factor driving abnormal β-catenin activities in HCC. Emerging evidence has demonstrated that obesity-induced metabolic pathways can deregulate chromatin modifiers such as histone deacetylase 8 to trigger undesired global epigenetic changes, thereby modifying gene expression program which contributes to oncogenic signaling. This review focuses on the aberrant epigenetic activation of Wnt/β-catenin in the development of NAFLD-associated HCC. A deeper understanding of the molecular mechanisms underlying such deregulation may shed light on the identification of novel druggable epigenetic targets for the prevention and/or treatment of HCC in obese and diabetic patients.Non-alcoholic fatty liver disease (NAFLD), characterized by fat accumulation in liver, is closely associated with central obesity, over-nutrition and other features of metabolic syndrome, which elevate the risk of developing hepatocellular carcinoma (HCC). The Wnt/β-catenin signaling pathway plays a significant role in the physiology and pathology of liver. Up to half of HCC patients have activation of Wnt/β-catenin signaling. However, the mutation frequencies of  Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world and the second leading cause of cancer-related mortality . In the Epigenetics refers to chromatin modifications exclusive of alterations in nucleotide sequences. It mainly includes DNA methylation, histone modifications and microRNAs (miRNAs), which can, respectively, activate or silence gene transcription , leading to diverse functional effects. Indeed, complex roles have been reported for a variety of chromatin modifiers, including but not limited to DNA methyltransferases (DNMTs), histone methyltransferases (HMTs), histone acetyltransferases (HATs) and histone deacetylases (HDACs) . It is nALOX25 and IRF5 are of interest because they are consistently hypermethylated in obesity and HCC samples [Although genomic study of NAFLD has been an established area of research for some time, the relevance of epigenomic factors such as DNA methylation in NAFLD development has only become apparent in recent years . For ins samples ,19.Recently, altered expression and activities of certain HDACs/HATs have been linked to deregulated histone acetylation and gene expression in NAFLD, leading to abnormal metabolism and cellular transformation. Several HDACs have been shown to be critical modulators in the pathophysiology of NAFLD. Among them, HDAC3 is the most well-studied epigenetic modifier that can govern both circadian metabolic activity and hepatic lipid homeostasis ,22,23. AMiRNAs have emerged as key regulators of metabolism . The metThe canonical or Wnt/β-catenin pathway is the best characterized Wnt pathway. Up to half of HCC patients have activation of Wnt/β-catenin signaling pathway ,32,33. WAPC, AXIN1/2 and CTNNB1 (encoding β-catenin) are common in HCC. These tumor-causative mutations lead to inappropriate stabilization of β-catenin, which persistently activates target genes associated with cell proliferation and transformation, such as cell cycle drivers cyclin D1 (CCND1) and c-Myc [Myc gene, which is a known Wnt target gene in cancer cells [An increasing body of evidence indicates that regulators controlling Wnt/β-catenin signaling are frequently dysregulated in human cancers owing to genetic and epigenetic defects . Mutationd c-Myc ,39. Altend c-Myc . The groer cells .CTNNB1, APC and AXIN gene mutations are classic defects that trigger chronic Wnt signaling in cancers [CTNNB1 mutation rate was 12%–16%, AXIN mutation rate was 8%–15%, and no mutation of APC was reported [Activation of Wnt/β-catenin signaling can drive the expression of specific oncogenes in various human cancers.  cancers . Howeverreported ,44. Thisreported . Indeed,reported .SFRP1, SFRP2, SFRP4 and SFRP5, correlates with constitutive activation of canonical Wnt/β-catenin signaling in liver cancers [SFRP5 was inversely correlated with its expression in NAFLD patients [The secreted frizzled-related protein (SFRP) family is a class of extracellular Wnt inhibitors that act at cell membrane to prevent Wnt-mediated induction of β-catenin . Promote cancers ,49. Amon cancers , and thepatients . These dSOX17 through promoter methylation is a frequent event in human cancers, and it contributes to the aberrant activation of Wnt/β-catenin signaling [Sox17 were more susceptible to high fat diet-induced hyperglycemia and diabetes [SOX17 via promoter methylation may promote Wnt activity and insulin secretion and thereby accelerate the progression from NAFLD to HCC.SOX17 is a nuclear protein that directly interacts with TCF/LEF to inhibit the transcription of Wnt signaling target genes. Epigenetic silencing of ignaling . Moreovediabetes . Functiodiabetes . Taken tDACT2 promoter methylation was inversely correlated with DACT2 expression, which could be restored by 5-aza-2′-deoxycytidine in HCC cell lines. Of clinical significance, reduced DACT2 expression was significantly related to promoter hypermethylation in 28 of 62 (45.16%) HCC patients, and the expression of DACT2 was inversely related to β-catenin expression in liver cancers [DACT2 promoter was reported in advanced NAFLD individuals compared with mild NAFLD patients [The cytosolic Wnt antagonists, such as members of the DACT gene family (DACT2 and DACT3), can antagonize Dvl and are central components of Wnt signaling . Zhang a cancers . Moreovepatients , which iDACT2, silencing of DACT3 in cancer cells is mediated by a bivalent histone modification that contains both repressive histone H3 lysine 27 trimethylation (H3K27me3) and activating H3K4me3, and such DACT3 ablation is not associated with promoter methylation [Distinct from hylation . Of notehylation , which iBased on previous studies, histone modification plays a key role in controlling the expression of Wnt inhibitors in HCC cells. Using chromatin immunoprecipitation microarray (ChIP-Chip) analysis, Cheng and colleagues discovered a panel of Wnt pathway inhibitors whose promoters were concordantly occupied by enhancer of zeste homolog 2 (EZH2) and H3K27me3 in HCC cells . EZH2 isAdditionally, Ezh2 was reported to directly suppress Wnt genes to facilitate adipogenesis in mice. The adipogenesis defects in cells with enzymatically inactive Ezh2 can be rescued by expression of adipogenic transcription factors PPARγ and C/EBPα, or inhibitors of Wnt/β-catenin signaling . Taken tAdditional studies demonstrated that EZH2 and HDACs work in concert at epigenetic level to reinforce the aberrant Wnt signaling activation in HCCs. Knock-down of EZH2 reduced the occupancy of HDAC1 at the promoters of Wnt antagonists in HCC cells . TreatmeAXIN2, NKD1, PPP2R2B and PRICKLE1) and promotes their silencing, and thereby increases the expression of a β-catenin target CCND1 that in turn induces p53/p21-mediated apoptosis and G2-M phase cell cycle arrest. Lentivirus-mediated silencing of HDAC8 in vivo was sufficient to reverse insulin resistance and reduce NAFLD-associated tumorigenicity. Furthermore, HDAC8 was directly up-regulated by the lipogenic transcription factor SREBP-1, and such positive relationship is highly consistent in dietary obesity models of NASH and HCC [Another class I HDAC, HDAC8, was also reported to physically interact with EZH2 to contribute to the activation of Wnt/β-catenin signaling during HCC development . HDAC8 h and HCC .Taken together, these studies provide links between histone modification and Wnt/β-catenin signaling in the development of NAFLD-associated liver cancers.Emerging evidence suggests the role of miRNAs in the regulation of key biological properties of HCC ,13,61. IWnt1 mRNA for suppression, as well as down-regulating the protein levels of Wnt1, β-catenin and TCF-4. It was previously shown that miR-122 was under-expressed in HCC relative to normal liver tissue, suggesting that loss of miR-122 in HCC might contribute to excess Wnt signaling [ob/ob mouse livers [miR-122 is the most abundant miRNA in the adult human livers. It can bind to the 3′-UTR of ignaling . There aignaling . miR-122ignaling . In addie livers . It has e livers . Anothere livers . SimilarCCND1 [miR-34a was reported to be a tumor suppressor in HCCs through regulation of Wnt/β-catenin pathway. Studies showed that ectopic miR-34a induces cell-cycle arrest and apoptosis by down-regulation of CCND1 ,70. In tCCND1 . In chroCCND1 .Another tumor suppressor miR-145 targets insulin receptor substrate-1 (IRS-1) in the insulin-like growth factor pathway and regulates resistin-induced insulin resistance . Down-reob/ob mouse livers compared with controls [However, there are several miRNAs that showed opposite expression patterns in metabolic disorder and HCC. MiR-214 is a tumor suppressor miRNA that can directly or indirectly targets β-catenin to inhibit cell growth by down-regulating c-Myc, cyclin D1, TCF-1 and LEF1 in HCC . Howevercontrols . FurtherIn this review, we have summarized the evidence that Wnt/β-catenin signaling is epigenetically dysregulated through multiple mechanisms in NAFLD-HCC . Of clin


To date, there is no effective therapy for patients with advanced/metastatic adrenocortical cancer (ACC). The activation of the Wnt/beta-catenin signaling is frequent in ACC and this pathway is a promising therapeutic target.To investigate the effects of the inhibition of the Wnt/beta-catenin in ACC cells.Adrenal (NCI-H295 and Y1) and non-adrenal (HeLa) cell lines were treated with PNU-74654 (5–200 μM) for 24–96 h to assess cell viability (MTS-based assay), apoptosis (Annexin V), expression/localization of beta-catenin , expression of beta-catenin target genes (qPCR and western blot), and adrenal steroidogenesis .CTNNB1/beta-catenin expression and increased beta-catenin target genes 48 h after treatment. No effects were observed on HeLa cells. In NCI-H295 cells, PNU-74654 decreased cortisol, testosterone and androstenedione secretion 24 and 48 h after treatment. Additionally, in NCI-H295 cells, PNU-74654 decreased SF1 and CYP21A2 mRNA expression as well as the protein levels of STAR and aldosterone synthase 48 h after treatment. In Y1 cells, PNU-74654 impaired corticosterone secretion 24 h after treatment but did not decrease cell viability.In NCI-H295 cells, PNU-74654 significantly decreased cell proliferation 96 h after treatment, increased early and late apoptosis, decreased nuclear beta-catenin accumulation, impaired Blocking the Tcf/beta-catenin complex inhibits the Wnt/beta-catenin signaling in adrenocortical tumor cells triggering increased apoptosis, decreased cell viability and impairment of adrenal steroidogenesis. These promising findings pave the way for further experiments inhibiting the Wnt/beta-catenin pathway in pre-clinical models of ACC. The inhibition of this pathway may become a promising adjuvant therapy for patients with ACC. The management of patients with adrenocortical carcinomas (ACCs) remains a challenge and patients with invasive, metastatic or recurrent disease have a poor prognosis , 2. AlthTP53 and CTNNB1 (the beta-catenin gene) in both adult and pediatric adrenocortical tumors (ACTs) [TP53 or CTNNB1 abnormalities [CTNNB1 mutations have been found in 15–36% and 6% of adult and pediatric ACTs, respectively [CTNNB1 mutations [CTNNB1 mutations has been recently reinforced. A large-scale high-resolution analysis study showed that variations in ZNRF3, which is a Wnt/beta-catenin pathway inhibitor, were the most common genetic defect found in a large number of ACC samples. ACCs presenting ZNRF3 variants showed transcriptional activation of beta-catenin target genes [CTNNB1 and ZNRF3 mutations or down regulation of Wnt/beta-catenin inhibitors are important for ACC pathogenesis. Therefore, inhibition of the Wnt/beta-catenin signaling is a rational option and may become a promising approach.Several genetic abnormalities have been found in these tumors, the most prominent being IGF2 overexpression –7 and mus (ACTs) –10. Tranmalities . Exon 3 ectively , 12–15. utations , 9. The et genes . Thus, aCTNNB1 mutations found in ACCs are located at residues involved in phosphorylation, which are essential sites for beta-catenin degradation by ubiquitin/proteasome signaling. Therefore, mutations in these sites lead to beta-catenin accumulation in the nucleus, where it binds with the T cell factor (Tcf) and enhances its transcriptional activity [CTNNB1 p.S45P mutation, thus representing a good in vitro model of ACC showing Wnt/beta-catenin pathway activation [activity . The NCIactivity . Remarkativation , 15. Higtivation . Among Ttivation .The PNU-74654 (PNU) compound is a non-FDA-approved drug which prevents that Tcf from binding to beta-catenin, acting as a Wnt/beta-catenin antagonist Figure . This smin vitro effect of PNU on adrenocortical tumor cells. Our results showed that inhibition of the Wnt/beta-catenin signaling resulted in a significantly decreased cell viability, increased apoptosis and impaired steroidogenesis.Taken together, these data suggest that the Wnt/beta-catenin pathway might be a potential targeted therapy for patients with ACC. Tcf/beta-catenin antagonism may be useful to treat patients with ACC exhibiting increased Wnt/beta-catenin signaling. In the present study, we assessed the CTNNB1 variation. Immunofluorescence analysis showed that, under basal conditions, beta-catenin is highly expressed in the cytoplasm and nucleus of NCI-H295 cells . Additionally, we identified an intronic TP53 variation at position c.74 + 38 (C > G rs1642785) in HeLa cells. These results are summarized in HeLa, a cervix carcinoma cell lineage, was employed as a non-adrenal cell line that did not carry the exon 3 p < 0.0001; Figure PNU impaired NCI-H295 cell viability. NCI-H295 cell viability was decreased 24 and 48 h after treatment with 100 and 200 μM PNU Figure  and 72 hp = 0.05; Figure p = 0.01; PNU did not decrease HeLa cell viability 96 h after treatment compared to vehicle  of late apoptotic cells 48 h after treatment Figure . PNU treCTNNB1 mRNA expression was decreased 48 h after PNU treatment with 50 , higher than recommended for good absorption (less than 140 Å2), characterizing this compound as poorly absorbed by cells [2 causing this compound to be very well absorbed by cells for a better effect on Wnt signaling inhibition .) [A previous study had shown that PKF115-584, currently called Calphostin C, dose-dependently inhibited beta-catenin-dependent transcription and H295R proliferation, and induced entry of cells into the S phase . Howeverby cells . On the  2015).) . In our et al. showed that Wnt/beta-catenin inhibition by PKF115-584 repressed the entry of H295R cells into the S phase and induced their apoptosis [lowSf1/Crebeta-catenin knockout mice, supporting the idea that the loss of beta-catenin in the adrenal cortex progressively contributes to the loss of adrenocortical tissue via apoptosis [In NCI-H295 cells, the analysis of apoptosis 48 h after PNU treatment showed a dose-dependent increase of apoptotic cells. These data support the proposal that inhibition of Wnt/beta-catenin signaling impairs cell proliferation mostly by increasing early and late apoptosis. In agreement, Doghman poptosis . In addipoptosis .in vitro and in a xenograft mouse model [We found reduced expression of beta-catenin at the mRNA and protein level 48 h after PNU treatment. In addition, we also found a reduction of nuclear beta-catenin accumulation in PNU-treated cells, as shown by immunofluorescence. Similarly, to previous data, our results show that shorter exposure to PNU 24 h) did not affect beta-catenin expression  h did no. Taken tse model .CCND1 and AXIN2. In agreement, it has been shown that the Wnt/beta-catenin pathway inhibition resulted in increased AXIN2 mRNA expression in endometrial cells [MYC mRNA, a well-known beta-catenin target gene, supports the hypothesis that MYC overexpression is common in many cancers but not in ACTs [TCF7 mRNA expression did not change in response to PNU treatment, supporting the hypothesis that this drug directly interferes with the Tcf/beta-catenin interaction [We also observed that PNU led to increased mRNA expression of the beta-catenin target genes al cells . Furtheral cells , 32. The in ACTs , 33. TCFeraction .SF1 and CYP21A2, both important genes required for steroidogenesis, suggesting that PNU directly impairs adrenal steroid secretion by inhibiting the initial steps of steroidogenesis.The majority of ACCs secrete excessive amounts of cortisol, androgens or both, resulting in significant morbidity associated with excess hormone production . Thus, aCYP11B2 gene. This result agrees with data reported by Berthon et al (2014), who showed that the inhibition of the Wnt pathway decreased hormone secretion, CYP11B2 and CYP21 expression after angiotensin stimulation [SF1 is required for StAR gene expression, which is an early and limiting step in normal steroidogenesis. Accordingly, the finding of impaired StAR protein levels after PNU treatment in NCI-H295 cells reinforces this hypothesis , 36. In mulation . Our finmulation . Mouse smulation . On the mulation .in vitro effects and the results of this study do not ensure that the same effect will be obtained in in vivo models. The use of agents suppressing this pathway in clinical practice must be very cautious in view of toxicity and side effects since the Wnt signaling is a ubiquitous signaling pathway [There are some possible limitations to the present study. We evaluated the effects of PNU only on immortalized tumor cell lines, which are known to have possibly accumulated additional genetic defects and chromosomal instability over time. However, the NCI-H295 lineage is a good surrogate model for the analysis of the effect of the Wnt/beta-catenin signaling inhibition on ACC since it harbors the p.S45P beta-catenin mutation, as confirmed in our own stock of cells. This mutation leads to the activation of the constitutive Wnt/beta-catenin pathway , 15. In  pathway .in vitro study demonstrated that blocking the Tcf/beta-catenin complex inhibited the Wnt/beta-catenin signaling as shown by decreased beta-catenin expression and nuclear accumulation, and impaired expression of beta-catenin target genes. These effects resulted in increased apoptosis, decreased tumor cell viability, and impairment of adrenal steroidogenesis. These promising findings may pave the way for further experiments inhibiting this pathway in pre-clinical models of ACCs. Taken together, these lines of evidence indicate that inhibitors of the Wnt/beta-catenin complex may become useful for the individualized treatment of patients with ACC presenting increased Wnt/beta-catenin signaling.In conclusion, the results of this This study was approved by the local Ethics Committee (HCFMRP-USP #43758/2013).®). The NCI-H295 human adrenal cell line was cultured in RPMI 1640 medium  supplemented with 2% fetal bovine serum , 1% ITS Premix (BD Biosciences) and 1% penicillin/streptomycin  and harvested weekly. The Y1 mouse adrenal and HeLa human non-adrenal cell lines were cultured in DMEM medium (Sigma Aldrich) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and harvested every 2–3 days. All cell lines were cultured in monolayer and maintained under standard conditions at 37°C with 5% CO2. For all experiments, cell lines were harvested at the third passage. NCI-H295 and HeLa cell lines were authenticated by analyzing the following STR markers: CSF1PO, D13S317, D16S539, D5S818, D7S820, THO1, TPOX and vWA  . HeLa ce and vWA .CTNNB1 and TP53 coding gene was performed to confirm the presence of previously described TP53 and CTNNB1 genetic variations in our own stock of NCI-H295 and HeLa cells. Genomic DNA was extracted from the cell lines using the QIAamp DNA Minikit . Exon 3 CTNNB1 and the TP53 coding gene were amplified by PCR and fragments were sequenced using the ABI Prism Big Dye Terminator v3.1 Cycle Sequencing Kit on an ABI 3130 instrument . PCR primers and conditions are available upon request.The analysis of exon 3 50) of PNU-74654 for each cell line is shown in The PNU-74654 compound (Sigma Aldrich) was resuspended in dimethyl sulfoxide  at stock concentrations of 31.2 mM. For working solutions, PNU-74654 was diluted 100X in growth medium with no serum deprivation and then diluted according to the required concentrations. The half-maximal inhibitory concentration  or 10, 50, 100 and 200 μM PNU-74654. After 24 and 48 h, medium supernatants were collected for adrenal steroid measurements. Adherent cells were fixed for immunofluorescence or harvested for RNA and protein isolation. At least three independent experiments were performed.Y1 cells were plated at 200,000 cells per well in 24-well plates for gene expression and corticosterone measurement. After 24 h, cells were treated with vehicle or 10, 50, 100 and 200 μM PNU-74654. After 24 and 48 h, medium supernatants were collected for corticosterone measurements and adherent cells were harvested for RNA isolation. At least two independent experiments were performed.HeLa cells were plated at 50,000 cells per well in 24-well plates for gene expression and protein analysis. After 24 h, cells were treated with vehicle or 50, 100 and 200 PNU-74654. After 24 and 48 h, adherent cells were harvested for RNA and protein isolation. At least two independent experiments were performed.For protein isolation, the cells were lysed after treatment by homogenization in 100 μL IP Lysing Buffer  and 1 μL of Halt Protease and Phosphatase inhibitor cocktail (Thermo Scientific). Protein concentration was measured by BCA protein assay . Equal amounts (20 μg) of protein were subjected to SDS-PAGE, transferred to nitrocellulose membranes, blocked in TBST-T containing 5% skim milk and probed with anti-beta-catenin , anti-TCF , anti-STAR  and anti-CYP11B2 . Anti-GAfter treatment with PNU-74654, medium was collected and cells were fixed in methanol for 3 minutes, washed 3 times with 0.01 M PBS, and incubated with 10% normal horse serum for 1 h for blocking. Sections were then incubated overnight at room temperature with anti-beta-catenin primary antibody . Next, cells were washed 3 times with 0.01 M PBS, incubated with secondary antibody Cy™5-conjugated AffiniPure donkey anti-mouse antibody  for 4 h and washed 3 times with 0.01 M PBS. For nuclear counterstaining, cells were incubated with 4′,6-diamidino-2-phenylidone  for 2 minutes, washed in 1X PBS, and slides were set with Fluoromount (Sigma-Aldrich). Expression and localization of beta-catenin were observed with a Leica TCS SP5 laser scanning confocal microscope  with fixed exposure time for all samples.To assess the beta-catenin cellular localization in the Y1 cell line, cells were seeded over a coverslip in a 24-well plate for 24 h and fixed in methanol for 3 minutes, washed 3 times with 0.01 M PBS, and incubated with Hydrogen Peroxide Block for 10 minutes followed by Super Block  solution for 30 minutes for blocking. Sections were then incubated overnight 4°C with anti-beta-catenin primary antibody . Next, cells were washed 3 times with 0.01 M PBS, incubated with Complement  for 10 min and washed twice with 0.01 M PBS. Cells were then incubated with HRP conjugate  for 15 min, washed 4 times with 0.01 M PBS and incubate with peroxidase substrate chromogen . Expression and localization of beta-catenin was observed with a Zeiss microscope .® according to the manufacturer's protocol (Life Technologies). RNA concentrations were quantified by spectrometry . RNA integrity was checked according to the 28S/18S ratio, with an acceptable range of 1.6 to 2.0 and confirmed by 1.2% agarose gel electrophoresis. mRNA was reverse transcribed from 500 ng of total RNA using the High Capacity cDNA Reverse Transcription kit and MultiScribe® enzyme (Life Technologies).Total RNA from NCI-H295 cells was isolated using TRIzol® assays  were used according to the manufacturer's recommendation using cDNA (diluted 1:5) as template. TaqMan® assays for NCI-H295 and HeLa cells: CTNNB1 (Hs00170025_m1), CCND1 (Hs00765553_m1), AXIN2 (Hs00610344_m1), MYC (Hs00153408_m1), TCF7 (Hs00175273_m1), NR5A1/SF1 (Hs00610436_m1) and CYP21A2 (Custom: AIVI3LV). TaqMan® assay for Y1 cells: Ctnnb1 (Mm01350391_m1). Relative expression values were determined by the 2−ΔΔCt method using GUSB (ID: 4326320E) as endogenous control.For quantitative Real-Time PCR (qPCR), TaqManCortisol, testosterone and androstenedione concentrations were measured in the supernatant of the NCI-H295 cell medium and corticosterone concentrations were measured in the supernatant of the Y1 cell medium by radioimmunoassay (RIA) as previously described . The eff® AQueous One Solution Cell Proliferation Assay , an MTS-based assay, was used to determine cell viability before and after treatment with PNU. Briefly, cells were plated on 96-well plates at a density of 20,000 cells/well (NCI-H295) or 10,000 cells/well, incubated for 24 h (Y1 and HeLa) and 48 h (NCI-H295) and then treated with 5, 10, 50, 100 and 200 μM PNU in triplicate. After 24, 48, 72 and 96 h of incubation for NCI-H295 cells, 24 and 48 h of incubation for Y1 cells and 48 and 96 h of incubation for HeLa cells, 20 μL of CellTiter 96® Aqueous Solution was added to each well and incubated for 1–3 h at 37°C with 5% CO2 according to the manufacturer's datasheet. Absorbance at 490 nm was obtained from a microplate reader (BioRad). At least three independent experiments were performed for each cell line.The CellTiter 96® Aqueous Solution was added to each well and incubated for 2 h at 37°C with 5% CO2 according to the manufacturer's datasheet. Absorbance at 490 nm was obtained from a microplate reader. Three independent experiments were performed.To analyze the impact of PNU under conditions of stimulation with Forskolin, NCI-H295 cells were plated on 96-well plates at a density of 20,000 cells/well, incubated for 48 h and then stimulated with 10 μM Forskolin (Sigma Aldrich) and treated with 5, 10, 50, 100 and 200 μM PNU in triplicate. After 96 h of stimulation/treatment, 20 μL of CellTiter 966 total cells and assayed according to manufacturer's recommendations .NCI-H295 cells were plated at 350,000 cells per well in 6-well plates in triplicate. After 48 h, cells were treated with vehicle  or with 10, 50 and 100 μM PNU. After 48 h, cells were harvested, triplicates were pooled, washed once in 0.01 M PBS and suspended in binding buffer (Sigma Aldrich) at a minimum of 1.10The procedure basically consists of the binding of annexin V-FITC to phosphatidylserine in the cell membranes, which are beginning the apoptotic process, and the binding of propydium iodide to cellular DNA in cells where the cell membrane has been totally compromised. The cells were incubated with annexin V-FITC and propydium iodide at room temperature for 10 min and then analyzed by flow cytometry. Annexin V-FITC is detected as green fluorescence and propydium iodide as red fluorescence. Results were analyzed with the BD CellQuest Prosoftware considering 10,000 events.of percentage and standard error (SEM) relative to vehicle. The level of significance was set at p ≤ 0.05 in all analyses. Continuous variables are reported as mean and standard error (SEM).Statistical analysis was performed using GraphPad Prism 6 software . One-way ANOVA followed by Dunnett's multiple comparison test was used to determine differences before and after treatment with all PNU concentrations. For analysis of hormone secretion, all values are reported as mean