MYCN is the most well-known prognostic marker of neuroblastoma risk classification, but still is only observed in 25% of cases. Recent evidence points to the cyclic adenosine monophosphate (cAMP) elevating ligand prostaglandin E2 (PGE2) and β-catenin as two novel players in neuroblastoma. Here, we aimed to define the potential role of PGE2 and cAMP and its potential interplay with β-catenin, both of which may converge on neuroblastoma cell behaviour. Gain and loss of β-catenin function, PGE2, the adenylyl cyclase activator forskolin and pharmacological inhibition of cyclooxygenase-2 (COX-2) were studied in two human neuroblastoma cell lines without MYCN amplification. Our findings show that PGE2 enhanced cell viability through the EP4 receptor and cAMP elevation, whereas COX-2 inhibitors attenuated cell viability. Interestingly, PGE2 and forskolin promoted glycogen synthase kinase 3β inhibition, β-catenin phosphorylation at the protein kinase A target residue ser675, β-catenin nuclear translocation and TCF-dependent gene transcription. Ectopic expression of a degradation-resistant β-catenin mutant enhances neuroblastoma cell viability and inhibition of β-catenin with XAV939 prevented PGE2-induced cell viability. Finally, we show increased β-catenin expression in human high-risk neuroblastoma tissue without MYCN amplification. Our data indicate that PGE2 enhances neuroblastoma cell viability, a process which may involve cAMP-mediated β-catenin stabilization, and suggest that this pathway is of relevance to high-risk neuroblastoma without MYCN amplification.Amplification of  MYCN, which is especially amplified in the most aggressive and often metastatic forms of neuroblastoma [MYCN has important prognostic value, amplification is only observed in about 25% of neuroblastoma cases and it remains largely to be defined what other factors contribute to high-risk neuroblastoma.Neuroblastomas are heterogeneous tumours that vary to a large extent in prognosis and disease outcome and are responsible for 15% of all childhood cancer deaths [blastoma ,3. Altho2 (PGE2) have been found increased in a variety of malignant tumours, including neuroblastoma [2 is produced by a multistep enzymatic process in which the rate-limiting step is mediated by COX enzymes. PGE2 binds to its membrane bound E-type prostanoid receptors, of which prostanoid receptors type 2 and 4 are known to couple to Gαs and are thereby able to increase intracellular cyclic adenosine monophosphate (cAMP) levels. cAMP is involved in the regulation of diverse cellular processes, including regulation of cytoskeletal dynamics, cellular differentiation, proliferation and programmed cell death in a variety of cells including neural-like cells [Expression of cyclooxygenase-2 (COX-2) and prostaglandin Eblastoma ,5 and phblastoma –9. PGE2 ke cells ,11.2, cAMP and β-catenin. β-catenin contributes to other malignancies such as hepatocellular carcinoma and colorectal carcinoma and its role in paediatric malignancies is well documented [MYCN non-amplified neuroblastoma cell lines and β-catenin target gene transcription is increased in neuroblastoma tumours without MYCN amplification [Of particular interest are recent research lines that focus on molecular interactions between PGEcumented . Also, icumented –15. Regafication .2 has been shown to enhance β-catenin nuclear localization via dissociation of GSK3β from axin by Gαs [Distinct pools of β-catenin exhibit distinct cellular functions. β-Catenin associates with membrane junctional complexes where it binds to cadherins and α-actin. Free cytosolic β-catenin is rapidly tagged for proteasomal degradation by a multiprotein destruction complex comprised of the kinases glycogen synthase kinase 3β (GSK3β), casein kinase 1 and adaptor proteins like axin2, which is the limiting component in the assembly of this complex –19. Stabn by Gαs  and by an by Gαs . Activatn by Gαs  and GSK3n by Gαs ,25,26.2 and β-catenin to cell proliferation and inhibition of apoptosis, independent of MYCN amplification.In this paper, we aim to identify the contribution of a molecular link between PGE2 at 37°C. Cells were washed with HBSS , dissociated from the plate with trypsin EDTA and seeded in appropriate cell culture plate format. Cells were serum-deprived for 24 hrs before stimulation. Inhibitors  or antagonists  were added 30 min. prior to stimulation with PGE2.Human neuroblastoma cell lines SK-N-AS and SK-N-SH were obtained from ATCC . Both cell lines are of epithelial morphology. Cells were maintained in DMEM  supplemented with 10% v/v heat-inactivated FCS, non-essential amino acids and antibiotics  in a humidified atmosphere of 5% CO2PO4, 8 g/l NaCl, 140 mg/l CaCl2, 100 mg/l MgCl2·6H2O, 100 mg/l MgSO4·7H2O, 90 mg/l Na2HPO4·7H2O, 1 g/l glucose, pH 7.4) and then incubated with 5% v/v AlamarBlue  followed by fluorescence spectrophotometry. Treated cultures were normalized to control cultures.Experiments were performed in 24-well cell culture plates. Prior to measurement, cells were washed with calcium containing HBSS  and stained with 0.05% Crystal Violet. Plates were photographed and confluency was quantified using ImageJ software.2 production was measured in culture medium from overnight cultures incubated with niflumic acid using the PGE2 ELISA assay from Cisbio .Cells were cultured in 24-well plates and PGE2, 10 mM HEPES). A total of 400,000 cells were resuspended in ice-cold binding buffer supplemented for 15 min. with 2 mM CaCl2, PI (1 μg/ml) and FITC-conjugated annexin V antibody . A total of 10,000 stained cells were analysed by Kaluza Flow Analysis Software .Cells were cultured on 60 mm plates and incubated for 24 hrs with designated stimuli. For cell cycle analysis, cells were incubated for 30 min. with 30 μM bromodeoxyuridine (BrdU). Cells were dissociated from the plate with accutase, fixed with 95% EtOH and DNA was denatured using 2M HCl. Cells were incubated with anti-BrdU FITC-conjugated antibody, propidium iodide (PI) and RNAse A. A total of 40,000 stained cells in FACS buffer  were analysed in FACSCalibur . ModFit LT software was used to determine the distribution of cells over the cell cycle phases designated G0/G1, S and G2/M. For detection of apoptosis, cells were washed with binding buffer .Total mRNA extraction was performed with the NucleoSpin RNA II Kit . cDNA was acquired using reverse transcription by AMV Reverse Transcriptase Kit . qPCR was performed with the Illumina Eco Personal qPCR System . Cycle parameters (30 sec. each): denaturation at 94°C, annealing at 60°C and extension at 72°C. Target genes were normalized to the geometric mean of reference genes GAPDH, SDHA and YWHAZ . Primer S33Y  [Cells grown to 60% confluence were transfected in serum- and antibiotics-free DMEM with plasmid DNA [TOPflash, FOPflash, renilla luciferase , β-cateninMA, USA) , pcDNA3 via the Dual Reporter luciferase assay system (Promega).TOPflash- or FOPflash-transfected cells were subjected to stimulation in serum-free DMEM for 16 hrs, and luciferase activity was assayed 3VO4, 50 mM NaF, β-glycerophosphate, 10% v/v glycerol, pepstatin A, leupeptin, apoprotinin) for 10 min. on ice and 15 min. with 0.1% v/v IGEPAL added. After centrifugation at 3000 × g for 3 min., the supernatant (cytosolic fraction) was collected and pellets were resuspended in ice-cold cell extraction buffer  for 30 min. After centrifugation at 14,000 × g for 10 min., supernatant (nuclear fraction) was collected.Cell were seeded in 100 mm cell culture dishes and dissociated with a cell scraper. Cells were incubated with hypotonic buffer . Equal amounts of protein were subjected to electrophoresis on polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% w/v BSA or 5% w/v milk in tris-buffered saline with 0.1% v/v Tween-20. Protein expression was determined by specific primary and horseradish peroxidase (HRP)-conjugated secondary IgGs. Antibodies used are listed in Table Cells were lysed in ice-cold RIPA buffer supplemented with phosphatase and protease inhibitors . Cells were blocked using 1% w/v BSA and 2% v/v donkey serum in CytoTBS-T . p-β-Catenin (ser675) IgG was applied overnight, secondary FITC-conjugated donkey anti-rabbit IgG  was applied for 3 hrs. Nucleus was visualized with 1 μg/ml Hoechst 33342 (Invitrogen). Slides were mounted with ProLong® Gold Antifade Reagent . Images were captured with a Leica DM4000 B Fluorescence microscope  equipped with a Leica DFC 345 FX camera.Cells were grown on LabTek II Chamber Slides , fixed with PFA and permeabilized with 0.3% w/v Triton X-100 in cytoskeletal buffer  and national ethical and professional guidelines . Tumour specimens were subdivided based on risk group assessment in low risk, medium risk and high risk according to Children's Oncology Group risk groups  SpectrumGreen/CEP2 SpectrumOrange Probe . The LSI N-MYC (2p24) probe hybridizes to the 2p24 region and contains sequences that flank both 5′ and 3′ ends of the MYCN gene. The CEP2 (2p11.1-q11.1) probe hybridizes to alpha satellite sequences specific to chromosome 2. Images were captured using a Leica DMRA2 Fluorescence microscope (Leica Microsystems) equipped with a Leica DC350F camera. The number of spots in 20 adjacent tumour cells was counted for MYCN and CEP2. When ratio between the two signals exceeded 2, the tumour was scored as MYCN amplified.Fluorescence 2O2 and 1 mM EDTA, pH 7.0. As a negative control, the primary antibodies were omitted. For each sample, expression was quantified by measuring the average staining intensity on a scale of 256 channels using ImageJ software in four different random selected regions within the tumour (using haematoxylin and eosin-stained sections). Differences between the groups were tested for significance using the Kruskal–Wallis test. All procedures were performed blind.For determination of β-catenin protein expression, deparaffinized tumour sections were incubated with anti-β-catenin IgG  overnight and subsequent with HRP-conjugated rabbit antimouse IgG . Staining was performed with 0.5 mg/ml 3,3′-diaminobenzidine in 30 mM imidazole containing 0.03% v/v H2, XAV939, AH6809 and forskolin were from Tocris Bioscience . Niflumic acid was from Cayman Chemical . All other chemicals were of analytical grade.BrdU, mouse anti-BrdU antibody, PI, L-161,982 and celecoxib were purchased from Sigma-Aldrich. 16,16-dimethyl-PGEn separate experiments. Normality and equal variance were evaluated by Shapiro–Wilk test and f-test. Statistical significance of differences was evaluated by Student's t-test and one-way or two-way anova followed by a Tukey multiple comparison test. Non-parametric data were evaluated with the Kruskal–Wallis test followed by Dunn's multiple comparison test. Differences were considered to be statistically significant when P < 0.05.Data represent means ± SEM, from MYCN non-amplified neuroblastoma cell viability, SK-N-AS cells were incubated for the indicated periods of time with the direct adenylyl cyclase activator forskolin or PGE2  induces cAMP elevation  and COX-2 inhibition with niflumic acid decreases PGE2 (6 pg/ml). Importantly, niflumic acid decreases dose-dependently viability of SK-N-AS cells . β-catenin not phosphorylated by GSK3β is stable and translocates to the nucleus. In this paper, we refer to this stable form of β-catenin as active β-catenin. Neuroblastoma cells stimulated with forskolin or PGE2 were analysed for protein levels of phosphorylated β-catenin and GSK3β. Forskolin and PGE2 induced phosphorylation of β-catenin (ser675), p-GSK3β (ser9) and concomitant higher levels of active, unphosphorylated, β-catenin , neuroblastoma cells were stimulated with forskolin or PGE2 after which p-β-catenin (ser675) was visualized by immunofluorescence  and induced accumulation of p-β-catenin (ser675) in (peri)nuclear regions  containing multiple TCF-binding sites or a construct with mutated TCF-binding sites (FOPflash). Either forskolin or PGE2 induced TCF reporter gene expression, as measured by luciferase activity in cell lysates . Transfection with β-cateninS33Y increased protein levels of β-catenin  β-catenin by enhancing the stability of axin2, the limiting factor of the APC/GSK3β/CK1 destruction complex that targets β-catenin for proteasomal degradation, with the tankyrase inhibitor XAV939 [2-induced cell viability, it did not affect neuroblastoma cell viability at a basal level  abrogations that do not accurately reflect the behaviour of other cancer cell lines originating from the same disease. We therefore repeated key experiments in a different human neuroblastoma cell line without ity Figs B. Specifity Figs C. SK-N-S939 Figs D. Thus, MYCN amplification as it has previously been reported that β-catenin expression is increased in neuroblastoma tumours and cell lines without amplification of MYCN [in situ hybridization was performed to identify amplification status of neuroblastoma specimens. We regarded tumour specimens that scored positive for MYCN amplification as a separate group of tumours. β-Catenin expression was determined by scoring the average staining intensity of the tumour. β-Catenin was found predominantly at the periphery of the cell body and within the neuropil, areas of the tumour that are comprised of non-myelinated axons, dendrites and synaptic dense regions. We did not observe β-catenin-positive nuclei. When high-risk tumours with MYCN amplification were compared to high-risk tumours without MYCN amplification, higher levels of β-catenin were found in high-risk tumours without MYCN amplification. Importantly, we observed higher levels of β-catenin in tumour specimens from high-risk tumours compared to expression in low- and medium-risk cases (P = 0.042)  Fig. C.MYCN. Amplification of MYCN is only observed in about one-fourth of neuroblastomas [2 and β-catenin to progression of neuroblastomas without MYCN amplification. Our findings in MYCN non-amplified neuroblastoma cell lines show that PGE2 enhances cell viability through the EP4 receptor, whereas this was attenuated by inhibition of PGE2 with specific COX-2 inhibitors. Elevating cAMP using forskolin mimicked the effects of PGE2 on cell viability. PGE2 and cAMP promoted GSK3β inhibition, phosphorylation of β-catenin at the PKA target residue ser675, β-catenin nuclear translocation and TCF-dependent gene transcription. Furthermore, we show that expression of a degradation-resistant β-catenin mutant enhances neuroblastoma cell viability and that inhibition of β-catenin with XAV939 prevents PGE2-induced viability. β-Catenin expression in tumour sections was increased in high-risk neuroblastoma without MYCN amplification compared to expression in low- or medium-risk sections. These data suggest that autocrine PGE2 enhances neuroblastoma cell viability via a mechanism involving cAMP-mediated β-catenin stabilization, and suggest that this pathway is of relevance to high-risk neuroblastoma without MYCN amplification.The development of neural crest-derived tissues is under tight control of molecular mechanisms that regulate proliferation, differentiation and programmed cell death. When these core molecular pathways become deregulated, malignancies such as neuroblastoma can develop. The most extensively defined factor contributing to neuroblastoma pathogenesis is amplification of the Myc oncoprotein family member lastomas ,3. In th2 levels and concomitant up-regulation of COX-2 are frequently observed in malignant tissues of epithelial origin [2 are involved in progression of neuroblastoma [2 functions by binding to its membrane bound E-type prostanoid receptors which are all expressed in neuroblastoma cells. Of the four receptor subtypes, EP2 and EP4 are Gαs-coupled and stimulate adenylyl cyclase activation and thus intracellular cAMP levels [2 incubation elevated cellular cAMP levels in neuroblastoma. Conversely, inhibiting endogenous PGE2 synthesis with COX-2 inhibitors decreased cAMP levels. The rise in cAMP levels, either through receptor-mediated adenylyl cyclase activation by PGE2 or via direct activation of adenylyl cyclase by forskolin, increased cell viability of neuroblastoma cells. Further, we demonstrate that enhancement of cell viability by PGE2 is mediated through the EP4 receptor as antagonism of the EP4 receptor using L-161,982 completely abolished the enhancement of cell viability by PGE2, whereas antagonism of the EP2 receptor using AH6809 did not affect this. In earlier studies, Rasmuson et al. demonstrated that exogenous PGE2 enhanced viability of MYCN-amplified SK-N-BE(2) neuroblastoma cells, suggesting that PGE2 is involved in neuroblastoma cell viability independent of amplification of MYCN [Increased PGEl origin ,40. Studblastoma , althougP levels . Indeed,in vitro [in vivo [2 production, there are several reports that indicate that accumulation of arachidonic acid, the precursor for PGE2 synthesis, is also involved. For instance, combined treatment with arachidonic acid and COX inhibitors results in a synergistic effect on cell survival [2, rescues neuroblastoma cells from decreased viability as a result of COX-2 inhibition. This is also reflected on intracellular cAMP levels, which are decreased by niflumic acid, but restored by simultaneous addition of exogenous PGE2.Inhibition of COX enzymes has been shown to inhibit neuroblastoma cell survival in vitro ,42 and t[in vivo . Indeed,[in vivo ,43, whilsurvival . However2 and β-catenin. In 2002, Fujino et al. showed that PGE2 stimulates TCF/Lef promoter activity through EP2 and EP4 receptor signalling [2 increased p-GSK3β (ser9) and elevated levels of unphosphorylated β-catenin at the GSK3β residues. In addition, we found increased nuclear localization of active β-catenin. These findings are in agreement with studies in different malignant cell models. In non-small cell lung cancer and colorectal cancer, PGE2 enhances β-catenin nuclear localization and inhibits GSK3β by phosphorylation which is dependent on cAMP and kinase activity of PKA [2 or forskolin. Subsequently, we observed a transient increase in p-β-catenin (ser675) at the (peri)nuclear regions. Importantly, we also found increased TCF-dependent gene transcription, which confirms earlier studies in HEK cells in which PKA-dependent p-β-catenin (ser675) resulted in increased TCF luciferase activity [2 and subsequent PKA activity was found in in vitro and in vivo models for colorectal cancer [2 synthesis using the COX-2 inhibitor celecoxib has been found to attenuate β-catenin stability, nuclear translocation and TCF luciferase activity in colorectal cancer [Of particular interest are emerging research lines that focus on molecular interactions between PGEgnalling . Downstrgnalling ,32,33. Iy of PKA ,36. Apary of PKA ,37,38. Iactivity ,37. Morel cancer ,34. Convl cancer , non-smal cancer , with coMYCN non-amplified neuroblastoma cell lines [S33Y). Importantly, the enhanced cell viability resulting from PGE2 was completely prevented by the tankyrase inhibitor XAV939. XAV939 prevents tankyrase-mediated degradation of axin2, thereby stabilizing axin2 which is the concentration-limiting component of the destruction complex that sequesters β-catenin and GSK3β. Thereby, XAV939 functions as an inhibitor of β-catenin activity [2-induced enhanced cellular viability. Recently, XAV939 has been shown to decrease neuroblastoma cell survival by inhibiting β-catenin transcriptional activity and decreasing expression of cyclin D1 in both MYCN-amplified and non-amplified cell lines [The contribution of β-catenin and TCF-dependent gene transcription in colorectal carcinoma has been well-established. In neuroblastoma, it has been shown that β-catenin expression is increased in ll lines . The resll lines . Moreovell lines . This, tMYCN amplification revealed particularly high expression in high-risk tumours. This further strengthens our hypothesis that β-catenin plays an important role in neuroblastoma, not only in vitro cell models but also in a clinically more relevant context. Other recent studies have also found increased expression of β-catenin in neuroblastoma, which was involved in maintaining a neuroblast-like phenotype of neuroblastoma cells and conferring resistance to chemotherapeutic agents [MYCN-amplified and non-amplified tumours. We are aware that based on our study, we cannot draw strong conclusions on the functional role of increased β-catenin in high-risk neuroblastoma tumours as expression of β-catenin was found predominantly at the cell periphery and the neuropil, where it exerts a different function than in the nucleus. However, during epithelial–mesenchymal transition, which is an early step in the metastatic cascade in cancer [2, β-catenin translocates to the nucleus and activates TCF/Lef-dependent transcriptional programmes, thereby enhancing survival and metastatic potential [Interestingly, investigation of β-catenin expression in neuroblastoma tumour sections without c agents ,49. Howen cancer , β-catenotential .The promising therapeutic potential of COX-2 inhibitors has resulted in multiple compounds that have reached the clinical market. These compounds were, however, rejected as a consequence of concerns regarding drug safety . Recent 2 is an important factor being involved in the regulation of neuroblastoma cell viability. cAMP activates PKA and thereby stabilizes β-catenin either directly or indirectly by inhibiting GSK3β, resulting in increased β-catenin activity and neuroblastoma cell viability. The increased expression of β-catenin in high-risk neuroblastoma, without MYCN amplification, suggests that β-catenin could play a major role in neuroblastoma. Thus, strategies that target either PGE2 production by the tumour (COX-2 inhibitors) or target the interaction between PGE2 and β-catenin warrant further investigation.Taken together, our results demonstrate that PGE


The purpose of this paper is to study the spatial agreement between visual field defects and ultra-wide field (UWF) fundus autofluorescence (FAF) in patients with birdshot chorioretinopathy (BSCR). The study is a retrospective, cross-sectional analysis of a university uveitis practice. Eight (8) eyes of five (5) patients with BSCR were included. Inclusion criteria were ability to fixate reliably. Goldmann visual fields (GVF) and UWF FAF were obtained, digitalized, and standardized. Analysis was performed by measuring areas of overlap of hypo-autofluorescent FAF lesions and GVF scotomas within the central 60°. Overlap was calculated as a percentage of the total area of FAF and GVF, respectively. Average areas were also calculated.The mean age of the subjects was 51 ± 12.28 years (range 38–69 years). 14 ± 23 % of the total lesion area identified as hypo-autofluorescent on FAF overlapped with scotoma. 28 ± 41 % of the GVF scotomas overlapped with hypo-autofluorescent FAF lesions. Average area of FAF hypo-autofluorescence was much larger (15.19 disc areas) than GVF (3.45 disc areas).There appear to be larger total areas of hypo-autofluorescence on FAF than scotoma evidenced by GVF and only a small amount of overlap. The finding suggests that GVF is relatively insensitive to anatomic loss, which can be detected using FAF. Further studies are required to assess whether this finding holds true for automated white-on-white perimetry. In addition, more selective psychophysical stimuli may have higher sensitivity in detecting early functional loss that accompanies anatomic damage. Birdshot chorioretinopathy (BSCR) is a form of inflammatory posterior uveitis characterized by typical clinical and imaging characteristics. This condition is associated with bilateral hypo-pigmented chorioretinal “birdshot” lesions affecting the peripapillary and posterior region –3. ExcelBSCR appears to have a chronic, progressive course with most patients developing functional loss over a long-term follow-up . Visual Evaluation of spatial agreement between UWF FAF and GVF has been performed in other retinal pathologies, such as retinitis pigmentosa . HoweverThe study was conducted at the retina and uveitis services of the Stanley M. Truhlsen Eye Institute, University of Nebraska Medical Center. Institutional Review Board (IRB) clearance was obtained and the study adhered to the tenets of the Declaration of Helsinki. All the study procedures were compliant as per the HIPAA act of 1996. This retrospective, observational case series included patients diagnosed with BSCR from January 2013 to December 2014. The diagnosis was established based on clinical findings, slit-lamp biomicroscopy, and indirect ophthalmoscopic visualization of characteristic yellow-orange ovoid chorioretinal lesions with mild vitritis, as per the International Consensus Conference on BSCR [All the patients underwent imaging studies including UWF color fundus photography, fluorescein angiography (FA), and FAF . Sixteen eyes of 8 patients were screened for inclusion in the study. The exclusion criteria were unreliable GVF testing, poor central fixation due to central visual loss and media opacity precluding adequate imaging. GVF testing was performed using Haag-Streit system, Bern, Switzerland, using V4e, I4e, I2e, and I1e test targets. A single, trained operator tested the visual fields to minimize the variability of the tests. Scotoma was defined using the V4e isopter.The GVF test results were scanned and digitalized using Adobe Photoshop Elements 11.0 . ImageJ software   was usedp value of <0.5 was considered to be statistically significant.The data analysis was performed using GraphPad Prism 6 . The area of scotomas on V4e isopter was calculated in terms of disc areas on both GVF and FAF. The percentage area of hypo-autofluorescence on FAF that overlapped with the scotomas on GVF was calculated. Similarly, the percentage area of the scotoma that overlapped with the FAF was calculated. Bland-Altman analysis was used to evaluate the agreement between the two techniques in detecting the areas involved by the disease process. Inter-observer agreement for the measured values was calculated using the intraclass correlation coefficient (ICC) test. A Data was collected from a total of 16 eyes from 8 patients. Eight eyes from 5 patients were excluded due to ungradable image quality, lack of patient cooperation, poor central fixation, and/or unreliable GVF testing. Eight (8) eyes of 5 patients  met the inclusion criteria and their clinical data and images were included in the analysis. All the patients included in the study were positive for HLA-A29. The mean age of all 8 subjects was 51 ± 12.28 years (range 38–69 years). The mean best-corrected visual acuity for the included eyes (converted to LogMAR units) measured 0.65 ± 0.39 units. The baseline demographic details of all the patients are listed in Table p = 0.0002).Of the area covered by hypo-autofluorescent lesions identified on UWF FAF, 14 % ±23 %) overlapped with scotoma on GVF. On the other hand, 28 % (±41 %) of the area of GVF scotomas overlapped with the hypo-autofluorescent lesions seen on UWF FAF. An example of the overlap analysis is shown in Fig. 3 % overlTwo distinct patterns of distribution of the hypo-autofluorescent lesions were noted on FAF imaging. The majority of eyes  demonstrated macular and extra-macular areas of hypo-autofluorescence. Placoid hypo-autofluorescence involving the macula was noted in one eye. The remaining eyes showed peripapillary areas of hypo-autofluorescence. Unlike the FAF, no consistent pattern of visual field defects was noted on GVF.BSCR is an uncommon form of posterior uveitis with protean clinical manifestations, often leading to difficulty in diagnosis , 16. TheUWF FAF imaging using Optos scanning laser ophthalmoscopy system provides a non-invasive technique to obtain panoramic, high-quality images in a short span of time , 24. TheVisual field loss is common among patients with BSCR. In a previous study by Thorne et al., 75 % of patients had abnormal field scores for the I4 isopter on GVF . QuantitIn the index study, the typical patterns noted on FAF included peripapillary hypo-autofluorescence and scattered macular and extra-macular areas of hypo-autofluorescence, which is consistent with what is reported in literature , 15, 21.precede functional loss detected by GVF. Thus, detection of hypo-autofluorescence on FAF may help the clinician in predicting the areas of the retina that may develop loss of function in the future. Such concept is consistent with the observation that significant structural loss at the level of photoreceptors/RPE may be required before functional loss can occur. Our study results may also be partially explained from those observed in glaucoma. Although BSCR and glaucoma are different diseases with very dissimilar pathophysiological mechanisms, the process of visual loss may share certain similarities. In both the conditions, there is slowly progressive visual field loss and involvement of retinal photoreceptors, retinal nerve fiber layer, and the ganglion cells. In studies performed among glaucoma subjects, GVF testing has been shown to be insensitive to early visual field loss with 75 % subjects showing evidence of visual field defects on automated perimetry 1 year before appearing on manual GVF testing [One of the reasons for the differences in the findings of FAF and GVF may be because changes in FAF may  testing . Similar testing . Thus, f testing . Similar testing , sparselParallels can be drawn from glaucoma studies to further explain the poor structure-function (FAF-GVF) correlation observed in our study. Evidence suggests that comparing the available imaging and diagnostic techniques may be associated with a number of confounding factors that may limit our understanding of structure-function correlation . Test-reAnother reason for the poor correlation between FAF and GVF may be related to the natural history of BSCR. Literature suggests that there may be a lack of correspondence among various imaging tests in eyes with BSCR , 17, 18.Limitations of this study include the retrospective nature of analysis and small sample size. Due to the requirement for fixation, the present study only included patients with the ability to maintain fixation and may not correlate well with patients who have advanced disease and can no longer fixate. The requirement for fixation may also be a source of inclusion bias as patients with higher grades of GVF loss were excluded from the study. Approximately half the eyes were excluded from the analysis due to ungradable image quality or poor patient cooperation/fixation. Such exclusion may represent a source of bias and may affect the overall generalizability of the results. An important aspect to consider is the scale of measurement on GVF and FAF techniques. For example, a loss of 3 dB on GVF represents 50 % loss of sensitivity, since it is a logarithmic scale. On the other hand, FAF has been considered as an all-or-none scale. The difference in scale may represent a limitation in our ability to accurately assess the structure-function relationship . MoreoveThe results of this pilot study underscores the need to follow patients with BSCR using multi-modal imaging as each technique may contribute different information about the disease status. It is relevant to combine structural and functional testing to enhance our understanding of the disease process and relationship between visual loss and underlying tissue damage in diseases such as BSCR.


Many scholars have found that high glucose can activate the Wnt signaling pathway. However, the effects of activation of this pathway in the presence of high glucose levels during the progression of diabetes still remained unclear. Here, we provide a review of the study on the effects of high glucose state on the Wnt/β-catenin signal pathway and the influence of Chinese medicine on it.Diabetes mellitus (DM) is a metabolic disease characterized by chronic hyperglycemia and a series of complications. The Wnt/ Many scholars have focused on its important role during embryonic development [β-catenin signaling can be promoted by the highly conserved gene Fezf2 to stimulate neuronal differentiation during forebrain development [β-catenin signal pathway in the presence of high glucose levels and the influence of Chinese medicine on it.Diabetes mellitus (DM) is a heavy burden for patients worldwide, and the number of affected individuals is growing significantly. By 2030, it is estimated that it will affect almost 552 million . High glelopment , and tumelopment . It has elopment . Recent elopment . This paβ-catenin), nuclear transcription factor (TCFS/LEF), and a series of proteins [β-catenin signaling pathway is closely related to diabetic nephropathy [The Wnt signal pathway mainly includes the following: extracellular factor (Wnt), transmembrane receptors (frizzled), cytoplasmic protein , and the protein kinase A pathway . It is kβ-catenin accumulation through interaction between Wnt protein and cytoplasmic proteins, which interacted with transcription factors and attached to the DNA sequence of -YCTTTGWW to regulate its downstream gene transcription. The noncanonical pathway improves the levels of intracellular calcium by activating different elements of heterotrimeric G proteins to activate the Wnt/Ca2 + channels [β-catenin is the key factor of the canonical Wnt signaling pathway, which is a cytosolic protein that is degraded by the ubiquitin proteasome system by glycogen synthase kinase 3 beta (Gsk-3β) phosphorylation. It forms a complex after combining with LEF/TCF and Smad4, thereby promoting the latter into the nucleus and regulating the expression of specific target genes. It is the main effector molecule cytoplasm within the classical Wnt approach and is also associated with the intercellular junctions and cadherin [β-catenin in the cytoplasm determines the activation and inhibition of the canonical Wnt pathway [β phosphorylation can result in β-catenin accumulation in the nucleus and activation of the Wnt pathway in the cytoplasm. Therefore, increased expression of β-catenin suggests the activation of Wnt/β-catenin signaling pathway, and inhibitors of Gsk-3β are viewed as an activator of Wnt/β-catenin signal pathway [The canonical pathway resulted in channels . Among tcadherin , 20. The pathway . Inhibit pathway , 23.β-catenin signaling pathway, but the activation of this pathway is a protective response of organization or an injury is still controversial. The possible relationship between high glucose and the activation of this pathway is shown in β in the Wnt/β-catenin signal pathway was phosphorylation inhibited at high glucose levels, and the activation pathway can inhibit the degradation of apoptosis nuclear DNA, thus playing a protective role in a variety of vascular endothelial cells. Yang et al. [β-catenin signaling pathway is upregulated, and β-catenin as the core of the upstream gene APC and downstream gene c-Myc was expressed and upregulated in the islet regeneration process to promote the regeneration of damaged pancreatic islet cell. Sun et al. [β-catenin. The results showed that the activation of Wnt/β-catenin signaling pathway can promote the healing of diabetic wounds. Some scholars showed that in mouse podocytes early diabetic podocyte injury was caused by upregulation of transient receptor potential cation channel 6 (TRPC6), which is regulated by the canonical Wnt signalling pathway. This indicates that the Wnt/β-catenin signalling pathway may potentially be active in pathogenesis of TRPC6-mediated diabetic podocyte injury [Many scholars have confirmed that high glucose levels can activate the Wnt/g et al.  reportedn et al.  used a se injury .β-catenin protein expression on cells. They also used RT-PCR assay for the detection of β-catenin mRNA. The results showed that high glucose can induce the increased expression of β-catenin in rat peritoneal mesothelial cells, indicating that high glucose levels can upregulate the expression of β-catenin induced peritoneal injury leading to fibrosis. The research of Hwang et al. [β-catenin signal pathway was closely related to the occurrence of diabetic nephropathy renal interstitial fibrosis and that blocking or inhibiting this pathway might be a new target for treatment of diabetic nephropathy. Yan et al. [β-catenin and Wnt-1 expression by indirect immunofluorescence and Western blot analysis, and results showed that, after 12 h of high glucose exposure, podocyte Wnt-1 and activation of β-catenin expression began to increase and reached its peak at 24 h. The Wnt/β-catenin signaling pathway may be involved in the phenotypic transformation of podocytes induced by high glucose levels. García-Jiménez et al. [β-catenin signal pathway in tumor cells at high glucose levels may be one reason for certain cancers in DM patients. Li et al. [β-catenin protein. Results showed that, after 12 W, the expression of retinal and microvascular β-catenin protein increased significantly, and the extent of this increase is associated with the duration of DM, thereby suggesting that the occurrence of β-catenin protein may be involved in early diabetic retinopathy. However, the research was only limited to the detection of β-catenin protein and still could not fully explain the role of Wnt/β-catenin pathway. Portal-Núñez et al. [β-catenin signal pathway change and analysis of β-catenin protein expression by immunohistochemical method change. The results showed that β-catenin expression in both osteoblasts and bone cells in DM rats decreased significantly, which was not due to a decrease in bone cell activity. In addition, they also found that, in the osteoblast related β-catenin signal transduction process, transfer of β-catenin to the nucleus was significantly reduced, thus leading to failure of intracellular signal transduction. It proved that dysregulation of the Wnt/β-catenin pathway was involved in the occurrence and development of DM osteoporosis. Wang et al. [β-catenin and its downstream target gene WISP-1 in Wnt signal pathway in STZ induced DM rats myocardial tissue by immunohistochemical method. The results showed that both increased, thereby suggesting that the activation of the pathway was involved in DM induced myocardial injury. There were also reports that the protein and mRNA levels of Wnt2, β-catenin, and c-Myc were progressively increased 4, 8, and 12 weeks following DM. However, the expression of the endogenous Wnt inhibitor Dickkopf-1 was increased after STZ injection and then decreased as diabetic cardiomyopathy developed. Jia et al. [β-catenin was upregulated in proximal tubular epithelial cells under DN conditions both in vitro and in vivo. Injection of LRP5 and LRP6 antibodies suppressed activation of the Wnt pathway and decreased the formation of extracellular matrix in DN animal models, suggesting that Wnt/β-catenin signaling might be involved in tubular-interstitial fibrosis in DN. It was also reported that Wnt/β-catenin/GSK3β signaling pathway is activated in the development of diabetic cardiomyopathy [β-catenin signaling. In addition, some researchers have found that enhanced proliferation, accompanied by increased aerobic glycolysis, was detected in colorectal epithelium of patients with diabetes. β-Catenin accumulation with altered phosphorylation correlated with the proliferative changes [On the other hand, Huo et al.  used difg et al.  and Roong et al.  showed tn et al.  reportedn et al.  culturedz et al.  reportedi et al.  set up tz et al.  also obsg et al.  detecteda et al.  found thmyopathy . Qi et amyopathy  found th changes .β-catenin activation is a key component of arteriosclerotic physiology, particularly in diabetic arteriosclerosis [β-catenin activity in the vascular cells.Besides, as is known to all, T2DM is often associated with atherosclerosis. Nowadays, a lot of data have demonstrated that clerosis . Gaudio clerosis  found thβ-catenin signaling in DM are very complex and still unclear now. At present, researches about the effects of this signaling pathway on DM and a series of complications were focused on the activity of the Wnt/β-catenin signaling pathway involved in the regulation of morphological changes and pathogenesis in cells, its effect on high glucose induced cells apoptosis via the promotion of caspase-3 and poly (ADP-ribose) polymerase cleavage, its influence on diabetic wound healing, and its effect on the regulation of bone and vascular and other metabolic processes. It has been suggested that sustained Wnt/β-catenin expression is essential for its protective role against cellular damage, while abnormal activation of Wnt/β-catenin results in adverse effects and promotes the progression of DM.Mechanisms of activated Wnt/β-catenin gene in glomerular mesangial cells and the effects of Rhein on it by RT-PCR method. The results showed that, in the basic state, mesangial cells express a certain amount of Wnt/β-catenin. After glucose stimulation, the expression of the Wnt/β-catenin gene was increased, thereby suggesting that Rhein may inhibit the high glucose induced proliferation of mesangial cells by downregulation of Wnt/β-catenin gene expression. Huang et al. [β-catenin, epithelial cell marker protein of E-cadherin, and mesenchymal cell marker protein α-SMA, and RT-PCR was used to detect the mRNA expression of β-catenin, epithelial cell marker protein of E-cadherin. The results showed that the final concentration of 100 μmol/L tanshinone IIA can be significantly reduced by the ectopic expression of β-catenin, and the expression of β-catenin in nuclear protein and mRNA decreased significantly at this concentration, thereby indicating that the Wnt/β-catenin signaling pathway is involved in the high glucose induced transdifferentiation of renal tubular epithelial cells. Tanshinone IIA could inhibit this process by downregulating the expression of the Wnt/β-catenin signaling pathway activity and protecting the kidneys. Deng and Fang [ Astragalus group, the diabetic nephropathy model group + losartan treatment group, and the diabetic nephropathy model group + Astragalus combined with losartan treatment group. Immunohistochemistry and FQ-PCR methods were used to detect protein expression as well as expression of Wnt4, β-catenin, and TGF-β1 mRNA in renal interstitium. The results showed that Astragalus can protect the kidney by downregulating the expression of Wnt4, β-catenin, and TGF-β1 in renal interstitium. Duan et al. [ Astragalus, Angelica, Gorgon fruit, oriental water plantain rhizome, Rhodiola, etc.) on diabetic nephropathy rats induced by STZ. They found that, at the end of 12 W, the expression of Wnt pathway inhibition factor-secreted frizzled related protein-1 in renal tubular-interstital cells increased compared with the control group, and β-catenin expression appeared in the cytoplasm or (and) nucleus. PCR results showed increases in the two mRNA expression levels. After Yishen Capsule treatment, the secreted frizzled related protein-1 in tubulointerstital cells further increased compared to the control, while β-catenin mRNA expression was reduced, which indicated that the Yishen Capsule could upregulate the Wnt pathway inhibiting factor, which played a role in renal protection in diabetic nephropathy rats.Chinese medicine has multitarget and multiangle effects, which has caused wide attention. Lv et al.  used theg et al.  divided and Fang  randomlyn et al.  observedβ-catenin signaling pathway was closely related to the neural stem cell proliferation and differentiation. There were also reports showing that dysregulation of the Wnt/β-catenin signaling pathway may be responsible for inefficient myelin repair after human nervous system lesions [β-catenin siRNA found that β-catenin played a key role in promoting the differentiation from neural stem cells into neurons in high-pressure oxygen in vitro [β-catenin signal transduction pathway expression in kidney of diabetic rats. They found that Wnt-1 and β-catenin expression increased in kidney of high-fat high-sugar induced type 2 diabetic rats. Compared with diabetic group, the level of serum creatinine, blood urea nitrogen, 24 h urine protein, mean glomerular area, and mean glomerular volume were decreased, renal histopathology was improved, and expression of Wnt-1 and β-catenin mRNA and protein was reduced in TGP group. These results showed Wnt/β-catenin abnormal activation in kidney of type 2 diabetic rats; TGP can improve kidney damage in diabetic rats and delay the development of diabetic nephropathy by inhibiting the Wnt/β-catenin signaling pathway [In addition, the study by Lange et al.  found th lesions . Osakada lesions  found thin vitro . Scholar pathway .β-catenin signaling pathway as a protein interaction network can be activated by high glucose levels. However, the relationship between the pathway and DM as well as the appearance of its complications is still unclear and needs future investigation to better clarify the accurate role. Currently, the study of the activated Wnt/β-catenin signaling pathway at high glucose levels is relatively more concentrated in animal experiments in vitro, and most of these experimental methods are used to detect the expression of the pathway's upstream or downstream protein and mRNA. Some parts of the comprehensive and systemic study of the pathway are still poorly understood. Regulation of specific genes on this pathway and the exact mechanism are also unclear. In addition, studies of the activation of this pathway under high glucose conditions are mainly focused on diabetic nephropathy, diabetic retinopathy, diabetic osteoporosis, diabetic cardiomyopathy, and so on. However, the effects of this pathway on diabetic peripheral neuropathy are still unknown, thereby suggesting that there are still parts of the activation of the Wnt/β-catenin pathway at high glucose levels that are worth exploring. Further investigation into the role of Wnt signaling during DM will functionally find novel therapeutic target for DM.With the improvement of living standards, DM as a metabolic syndrome has a significant influence in the global scope, not to mention its peculiar set of complications. Many scholars have been exploring its pathogenesis, but that does not encompass the whole picture. The Wnt/At the same time, there are few studies on the effects of traditional Chinese medicine on regulating this pathway at high glucose levels. We look forward to these studies, as long as they reveal the mechanism of diabetes and its complications from a new angle, make full use of the multidirection, multitarget role of Chinese medicine, and explore the exact effective components from the numerous complexes. The goal is to develop more effective new measures and methods for treatment of diabetes and its complications as well as its prevention so that we can relieve the physical and mental suffering of diabetic patients.


WNT5B, a component of the noncanonical/planar cell polarity Wnt pathway, it upregulated its secretion. Furthermore, TGFβ1 not only decreased WNT2B expression, but also inhibited FTRI-induced photoreceptor differentiation of hMSC, as determined by expression of the photoreceptor markers NR2E3, RHODOPSIN, and RECOVERIN. Inhibition of TGFβ1 signaling by an ALK5 inhibitor prevented TGFβ1-induced changes in the expression of the two Wnt ligands examined. More importantly, inhibition of the canonical WNT signaling by XAV-939 prevented FTRI-induced photoreceptor differentiation. These observations suggest that TGFβ may play a key role in preventing neural differentiation of hMSC and may constitute a potential target for induction of endogenous regeneration of the human retina.Müller glia are responsible for the retina regeneration observed in zebrafish. Although the human retina harbors Müller glia with stem cell characteristics, there is no evidence that they regenerate the retina after disease or injury. Transforming growth factor-β (TGFβ) and Wnt signaling regulate retinal neurogenesis and inflammation, but their roles in the neural differentiation of human Müller stem cells (hMSC) are not known. We examined hMSC lines in vitro for the expression of various Wnt signaling components and for their modulation by TGFβ1, as well as the effect of this cytokine on the photoreceptor differentiation of these cells. Culture of hMSC with a combination of factors that induce photoreceptor differentiation of hMSC , markedly upregulated the expression of components of the canonical Wnt signaling pathway, including WNT2B, DKK1, and active β-CATENIN. Although FTRI did not modify mRNA expression of  Althoug neurons . Unlike  neurons . The rea neurons . Many of neurons , which mTGFβ signaling mediated through Smad 2/3, which is controlled by transcriptional corepressors such as Tgif1 and Six3b, has been demonstrated to regulate Müller glia-derived photoreceptor regeneration in the adult zebrafish . In addiAlthough interaction of these signaling pathways in fish and amphibians as well as small mammals during development and adult regeneration are documented , there i10 concentrations ranging from 0.1 to 100 ng/mL. To investigate the effect of TGFβ1 on the neural differentiation of hMSC, cells were cultured for 7 days in flasks coated with basement membrane protein (bMP) (Sigma-Aldrich) in the presence or absence of TGFβ1 (50 ng/mL) and factors known to induce the expression of photoreceptor cell markers as previously published by our group [Human Müller stem cell lines (hMSC) developed in our laboratory were cultured as previously described . Brieflyur group . Brieflyur group . In seleTotal RNA was isolated using the RNeasy Mini Plus Kit (Qiagen) according to the manufacturer's instructions. Quantification of total RNA was performed using a NanoDrop spectrophotometer (Thermo Scientific). Reverse transcription (RT) was performed using 1 μg RNA as per the manufacturer's instructions (Life technologies). Polymerase chain reaction (PCR) was then performed using the primer sequences shown in www.jacksonimmuno.com/) for 1 h at room temperature. The blots were visualized using the enhanced chemiluminescence advanced detection reagent ECL2 (Thermo Scientific) and a Fujifilm Imager .Cell lysates were prepared using RIPA buffer (Thermo Scientific) containing protease inhibitor cocktail (Sigma). Gels and buffer systems  were used for western blot analysis, as previously described ,21 BriefThe hMSC were grown for 5–6 days in DMEM with 2% FCS on bMP-coated flasks in the presence or absence of either human recombinant TGFβ1 (50 ng/mL) or FTRI to induce photoreceptor differentiation as indicated above . SupernaThe hMSC were grown for 7 days in DMEM supplemented with 2% FCS on bMP-coated glass coverslips placed in 24-well plates in the presence or absence of TGFβ1 and/or inhibitors. After 6–7 days cells were fixed in 4% PFA for 20 min. Slides were blocked for 1 h at room temperature using Roche blocking reagent . Primary antibodies against rabbit anti-NR2E3 , rabbit anti-recoverin  were diluted in blocking reagent and incubated overnight at 4°C. Primary antibody labeling was detected using donkey anti-rabbit or anti-mouse antibodies labeled with AlexaFluor 488  for 2 h at room temperature. DAPI was used to counterstain the cell nuclei, and slides were mounted with VECTASHIELD . Fluorescent images were captured with identical exposure times using a Zeiss LSM710 confocal microscope and identically processed using Carl Zeiss Zen imaging software (Carl Zeiss Microscopy GmbH).t-test or one-way repeated measures ANOVA. The standard error of the mean was plotted as error bars on bar charts and a probability of <0.05 was considered to be significant.For PCR and western blots, the integrated optical density of each band was calculated using ImageJ software . The optical density of each band was normalized by dividing the optical density of the sample by the optical density of its corresponding control gene (β-actin) band. Histograms were generated to represent the pixel intensities of each band. For ELISA, a standard curve was plotted of diluted standard solutions for each experiments  using Curve Expert 1.3 software. The concentration of proteins in each of the samples was identified by extrapolation to the generated curve. Statistical analysis of all results was carried out using Graphpad Prism 5 software. Statistical differences were calculated using paired Student's WNT2B, WNT3A, WNT5B, WNT11, FZD1, FZD4, FZD7, and β-CATENIN under baseline conditions. Transcripts for WNT8B and FZD5 were not detected despite the use of three different primers and variations in assay parameters  in the expression of this gene, without showing significant differences among them (P < 0.05) in intracellular WNT2B protein levels as compared with cells cultured in medium alone. Interestingly, the levels of WN2B ligand present in culture supernatants were minimally detected in both control and TGFβ1-treated cells (below 1 pg/mL) and there were no differences between the two conditions . In agreement with the mRNA findings, culture of hMSC with TGFβ1 caused a significant increase (P < 0.05) in the intracellular levels of WNT5B protein as compared with cells cultured in medium alone  (DKK1 (P < 0.001) and secreted DKK1 protein (P < 0.05) compared to cells cultured with medium alone (DKK1 mRNA (P < 0.05) (P < 0.05) (Examination of the ratio of phosphorylated β-catenin over β-catenin protein expressions showed that the levels of phosphorylated β-catenin (which indicates that β-catenin is targeted for degradation) were increased by TGFβ1 in hMSC ( < 0.05) . Furtherum alone . To test < 0.05) , recombi < 0.05) .These findings indicate that different adult hMSC lines express various components of the Wnt signaling pathway and that TGFβ1 downregulates the canonical Wnt signaling ligand WNT2B as well as the active form of β-catenin, which are important for canonical Wnt signaling. Furthermore, upregulation of the noncanonical Wnt ligand WNT5B by TGFβ1 may indicate the potential of this cytokine to inhibit the canonical Wnt signaling pathway.WNT2B (P < 0.001), but did not modify the mRNA expression of WNT5B (P = 0.46) (P < 0.05) (P < 0.05) (DKK1 (P < 0.05)  . Given t = 0.46) . A signi < 0.05) . In addi < 0.05) . Further < 0.05) .These observations suggest that opposite to that seen with TGFβ1 alone, photoreceptor differentiation of hMSC induced by FTRI caused upregulation of the canonical Wnt signaling pathway in hMSC. This suggests that FTRI promotes signaling in hMSC through the Wnt canonical pathway.Having shown that photoreceptor differentiation of hMSC promotes activation of the canonical Wnt signaling pathway and that TGFβ1 downregulates canonical Wnt signaling components while upregulating the noncanonical WNT5B ligand  and 2, wP < 0.001) in WNT2B mRNA expression, as compared to hMSC cultured with FTRI alone (WNT5B mRNA expression (P < 0.01) as compared to cells cultured in the presence of FTRI alone (NR2E3 (P < 0.001), RECOVERIN (P < 0.05), and RHODOPSIN (P < 0.001) as compared to hMSC cultured with FTRI alone (NR2E3 (P < 0.01) and RECOVERIN (P < 0.01) when cells were cultured with FTRI in the presence of TGFβ1, as compared to cells cultured with FTRI alone . However, addition of SB431542 markedly inhibited this effect (P < 0.05) (WNT2B mRNA by TGFβ1 (P = 0.91)  was inhibited by addition of SB431542 (P < 0.01) (P = 0.14)  . Unlike  = 0.91) . This su < 0.01) . This co = 0.14) . These o10 concentrations of this compound caused a gradual increase in the expression of phosphorylated β-catenin. Although significant increase was only observed with concentrations above 10 nM (P < 0.05) (NR2E3 (P < 0.01) and RECOVERIN (P < 0.05) (P < 0.01) and RECOVERIN (P < 0.05) proteins in hMSC cultured with FTRI in the presence of XAV-939 (P < 0.01)  . Additio < 0.05) . This in < 0.01) . Taken tMüller glia, which are responsible for the spontaneous retina regeneration observed in zebrafish ,26, haveActivation of the TGFβ and Wnt signaling pathways require the expression of specific receptors on the cell surface, and as previously shown, mammalian Müller glia express TGFβ and Wnt receptors and their ligands ,33–35, fwww.omim.org/entry/606361) in mouse mammary cells, results in increased Wnt/β-catenin activity [wnt5b in zebrafish causes downregulation of the canonical Wnt target axin2, with consequent inhibition of tail fin regeneration [WNT2B and upregulation of WNT5B, is in accordance with the reported functions of this ligand in promoting the activation of the canonical Wnt signaling pathway in other species. This suggests that by increasing WNT5B expression, TGFβ1 may potentiate its inhibitory effect that prevents photoreceptor differentiation of hMSC. That FTRI upregulates the expression of WNT2B strongly suggests that the effect of these factors on the photoreceptor differentiation of hMSC may be exerted through the activation of the canonical Wnt signaling pathway.It has been documented that loss of TGFβ or Wnt5a  in cells cultured with FTRI, prevented photoreceptor differentiation of these cells. Activation of canonical Wnt signaling is associated with the maintenance and proliferation of retinal progenitors in the embryonic chick and mouse retina , while ller glia . Wnt sigler glia , and conler glia .To summarize, we have demonstrated that differentiation of hMSC into photoreceptors in vitro is dependent on the activation of the canonical Wnt signaling pathway and that TGFβ, which is highly upregulated during gliosis , modifie


Renal fibrosis is an inevitable outcome of end-stage chronic kidney disease. During this process, epithelial cells lose E-cadherin expression. β-Catenin may act as a mediator by accumulation and translocation to the nucleus. Studies have suggested that CIP4, a Cdc42 effector protein, is associated with β-catenin. However, whether CIP4 contributes to E-cadherin loss in epithelial cells by regulating β-catenin translocation is unclear. In this study, we investigated the involvement of CIP4 in β-catenin translocation. Expression of CIP4 was upregulated in renal tissues of 5/6 nephrectomized rats and mainly distributed in renal tubular epithelia. In TGF-β1-treated NRK-52E cells, upregulation of CIP4 expression was accompanied by reduced expression of E-cadherin. CIP4 overexpression promoted the translocation of β-catenin to the nucleus, which was accompanied by reduced expression of E-cadherin even without TGF-β1 stimulation. In contrast, CIP4 depletion by using siRNA inhibited the translocation of β-catenin to the nucleus and reversed the decrease in expression of E-cadherin. The interaction between CIP4 and β-catenin was detected. We also show that β-catenin depletion could restore the expression of E-cadherin that was suppressed by CIP4 overexpression. In conclusion, these results suggest that CIP4 overexpression represses E-cadherin expression by promoting β-catenin translocation to the nucleus. Cdc42 (cell division cycle 42 (GTP binding protein)) is a member of the Rho family of small GTPases and is an important regulator of actin polymerization and cytoskeleton reorganization. Cdc42-interacting protein 4 (CIP4), a downstream effector of Cdc42, is a 545-amino acid protein that can bind to Cdc42 and Wiskott-Aldrich syndrome protein (WASP) through a Cdc42-binding domain and an SH3 domain at the carboxyl terminus, and also contains a FER/CIP4 homology (FCH) domain, through which it interacts with tubulin ,2,3. RhoSlug, a member of the Snail family, repressing the expression of E-cadherin [Recent studies showed that TGF-β1 may induce loss of E-cadherin expression in certain epithelial cells . E-cadhecadherin ,13. in vitro.There is evidence that CIP4 has a physical connection with β-catenin and is critical for cell–cell adhesion in renal cell carcinoma . HoweverWe first examined the distribution of CIP4 in fibrotic renal tissue in the 5/6 subtotal nephrectomy model. Masson staining revealed glomerular sclerosis and interstitial fibrosis in the renal tissues of the 5/6-nephrectomized rats, but not in the renal tissues of the sham-operated rats A,B. Immup < 0.05), while there were no differences between the two groups in β-catenin expression . There were no differences between these two groups in β-catenin expression . TGF-β1 has been reported to cause reduced expression of E-cadherin in various types of epithelial cells, which is relevant to the pathogenesis of renal fibrosis ,11,14. Wpression B. in vivo and TGF-β1 treated NRK-52E cells in vitro. This was accompanied by decreased expression of E-cadherin, but not of β-catenin. Some studies have suggested that nuclear translocation of β-catenin could be observed during fibrosis in addition to the loss of E-cadherin expression [We detected increased expression of CIP4 in both renal tissue of 5/6 nephrectomized ratspression ,14. We np < 0.05). We then investigated protein expression of β-catenin, a component of the intercellular adhesive junction [p < 0.05)  D.p < 0.05).Due to the results above, we wondered whether CIP4 depletion could affect E-cadherin expression. We examined E-cadherin expression after CIP4 depletion by transfection with CIP4-siRNA. We found that CIP4 expression was only 50% of the control after CIP4 depletion A. E-cadhp < 0.05; Nuclear translocation and accumulation of β-catenin are known to activate specific target genes ,13. In op < 0.05), and neither the expression of E-cadherin nor that of CIP4 was different from that of parental cells or control siRNA-transfected cells  and diabetic nephropathy, resulting in tissue fibrosis ,20,21. Wcadherin ,23,24. T et al. [Snail protein levels, which engage in the transcriptional processes of epithelial phenotype switching. β-Catenin also translocates to the nucleus and forms a complex with T cell factor (TCF); the complex then binds to the target gene Slug and represses the E-cadherin promoter [We postulated that CIP4 regulates E-cadherin expression by promoting translocation of β-catenin to the nucleus. β-Catenin is a mediator that regulates cadherin expression, as noted above. Yook et al.  showed tpromoter . As we kOur study thus provides evidence that nuclear translocation of β-catenin can regulate the expression of E-cadherin in epithelial cells, and that CIP4 binds with β-catenin and potentially mediates translocation of β-catenin to the nucleus where finally it regulates the expression of E-cadherin. From these findings, we propose in the future to identify the specific region of the CIP4-β-catenin interaction and the underlying mechanism by which CIP4 overexpression promotes nuclear translocation of β-catenin, and whether or not over-expressed CIP4 regulates the expression E-cadherin through Sail gene family. Further studies are also needed to evaluate CIP4 as a therapeutic target in alleviating the development of chronic kidney diseases. In addition, CIP4 is an interacting protein of Cdc42, a member of Rho-like GTPases . The potIn summary, we found that CIP4 modulates the expression of E-cadherin in NRK-52E cells by regulating the nuclear translocation of β-catenin. Furthermore, E-cadherin expression was promoted by CIP4 overexpression and inhibited by CIP4 depletion. Our findings support the importance of CIP4 in regulating E-cadherin expression, and that CIP4 may be a potential therapeutic target in the treatment of chronic kidney diseases.The use and care of animals employed in our renal fibrotic model were in accordance with guidelines for animal care at our university and all relevant laws of China. This study was approved by the ethics committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Male adult Sprague-Dawley rats weighing 150 to 200 g were purchased from the Tongji Laboratory Animal Center . Each rat was housed in our animal facility, received humane care under pathogen-free conditions, and was fed a standard laboratory diet with unlimited access to water.n = 12) and a sham-operated group (n = 12). Briefly, the animals were anesthetized with 2% pentobarbital sodium (50 mg/kg body weight) by intraperitoneal injection. Body temperature was monitored and maintained at 37 °C. A dorsoventral incision parallel to the spinal cord was made to expose the left kidney, and the upper and lower poles (two-thirds) of the left kidney were removed. Subsequently, the right kidney was exposed and removed one week later. For the sham operation, animals underwent the same surgical procedure as above without removing any renal mass. All rats were euthanized 20 weeks later, after nephrectomy. Kidneys were immediately excised and fixed with 4% paraformaldehyde. Renal tissues were stained with Masson’s trichrome for histological examination.The rats were randomly divided into a 5/6 nephrectomized group .Cells of the normal rat renal tubular epithelial cell line NRK-52E  were cultured in Dulbecco’s modified Eagle’s medium/high glucose  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin. These cells were maintained in 5% CO3VO4·10H2O, 2 mM NaF, 2 mM Na2P2O4·10H2O, 10 mM β-glycerophosphate disodium salt (pH 8.0) and protease inhibitor cocktail . Nuclear proteins were obtained with a nuclear and cytoplasmic protein extraction kit . Protein concentration was determined by the Bradford protein assay. A 100-μg aliquot of protein was then subjected to SDS-polyacrylamide gel electrophoresis for separation and transferred onto polyvinylidene difluoride membranes. After transfer, the membranes were blocked with 5% non-fat milk and blotted. Primary antibodies were directed against CIP4 , β-catenin , E-cadherin and Lamin A/C , and GAPDH and β-actin . The bound antibody complexes were visualized by enhanced chemiluminescence , and quantitated by volume densitometry using Quantity One software  with normalization to one of GAPDH, β-actin (Actb), or Lamin A/C.Cells were lysed in buffer  according to the manufacturer’s instructions. Cells were plated at 90% confluence in 6-well plates without antibiotics one day before transfection. These cells were washed and seeded in growth medium with Zeocin for 14 days to enable selection. Zeocin-resistant clones were then harvested and analyzed.To silence the expression of CIP4 and β-catenin, short-interfering RNA (siRNA) specific for CIP4 and β-catenin of rats, and control siRNA, were purchased from Ribobio . NRK-52E cells were transfected with siRNA by using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were analyzed 72 h after transfection.Cells were lysed as described above. Equal volumes of cell lysates were pre-incubated with anti-CIP4 antibodies  at 4 °C for 1 h and then incubated overnight with 20 μL Protein A/G beads (Santa Cruz Biotechnology). The beads were then washed 4× with lysis buffer, boiled in Laemmli loading buffer, and protein was collected and immunoblotted with anti-β-catenin and anti-CIP4 antibodies as described above.For immunohistochemical studies, paraffin sections were incubated with primary anti-CIP4 antibody at 4 °C overnight. The sections were then incubated with biotinylated goat anti-rabbit IgG antibody as the secondary antibody, and antibody reactions were visualized by using diaminobenzidine . Images from the microscope were captured with a Nikon DXM1200 digital camera.t-tests and one-way analysis of variance (ANOVA) followed by a post-hoct-test. Analyses were performed using SPSS software (version 15.0). Significance was assessed at p < 0.05.Results are expressed as mean ± s.d. Data were analyzed by


PLOS Computational Biology.This is a “Topic Page” article for self-replicating molecules connected in a cyclic, autocatalytic manner. It was introduced in an ordinary differential equation (ODE) form by the Nobel Prize winnerManfred Eigen in 1971 [Peter Schuster [error threshold problem encountered during modelling of replicative molecules that hypothetically existed on the primordial Earth (see: abiogenesis). The hypercycle is a special case of the replicator equation [information capacity, and selection against parasitic branches.A hypercycle is an abstract model of organization of Schuster ,3. It waequation . The mosmacromolecules. In the hypercycle, all molecules are linked such that each of them catalyses the creation of its successor, with the last molecule catalysing the first one. In such a manner, the cycle reinforces itself  (nucleotide sequences I (I stands for intermediate) and the same number of polypeptide chains E (E stands for enzyme). Sequences I have a limited chain length and carry the information necessary to build catalytic chains E. The sequence Ii provides the matrix to reproduce itself and a matrix to build the protein Ei. The protein Ei gives the catalytic support to build the next sequence in the cycle, Ii+1. The self-replicating sequences I form a cycle consisting of positive and negative strands)that periodically reproduce themselves. Therefore, many cycles of the +/− nucleotide collectives are linked together by the second-order cycle of enzymatic properties of E, forming a catalytic hypercycle. Without the secondary loop provided by catalysis, I chains would compete and select against each other instead of cooperating. The reproduction is possible thanks to translation and polymerization functions encoded in I chains. In his principal work, Manfred Eigen stated that the E coded by the I chain can be a specific polymerase or an enhancer) (or a silencer)) of a more general polymerase acting in favour of formation of the successor of nucleotide chain I [n must catalyse I1 formation for some integer n > 1.Analysis of potential molecules that could form the first hypercycles in nature prompted the idea of coupling an information carrier function with enzymatic properties. At the time of the hypercycle theory formulation, enzymatic properties were attributed only to slation) . The pro chain I . MoreoveRNA matrices existing among the quasispecies. This RNA-dependent RNA polymerase catalysed the replication of sequences that had specific motifs recognized by this replicase. The other RNA matrices, or just one of their strands, provided translational products that had specific anticodons and were responsible for unique assignment and transportation of amino acids.During their research, Eigen and Schuster  also conAnother concept devised by Eigen and Schuster was a model in which each RNA template’s replication was catalysed by its own translational product; at the same time, this RNA template performed a transport function for one amino acid type. Existence of more than one such RNA template could make translation possible.abundances).Nevertheless, in both alternative concepts, the system will not survive due to the internal competition among its constituents. Even if none of the constituents of such a system is selectively favoured, which potentially allows coexistence of all of the coupled molecules, they are not able to coevolve and optimize their properties. In consequence, the system loses its internal stability and cannot live on. The reason for inability to survive is the lack of mutual control of constituent dynamics of the elementary hypercycle can be modelled using the following differential equation [The equation :x˙i=xi, which guarantees that the total concentration is constant. Production and degradation rates are expressed in numbers of molecules per time unit at unit concentration (xi = 1). Assuming that at high concentration x the term ki can be neglected, and, moreover, in the hypercycle, a template can be replicated only by itself and the previous member of the cycle, the equation can be simplified to:In the equation above, i (with concentration xi) and polypeptides Ei (with concentration yi) [kinetics of nucleotide synthesis follows a Michaelis–Menten-type reaction scheme in which the concentration of complexes cannot be neglected. During replication, molecules form complexes IiEi-1 (occurring with concentration zi). Thus, the total concentration of molecules (xi0 and yi0) will be the sum of free molecules and molecules involved in a complex:A hypercycle with translation consists of polynucleotides Ition yi) . It is aThe dynamics of the hypercycle with translation can be described using a system of differential equations modelling the total number of molecules:cE and cI are total concentrations of all polypeptides and all polynucleotides, ϕx and ϕy are dilution fluxes, ki is the production rate of polypeptide Ei translated from the polynucleotide Ii, and fi is the production rate of polynucleotide Ii synthesised by the complex IiEi-1 (through replication and polymerization).In the above equations, origin of the genetic code.Coupling nucleic acids with proteins in such a model of hypercycle with translation demanded the proper model for the origin of translation code as a necessary condition for the origin of hypercycle organization. At the time of hypercycle theory formulation, two models for the origin of translation code were proposed by Crick and his collaborators . These wEigen made several assumptions about conditions that led to the formation of the first hypercycles . Some ofI—which is relatively easy to achieve, taking into account the quasispecies properties—is that the one chain I improves the synthesis of the similar chain I’. In this way, the existence of similar sequences I originating from the same quasispecies population promotes the creation of the linkage between molecules I and I’.In the process described above, the fact that the first hypercycles originated from the quasispecies population (a population of similar sequences) created a significant advantage. One possibility of linking different chains equilibrium or a state with oscillating concentrations of each type of chain I, but with the total concentration of all chains remaining constant. In this way, the system consisting of all chains can be expressed as a single, integrated entity. During the formation of hypercycles, several of them could be present in comparable concentrations, but very soon, a selection of the hypercycle with the highest fitness value will take place [environment, and the selection based on it is very sharp. After one hypercycle wins the competition, it is very unlikely that another one could take its place, even if the new hypercycle would be more efficient than the winner. Usually, even large fluctuations in the numbers of internal species cannot weaken the hypercycle enough to destroy it. In the case of a hypercycle, we can speak of one-for-ever selection, which is responsible for the existence of a unique translation code and a particular chirality [After formation, a hypercycle reaches either an internal ke place . Here, thirality .Eörs Szathmáry and Irina Gladkih showed that an unconditional coexistence can be obtained even in the case of a non-enzymatic template replication that leads to a subexponential or a parabolic growth [The above-described idea of a hypercycle’s robustness results from an exponential growth of its constituents caused by the catalytic support. However, c growth . This coFrom the mathematical point of view, it is possible to find conditions required for cooperation of several hypercycles. However, in reality, the cooperation of hypercycles would be extremely difficult, because it requires the existence of a complicated multi-step biochemical mechanism or an incorporation of more than two types of molecules. Both conditions seem very improbable; therefore, the existence of coupled hypercycles is assumed impossible in practice.Inew created by the mutation must be better recognized by one of the hypercycle's members Ii than the chain Ii+1 that was previously recognized by it. Secondly, the new member Inew of the cycle has to better catalyse the formation of the polynucleotide Ii+1 that was previously catalysed by the product of its predecessor Ii. In theory, it is possible to incorporate into the hypercycle mutations that do not satisfy the second condition. They would form parasitic branches that use the system for their own replication but do not contribute to the system as a whole. However, it was noticed that such mutants do not pose a threat to the hypercycle, because other constituents of the hypercycle grow nonlinearly, which prevents the parasitic branches from growing.Evolution of a hypercycle ensues from the creation of new components by the mutation of its internal species. Mutations can be incorporated into the hypercycle, enlarging it if, and only if, two requirements are satisfied. First, a new information carrier Cl is the total concentration of all polynucleotide chains belonging to a hypercycle Hl, C is the total concentration of polynucleotide chains belonging to all hypercycles, ql is the rate of growth, and ϕ is a dilution flux that guarantees that the total concentration is constant. According to the above model, in the initial phase, when several hypercycles exist, the selection of the hypercycle with the largest ql value takes place. When one hypercycle wins the selection and dominates the population, it is very difficult to replace it, even with a hypercycle with a much higher growth rate q.According to the definition of a hypercycle, it is a nonlinear, dynamic system, and, in the simplest case, it can be assumed that it grows at a rate determined by a system of quadratic differential equations. Then, the competition between evolving hypercycles can be modelled using the differential equationmembrane [compartments, a genome integration of the hypercycle can proceed by linking its members into a single chain, which forms a precursor of a genome. After that, the whole individualized and compartmentalized hypercycle can behave like a simple self-replicating entity. Compartmentalization provides some advantages for a system that has already established a linkage between units. Without compartments, genome integration would boost competition by limiting space and resources. Moreover, adaptive evolution requires the package of transmissible information for advantageous mutations in order not to aid less-efficient copies of the gene. The first advantage is that it maintains a high local concentration of molecules, which helps to locally increase the rate of synthesis. Secondly, it keeps the effect of mutations local, while at the same time affecting the whole compartment. This favours preservation of beneficial mutations, because it prevents them from spreading away. At the same time, harmful mutations cannot pollute the entire system if they are enclosed by the membrane. Instead, only the contaminated compartment is destroyed, without affecting other compartments. In that way, compartmentalization allows for selection for genotypic mutations. Thirdly, membranes protect against environmental factors because they constitute a barrier for high-weight molecules or UV irradiation. Finally, the membrane surface can work as a catalyst.Hypercycle theory proposed that hypercycles are not the final state of organization, and further development of more complicated systems is possible by enveloping the hypercycle in some kind of membrane ,3. AfterDespite the above-mentioned advantages, there are also potential problems connected to compartmentalized hypercycles. These problems include difficulty in the transport of ingredients in and out, synchronizing the synthesis of new copies of the hypercycle constituents, and division of the growing compartment linked to a packing problem.Carsten Bresch and coworkers raised an objection that hypercyclic organization is not necessary if compartments are taken into account [In the initial works, the compartmentalization was stated as an evolutionary consequence of the hypercyclic organization.  account . They pr account . This pr account  that ass account . Neverthdirected evolution [Watson-Crick pairing and, therefore, may provide greater sequence generality. Various other experiments have shown that, besides bearing polymerase properties, ribozymes could have developed other kinds of evolutionarily useful catalytic activity, such as synthase, ligase, or aminoacylase activities [peptide bonds might have been crucial to inventing translation. An RNA ligase, in turn, could link various components of quasispecies into one chain, beginning the process of a genome integration. An RNA with a synthase or a synthetase activity could be critical for building compartments and providing building blocks for growing RNA and protein chains as well as other types of molecules. Many examples of this kind of ribozyme are currently known, including a peptidyl transferase ribozyme [nucleotide synthetase [acylation reaction and makes it the smallest ribozyme that has been discovered. It supports a peptidyl-RNA synthesis that could be a precursor for the contemporary process of linking amino acids to tRNA molecules. An RNA ligase’s catalytic domain, consisting of 93 nucleotides, proved to be sufficient to catalyse a linking reaction between two RNA chains [acyltransferase ribozyme 82 nucleotides long was sufficient to perform an acyltransfer reaction [ligation of smaller RNA chains performed by the first RNA ligases resulted in a longer chain with the desired catalytically active polymerase domain.At the time of the hypercycle theory formulation, ribozymes were not known. After the breakthrough of discovering RNA’s catalytic properties in 1982 ,25, it wvolution . Neverthvolution . While ivolution . It was tivities . Ribozymribozyme , a ligasribozyme ,35, and nthetase . A transnthetase , which iA chains . Similarreaction . Altogetreaction —they didreaction . It is mautocatalytic set [Azoarcus group I intron ribozyme that, when fragmented, has an ability to self-assemble by catalysing recombination reactions in an autocatalytic manner. They mutated the three-nucleotide-long sequences responsible for recognition of target sequences on the opposite end of the ribozyme  as well as these target sequences. Some genotypes could introduce cooperation by recognizing target sequences of the other ribozymes, promoting their covalent binding, while other selfish genotypes were only able to self-assemble. In separation, the selfish subsystem grew faster than the cooperative one. After mixing selfish ribozymes with cooperative ones, the emergence of cooperative behaviour in a merged population was observed, outperforming the self-assembling subsystems. Moreover, the selfish ribozymes were integrated into the network of reactions, supporting its growth. These results were also explained analytically by the ODE model and its analysis. They differ substantially from results obtained in evolutionary dynamics [oligomers, could be sufficient at the pre-life stage to spark off life. This could happen because coupling the synthesis of short RNA fragments by the first ribozymal polymerases to a system capable of self-assembly not only enables building longer sequences but also allows exploiting the fitness space more efficiently with the use of the recombination process. Another experiment performed by Hannes Mutschler et al. [Forty years after the publication of Manfred Eigen’s primary work dedicated to hypercycles, Nilesh Vaidya and colleagues showed experimentally that ribozymes can form catalytic cycles and networks capable of expanding their sizes by incorporating new members . Howeverytic set . Earlierytic set . In theidynamics . Accordir et al.  showed tr et al. . Apart fJohn Maynard Smith raised an objection that the catalytic support for the replication given to other molecules is altruistic [Karl Sigmund [stochastic events and break the positive feedback loop that sustains the hypercycle. The extinction of the hypercycle then follows. It was also emphasized that a hypercycle size of up to four is too small to maintain the amount of information sufficient to cross the information threshold.The hypercycle concept has been continuously studied since its origin. Shortly after Eigen and Schuster published their main work regarding hypercycles, truistic . Therefo Sigmund  indicatePaulien Hogeweg, and later Nobuto Takeuchi, studied the replicator equations with the use of partial differential equations [Robert May, who noticed that an emergent rotating spiral wave pattern, which was observed during computational simulations performed on cellular automata, proved to be stable and able to survive the invasion of parasites if they appear at some distance from the wave core [Several researchers proposed a solution to these problems by introducing space into the initial model either explicitly ,13,46,47quations  and cellquations , methodsquations ,49. Theyave core . UnfortuAnother model based on cellular automata, taking into account a simpler replicating network of continuously mutating parasites and their interactions with one replicase species, was proposed by Takeuchi and Hogeweg  and exhiThe version history of the text file and the peer reviews (and response to reviews) are available as supporting information in S1 Text(XML)Click here for additional data file.S2 TextHuman-readable versions of the reviews and authors' responses are available as comments on this article.(XML)Click here for additional data file.