However, its prognostic significance for patients with CRC remains controversial.β-catenin expression and clinicopathological features and prognosis was analyzed.Identical search strategies were used to search relevant literatures in the PubMed, Embase and Web of Science databases. The correlation between β-catenin overexpression in the nucleus was significantly associated with disease free survival (DFS)  and overall survival (OS) for CRC patients . However, there was no significant association between β-catenin expression in the cytoplasm and OS . The combined odds ratio (OR) of β-catenin in the nucleus indicated that β-catenin overexpression was associated with advanced stage CRC  and metastasis of CRC . β-catenin overexpression in the nucleus had no correlation with the tumor site (colon or rectum), differentiation grade, lymph node status or depth of invasion. The pooled ORs were 1.09 , 1.27, 0.71 and 0.82.A total of 18 studies met the inclusion criteria, which comprised 3665 cases. Meta-analysis suggested that β-catenin overexpression in the nucleus, rather than in the cytoplasm, appeared to be associated with progress disease and a worse prognosis for CRC patients.This study showed that  Colorectal cancer (CRC) is the third most common human malignancy and the second highest cause of cancer-related death worldwide β-catenin protein in the nucleus. β-catenin maintains a low cytoplasmic concentration through the destruction complex when the Wnt signaling pathway is unactivated. Otherwise, the destruction complex is dissolved and β-catenin accumulates in the cell and undergoes translocation to the nucleus, where it activates the expression of target genes, such as CyclinD1, c-Myc, CD44 and Survivin, in conjunction with the TcF/LEF transcription molecule family. Thus, the level of β-catenin in the nucleus is an indicator of an active Wnt signaling pathway or CCICs β-catenin in the nucleus is expected to be a useful biomarker associated with disease progression and poor prognosis in CRC. Moreover, some researchers suggest that the accumulation of cytoplasmic β-catenin serves as a predictor of metastasis of CRC β-catenin detected by immunohistochemistry and patient survival are highly variable and contradictory. Therefore, it is necessary to analyze the data on β-catenin and CRC to draw a reasonable conclusion about its prognostic significance.Increasing evidence suggests that the existence of a small subset of bulk cancer cells, termed cancer initiating cells (CICs), are responsible for tumor progression, therapy resistance and disease relapse. Self-renewal and multilineage differentiation potential are two main traits of CICs. Since O’Brien and Ricci-Vitiani identified a CD133 positive subtype as colorectal cancer initiating cells (CCICs) in 2007, the molecular mechanisms sustaining CCICs have been slowly understood β-catenin expression and the prognosis of corresponding patients. The results showed that overexpression of β-catenin in the nucleus, rather than in the cytoplasm, was associated with progress disease and worse prognosis. The meta-analysis suggested that postoperative detection of β-catenin expression in CRC would help us develop better therapy strategies, distinguish high risk populations from the patients undergoing surgery and make better follow-up plans.In this study, we conducted a meta-analysis to investigate β-catenin,” “Axin Signaling Complex,” “Wnt Signaling Pathway,” “Colorectal Neoplasms,” “Colorectal Cancer” and “prognosis.” The citation lists associated with all the studies were used to identify additional eligible studies. The reviews and bibliographies were also manually inspected to find related articles.We carried out a search of the PubMed, Embase and Web of Science databases using the following terms and all possible combinations: “β-catenin expression evaluated in the human CRC tissues; (2) evaluation of the relationships between β-catenin expression and CRC pathological features or prognosis; (3) β-catenin expression examined by immunohistochemistry; (4) English language publications; and (5) sufficient information provided to estimate the hazard ratio (HR) or odds ratio (OR) and their 95% confidence intervals (CIs).The search results were included in our meta-analysis if they met the following inclusion criteria: (1) The following articles were excluded: (1) letters, case reports, reviews, and conference abstracts without original data; (2) non-English language articles; (3) articles from which the relevant data could not be extracted; and (4) overlapping articles or ones with duplicate data.β-catenin positive, relationship between β-catenin and survival and adjuvant therapy condition of patients. Controversial problems were arbitrated by the third investigator (XJH). Study quality was assessed independently by two investigators (HX and JMY) according to the Newcastle–Ottawa quality assessment scale All data were extracted independently by two authors (HX and JMY). For each study, the following characteristics were extracted: first author’s name, publication date, number of patients, gender of patients, tumor site, tumor stage, research technique used, antibody source, definition of β-catenin expression and pathological features into single categories: T1 and T2 stages, T3 and T4 stages, and well and moderate differentiation. ORs with 95% CIs were used to evaluate the association between β-catenin expression and clinicopathological factors, such as differentiation grade, Dukes’ stages, depth of invasion, lymph node status and metastasis. Survival data were extracted or calculated according to the methods described by Parmar http://digitizer.sourceforge.net/). HR and its variance were used to estimate the impact of β-catenin expression on OS and DFS. Heterogeneity across studies was evaluated using a Chi-square-based Q statistical test 2 statistic was also calculated to quantify the proportion of the total variation due to study heterogeneity We combined the data on β-catenin expression and pathological features or disease free survival (DFS)/overall survival (OS) investigated. Immunohistochemistry was used to detect β-catenin expression in all the publications, but the sources of primary antibodies came from different companies.We found 168 studies potentially eligible for inclusion based on the title Figure 1Each of the 18 eligible studies included in our meta-analysis was assessed for quality according to the Newcastle–Ottawa Scale (NOS). NOS assessed eight items of methodology, which were categorized into the three dimensions of selection, comparability, and outcome. A maximum score of 1 was awarded for each item with the exception of the item related to comparability that allowed for scores of 2. For quality, scores ranged from 0 (lowest) to 9 (highest), and studies with scores of 6 or more were rated as high quality. Fourteen of the included studies obtained scores of 6 or more in methodological assessment, indicating that they were of high quality Table 1.β-catenin expression in the nucleus with OS. The pooled HR was 1.55  (2 71.5% P = 0.000). Three studies assessed the association of β-catenin expression in the nucleus with DFS; the pooled HR was 1.87  (2 0% P = 0.412). These results suggested that β-catenin overexpression in the nucleus was significantly correlated with a worse prognosis of CRC and that β-catenin overexpression in the nucleus was an independent prognostic factor in CRC. We assessed three eligible studies and found that there was no significant association between β-catenin overexpression in the cytoplasm with OS; the combined HR was 1.04  without heterogeneity (I2 31.3% P = 0.233)  without heterogeneity (I2 0% P = 0.414) (2 0% P = 0.45) when the definition of β-catenin positive was a percentage  (2 79.6% P = 0.008 and I2 56.4% P = 0.076). It indicated that the differences of patient ethnicity and evaluation standards contributed to heterogeneity in the results.To explain the heterogeneity in OS, subgroup analysis was performed by the study location, source of primary antibodies, definition of β-catenin overexpression in the nucleus, rather than in the cytoplasm, influenced survival of CRC patients.Taken together, these results suggested that β-catenin expression in the nucleus with Dukes’ stages. The pooled OR was 0.71  (2 40.5% P = 0.121). This result suggested that β-catenin overexpression in the nucleus was associated with the progression of CRC. Five studies assessed the correlation of β-catenin overexpression in the nucleus with metastasis. The pooled OR was 0.49 , indicating that β-catenin overexpression in the nucleus was associated with metastasis of CRC (β-catenin overexpression in the nucleus had no relation with the tumor site (colon or rectum), differentiation grade, lymph node status or depth of invasion. The pooled ORs were 1.09 , 1.27 , 0.71 and 0.82  .Seven studies evaluated the correlation of β-catenin overexpression in the cytoplasm and Dukes’ stages and lymph node status. The combined ORs were 0.87  and 0.78 .There was no significant association between S4) for the studies included in our meta-analysis.Egger’s test indicated that there was no evidence of significant publication bias after assessing the funnel plot  family of transcription factors. Under physiological conditions, Wnt activity is crucial for intestinal stem cells and crypt homeostasis. However, Wnt signaling also plays a key role in CCICs maintenance, which is the origin of tumor progression, therapy resistance and disease relapse.β-catenin accumulation, cell-cycle arrest and epithelial–mesenchymal transitions (EMT). Notably, nuclear β-catenin was predominantly observed in the invasive front of CRC tissue. These observations concur with our finding that β-catenin overexpression in the nucleus was significantly associated with metastasis and worse prognosis of CRC. Some studies have also shown that the accumulation of β-catenin in the cytoplasm and nuclear translocation were inseparable processes and that some mechanisms were involved in the nuclear cytoplasmic shuttling of β-cateninβ-catenin cytoplasmic data and found that β-catenin overexpression in the nucleus, rather than in the cytoplasm, influenced the survival of CRC patients. In fact, some investigators have argued that overexpression of β-catenin in the nucleus, rather than in the cytoplasm, might reflect β-catenin transactivating activity β-catenin was not accompanied by nuclear accumulation β-catenin overexpression in the cytoplasm had no relationship with the prognosis of CRC.It has been shown that migrating cancer stem cells (MCSCs), which play a crucial role in the metastasis of CRC, usually undergo nuclear 2 71.5% P = 0.000). When the analysis was limited to studies of Asia, heterogeneity was not detected (I2 0% P = 0.414). Heterogeneity was also not detected (I2 0% P = 0.45) when the analysis was limited to studies that defined β-catenin positive using a percentage. However, when the analysis of OS was limited to studies with primary antibodies from the same company and with adjuvant therapy, heterogeneity still existed (I2 79.6% P = 0.008 and I2 56.4% P = 0.076). This finding suggested that primary antibodies and adjuvant therapy did not contribute to heterogeneity in the results. These results suggest that the heterogeneity in this study could be partially explained by patient ethnicity and evaluation standards. Although meta-analysis is robust, certain limitations exist in this study. First, the study included in our meta- analysis was restricted only to articles published in English, which probably provided additional bias. Second, methodological differences of immunohistochemistry may contribute to heterogeneity. We could not perform subgroup analysis to explore this influence because few studies offered the concrete data. Third, HRs calculated from data or extrapolated from survival curves might be less reliable than direct analysis of variance.In this meta-analysis, we dealt with highly significant heterogeneity between the 18 studies. This heterogeneity could potentially affect the meta-analysis results. We only included studies that used immunohistochemistry to reduce heterogeneity as much as possible. However, the source and dilution of primary antibodies, evaluation standards, study location and adjuvant therapy conditions were quite different across studies, creating significant heterogeneity. Accordingly, we used random effects models to analyze the data, but the models did not identify the source of heterogeneity. To clarify the source of heterogeneity in this study, we performed stratified analysis according to study location, source of primary antibodies, evaluation standards and adjuvant therapy condition. When the analysis of OS was performed without consideration of these other factors, heterogeneity was detected  in the meta-analysis: the relationship between the effect size of individual studies  and the precision of the study estimate .(TIF)Click here for additional data file.Figure S2Funnel plot to assess publication bias. Egger’s publication bias plot showed no publication bias for studies regarding β-catenin expression in the nucleus and overall survival (OS) in the meta-analysis.(TIF)Click here for additional data file.Figure S3Funnel plot to assess publication bias. Egger’s publication bias plot showed the presence of publication bias for studies regarding β-catenin expression in the nucleus and Dukes’ stages in the meta-analysis.(TIF)Click here for additional data file.Figure S4Funnel plot to assess publication bias. Egger’s publication bias plot showed no publication bias for studies regarding β-catenin expression in the nucleus and metastasis in the meta-analysis.(TIF)Click here for additional data file.Table S1PRISMA 2009 checklist.(DOC)Click here for additional data file.


To this end, HERG was expressed in Xenopus oocytes with or without ß-catenin and the voltage-gated current determined utilizing the dual electrode voltage clamp. As a result, expression of ß-catenin markedly upregulated HERG channel activity, an effect not sensitive to inhibition of transcription with actinomycin D (10 µM). According to chemiluminescence, ß-catenin may increase HERG channel abundance within the oocyte cell membrane. Following inhibition of channel insertion into the cell membrane by brefeldin A (5 µM) the decay of current was similar in oocytes expressing HERG together with ß-catenin to oocytes expressing HERG alone. The experiments uncover a novel function of APC/ß-catenin, i.e. the regulation of HERG channels.The multifunctional protein ß-catenin governs as transcription factor the expression of a wide variety of genes relevant for cell proliferation and cell survival. In addition, ß-catenin is localized at the cell membrane and may influence the function of channels. The present study explored the possibility that ß-catenin participates in the regulation of the HERG K The multifunctional protein ß-catenin is involved in the regulation cell proliferation and tumor growth ß-catenin may enter the nucleus and stimulate the expression of several genes important for cell proliferation 2+-activated K+ channels + channels + channels +/K+ ATPase ß-catenin has been shown to interact with and/or modulate the activity of large-conductance CaXenopus oocytes with or without the expression of ß-catenin. The results reveal that the coexpression of ß-catenin leads to marked upregulation of HERG activity by enhancing the plasma membrane abundance of the channel protein.The present study explored the possibility that ß-catenin participates in the regulation of the human ether-a-go-go  channel, which is critically important for the shaping of the cardiac action potential 1–530For generation of cRNA, constructs were used encoding human β-catenin Xenopus oocytes were prepared as previously described 1–530 and/or N-cadherin and on the same day with 7.5 ng cRNA encoding HERG. Standard two electrode voltage clamp recordings were performed 3 days after HERG injection 2 1.8, MgCl2 1 and HEPES 5 (pH 7.4 with NaOH). Pipettes were filled with 3 M KCl and had resistances of 0.5–1.0 MΩ. Experiments were performed with a Geneclamp 500B amplifier  and a Digidata 1322A interface . Data acquisition was achieved with pCLAMP 9.02 .For voltage clamp analysis, Xenopus oocytes treated with 10 µM actinomycin D one day before measurement to disrupt gene transcription. To discriminate between alterations of insertion and retrieval of HERG channel protein from the plasma membrane, the insertion was inhibited by brefeldin A Where indicated, the experiments were performed in To determine HERG cell surface expression by chemiluminescence Data are provided as arithmetic means ± SEM; n represents the number of oocytes or cells investigated. All oocyte experiments were repeated with at least three batches of oocytes; in all repetitions, qualitatively similar data were obtained. As different batches may yield different expression levels and currents, comparisons have always been made within the same batches of oocytes. All data were tested for significance by using ANOVA. Results with P<0.05 were considered statistically significant.Xenopus oocytes with 10 µM actinomycin one day before measurement to prevent ß-catenin-dependent gene expression . The additional expression of ß-catenin was followed by a marked increase in the tail current . pression .Binding of a specific antibody and subsequent determination of HA-dependent surface chemiluminescence was employed to determine the effect of ß-catenin on oocytes expressing HA-tagged HERG. As shown in Xenopus oocytes expressing HERG together with ß-catenin as fast as in Xenopus oocytes expressing HERG alone. This observation discloses that ß-catenin does not delay the retrieval of HERG protein from the membrane.To discriminate between increased HERG protein insertion into and delayed retrieval of HERG protein from the plasma membrane, additional experiments were performed in the presence of Brefeldin A (5 µM) which prevents the insertion of novel proteins advancing from the Golgi apparatus into the cell membrane Xenopus oocytes with or without additional expression of the truncated mutant ß-catenin1–530. As shown in Xenopus oocytes.To determine, whether the effect on HERG channels requires full-length ß-catenin, HERG channels were expressed in The present observations uncover a completely novel function of ß-catenin, i.e. the regulation of HERG channel activity. ß-catenin did not alter HERG channel kinetics but apparently increased the HERG protein abundance within the cell membrane. The effect is apparently not due to delayed retrieval of channel protein from the cell membrane but may result from enhanced insertion of HERG channel protein into the cell membrane.The effect of ß-catenin did not result from its function as a transcription factor, as it was not significantly modified by suppression of transcription with actinomycin. Moreover, the experiments were performed following heterologous HERG expression. Thus, the functional expression of HERG did not depend on genomic regulation of the channels. The present observations did not define the mechanisms underlying ß-catenin sensitivity of HERG channel activity. ß-catenin has previously been shown to interact with channel proteins 1 receptors with subsequent activation of protein kinase C linked to the PKC pathway in ventricular myocytes The ß-catenin-sensitive HERG channel activity may, at least in theory, impact on the cardiac action potential during cardiac hypertrophy. Enhanced HERG activity was expected to accelerate the repolarization of ventricular muscle cells, shorten the action potential thus favouring reentry. Cardiac hypertrophy is facilitated by decreased activity of the glycogen synthase kinase-3 beta (GSK3ß) ß-catenin is regulated by the Wnt pathway HERG channels are further implicated in the regulation of tumor growth + channel HERG.In conclusion, the present observations provide compelling evidence that ß-catenin upregulates the voltage-gated K


Aspergillus niger (A. niger) and Penicillium amagasakiense (P. amag.), which have both had their respective genes isolated and sequenced. GOx from A. niger is known to be more stable than GOx from P. amag., while GOx from P. amag. has a six-fold superior substrate affinity (KM) and nearly four-fold greater catalytic rate (kcat). Here we sought to combine genetic elements from these two varieties to produce an enzyme displaying both superior catalytic capacity and stability. A comparison of the genes from the two organisms revealed 17 residues that differ between their active sites and cofactor binding regions. Fifteen of these residues in a parental A. niger GOx were altered to either mirror the corresponding residues in P. amag. GOx, or mutated into all possible amino acids via saturation mutagenesis. Ultimately, four mutants were identified with significantly improved catalytic activity. A single point mutation from threonine to serine at amino acid 132  led to a three-fold improvement in kcat at the expense of a 3% loss of substrate affinity (increase in apparent KM for glucose) resulting in a specify constant (kcat/KM) of 23.8 (mM−1 · s−1) compared to 8.39 for the parental (A. niger) GOx and 170 for the P. amag. GOx. Three other mutant enzymes were also identified that had improvements in overall catalysis: V42Y, and the double mutants T132S/T56V and T132S/V42Y, with specificity constants of 31.5, 32.2, and 31.8 mM−1 · s−1, respectively. The thermal stability of these mutants was also measured and showed moderate improvement over the parental strain.Glucose oxidase (GOx) is an enzymatic workhorse used in the food and wine industries to combat microbial contamination, to produce wines with lowered alcohol content, as the recognition element in amperometric glucose sensors, and as an anodic catalyst in biofuel cells. It is naturally produced by several species of fungi, and genetic variants are known to differ considerably in both stability and activity. Two of the more widely studied glucose oxidases come from the species  Aspergillus niger (A. niger) and Penicillium amagasakiense (P. amag.), is a holoenzyme consisting of two identical 80 kDa subunits which require a cofactor  for activity. Although much work has been done in the field of directed enzyme evolution in general kcatWe selected GOx (1.1.3.4) for rational redesign employing genetic engineering techniques for improvement of specific activity and affinity towards the substrate without sacrificing stability. GOx, produced primarily by filamentous fungi such as A. niger GOx is more stable than GOx from P. amag., the latter enzyme has a six-fold higher substrate affinity (reflected as a lower KM) and nearly four-fold higher catalytic rate (kcat) at pH 7 In this work a comparison of the sequence and structure-function similarity in GOx and enzymes homologous to it from various organisms was conducted in order to identify sequence variations within conserved regions across organisms which could provide logical targets for mutagenesis. This design stage was performed using the BLASTP search engine to search NCBI’s non-redundant protein database A. niger were selected for site-directed mutagenesis to mirror the comparable residues from Penicillium, or for saturation mutagenesis. In the first region of the FAD-binding domain, the site of ADP-binding and the most conserved region of the FAD-binding site, nine residues are not conserved between A. niger and P. amag. Eight were targeted for site-directed mutagenesis as follows : serine to alanine in position 38 (S38A), V42T, T56V, N65K, S67K, S73K, S75F, and H100Q. The active site of GOx is formed by tyrosines (Y90 and Y537), threonine (T132), arginine (R534), asparagine (N536), and two histidine residues (H538 and H581) . With the exception of the two histidines, none of these residues is conserved across the glucose-methanol-choline (GMC) oxidoreductase family P. amag. structure. All of the mutations, both site-directed and saturation, were initially done independently of each other. Based on these observations, 15 amino acids in the FAD- and substrate-binding sites of A. niger GOx gene, designed for optimized codon efficiency in Saccharomyces cerevisiae (S. cerevisiae), was constructed by DNA 2.0 . The pYES-DEST52 Gateway® Vector  was used for cloning the GOx gene into yeast. This galactose-inducible S. cerevisiae expression vector includes a c-terminal hexa-histidine tag and a V5 antibody epitope. The native amino-terminal signal peptide sequence flagging GOx for secretion was preserved. The recombinant vector was then used to transform the S. cerevisiae strain INVSc1 . Mutations to this construct were made using the QuikChange® Multi Site-Directed Mutagenesis Kit . Saturation mutagenesis was achieved by using mixtures of mutagenic oligonucleotides (degenerate oligos).A synthetic version of the 2PO4 buffer, pH 8.0, to remove residual amino acids. Purification was carried out under native conditions as described in the manufacture’s protocol with slight modifications. Briefly, 1 mL of Ni-NTA resin was added to polypropylene columns, washed and then equilibrated with 50 mM NaH2PO4 buffer containing 20 mM imidazole (native binding buffer). Concentrated GOx samples were diluted to 6 mL volumes using 3 mL 18 MΩ water  followed by 3 mL of native binding buffer. Samples were incubated for 1 hour at 25°C with gentle mixing. After washes, the purified GOx was eluted off the resin using 1.5 mL of an elution buffer consisting of 50 mM NaH2PO4, 250 mM imidazole, pH 5.0. The resin was incubated in the elution buffer for 15 minutes with mixing.Purification of the secreted recombinant GOx was achieved by using a tetradentate nickel chelate (Ni-NTA) purification system (Invitrogen). After 24-hour growth in 50 mL of YP-Gal induction media, the soluble His-tagged GOx was harvested by centrifugation at 1400×g. The media was subsequently dialyzed with 50 mM NaHThe presence of the hexa-histidine and V5 epitope tags allowed for the use of affinity chromatography and immunodetection as tools to evaluate the presence of recombinant GOx in the purified sample. Post-purification concentration was measured and an aliquot was removed for SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Samples were run on 10–20% Tris-Glycine gels for 1 hour. Proteins were then transferred onto a nitrocellulose membrane followed by immunodetection using an anti-V5 (C-term)-AP antibody and a chromogenic immuno-detection kit . After blocking with a buffer saline-detergent-casein solution (supplied) the membrane was incubated with 15 mL of a 1∶2000 dilution of anti-V5 (C-term)-AP primary antibody. An alkaline phosphatase (AP) conjugated anti-IgG antibody (supplied) was used to visualize the Magic Mark Protein standard ladder. After washing, the membrane was incubated in 10 ml of BCIP/NBT chromogenic substrate for alkaline phosphatase.2PO4 again using 30 kDa MWCO centrifugal filers. GOx concentration in filtered, concentrated, and buffer exchanged culture media samples was determined via SDS-PAGE against known concentrations of GOx from A. niger. using Sypro Ruby Red  as protein stain. An immunoaffinity assay was used to determine the band that corresponded to GOx (as above), and ImageQuant software  was used to correlate band intensity to protein concentration.Each clone was inoculated into 50 mL of YP-Gal induction media and incubated for 24 hours at 30°C. To prepare recombinant GOx secreted into spent growth media for activity assay, Then the cultures were briefly centrifuged to pellet the cells, which were then discarded. The collected media was then filtered and concentrated using 30 kDa molecular weight cut-off (MWCO) Centricon Plus-70 centrifugal filters  to an end volume of 0.5 mL. The retained high mass fraction was then buffer exchanged with 50 mM NaHAmplex Red Glucose Oxidase Assay (Invitrogen) was used according to the manufacturer’s protocol. A second colorimetric assay method using 2, 2′-azino-bis [3-ethyl-benzothiazoline-6-sulfonic acid]  was modified from Sun, et al Electrochemical initial rate measurements were performed on a PGZ 402 VoltaLab potentiostat  and were measured versus a micro Ag/AgCl reference  and a platinum (Pt) wire counter electrode. A rotating disk electrode system  was used in conjunction with the 3 mm diameter Pt working electrode . Polystyrene 24 well plates (3.5 mL well volume) were used as the electrochemical cell .2SO4 from −200 to +1200 mV vs. Ag/AgCl at 100 mV s−1 until a stable voltammogram was obtained with well defined hydrogen and oxygen absorption and desorption waves . Afterwards the electrode was thoroughly rinsed and sonicated for several seconds in 18 MΩ water. The electrode was further conditioned in 1 mM ferricyanide, 1 mM ferrocyanide, 0.1 M KCl, in 10 mM sodium phosphate buffer (NaPB), pH 7.0, via 0.25 second chronopotentiometric steps from −0.5 µA to +0.5 µA for a total of 500 cycles. This was followed by 10 cyclic voltammograms in the same solution from −100 mV to +500 mV at 100 mV s−1. The anodic and cathodic potential peak separation (?Ep) of the final sweep was used to determine whether the electrode had been adequately cleaned. If the ?Ep exceeded 80–90 mV, the cleaning process was repeated starting with H2SO4 cleaning. Following final conditioning, the electrode was thoroughly rinsed in 18 MΩ water.Prior to each initial rate assay the Pt electrode was thoroughly cleaned to ensure a consistent electrode surface. The Pt electrode was rinsed in 18 MΩ water followed by electrochemical cleaning via potential cycling in 1 M HFor enzymatic activity assay, the Pt electrode was immersed with the reference and counter electrodes in 1.775 mL of 100 mM NaPB, pH 6.8, containing a given concentration of glucose. The Pt electrode was rotated at 6000 rpm and a chronoamperometric step to +700 mV vs. Ag/AgCl was applied. The system obtained a pseudo steady state current response within 150 seconds after which 25 µL of the enzyme solution was injected into the well.To determine the thermal stability of the mutant and commercial enzymes, tubes of enzyme were incubated in the ABTS assay buffer at 50°C for 48 hours. Every two hours an aliquot was removed from each tube and an ABTS kinetic assay was performed, as described above. Absorbance measurements were taken every minute for ten minutes, and each set of measurements was done in triplicate.E. coli was abandoned due to the formation of inclusion bodies, and the difficulty in reconstituting the functional enzyme from them.Recombinant GOx secreted by yeast into culture media was harvested, affinity purified, ran on a SDS-PAGE gel, and visualized by Western blotting with anti-V5-AP antibodies using the WesternBreeze Chromogenic Immuno-detection, as shown in GOx catalyzes the oxidation of β-D-glucose to D-glucono-1,5-lactone and hydrogen peroxide, using molecular oxygen as the electron acceptor as shown in the following two half-reactions:β-D-glucose + GOx:FAD + H2O ↔ GOx:FADH2+ gluconolactone +2H+(1)2+ O2+2H+ → GOx:FAD + H2O2(2)GOx:FADHIn the interest achieving higher throughput, in all assays concentrated, buffer exchanged, partially purified protein from spent culture media was used instead of protein purified to homogeneity via nickel-affinity column chromatography. Using immunoaffinity labeling, band in SDS-PAGE gels corresponding to GOx in such samples was clearly identified. Protein concentration was quantified from the band intensity using ImageQuant software  as described in the Methods section. All measurements were performed in triplicate to insure accuracy and reproducibility.A. niger and P. amag. enzymes is near 6, both retain nearly 90% activity at pH 7 Two different assay methods were used for screening of mutants: colorimetric and electrochemical. Both methods rely on the generation of hydrogen peroxide produced as a by-product of GOx turnover. Though the optimal pH for both the 2 = 0.9971). The ABTS colorimetric assay also relies on the coupled GOx-peroxidase turnover yielding polymerized ABTS and a standard curve with GOx concentrations ranging from 0–5 mU/ml was also generated using this assay (2 = 0.9964). The Amplex Red assay exhibited a higher sensitivity of 0.224 (Abs a.u./mU GOx) compared with 0.171 measured from the ABTS assay.The Amplex Red Glucose Oxidase Assay provides a sensitive coupled enzyme method for the detection of GOx activity based on peroxidase turnover of hydrogen peroxide produced by GOx in the presence of the Amplex Red reagent . For the Amplex Red GOx assay, a standard curve with GOx concentrations ranging from 0–5 mU/ml was constructed and is presented in When employing the Amplex Red and ABTS GOx activity assays to evaluate the activity of GOx mutants, disagreement between the two different colorimetric assays was observed. Typical results for the Amplex Red and ABTS activity assays on GOx mutant strains are shown in Promising mutants identified by colorimetric assays were further characterized by an electrochemical assay which yielded precise enzymatic kinetic rate information. Hydrogen peroxide can be directly oxidized at a positively charged platinum electrode yielding one molar equivalent of dioxygen, and two molar equivalents of electrons and protons:2O2 → O2+2e- +2H+ (3)H2O2 oxidation on Pt The measured current is proportional to the concentration of hydrogen peroxide in solution. The electrochemical measurement of hydrogen peroxide produced by GOx provides a very useful and sensitive method for studying the kinetic properties of GOx. The electrochemical assay chosen is well characterized 2 = 0.9980). The assay sensitivity of 84.3 nA/µmolar H2O2 was similar to the 84.5 nA/µmolar H2O2 value reported in an earlier work A hydrogen peroxide calibration curve was obtained by measuring the current response for eleven different concentrations of peroxide. Runs were randomized and 3 replicates were performed. A highly reproducible linear current response was observed for concentrations of 1 µM through 1 mM peroxide (v ([H2O2]/min), was obtained from the slope of this current response over the first second (or less) from injection. A detection limit of 1.7 mUnit GOx/mL was achieved, and was adequate for assaying GOx expressed in the yeast system of this work.Experimental conditions for the GOx activity assay were identical to those described above for the peroxide current response with the exception that glucose at various concentrations was included in the electrolyte solution, instead of hydrogen peroxide. Following application of the positive bias to the rotating Pt electrode and allowing the current to stabilize, 25 µL of a solution containing GOx was injected into the electrochemical cell. Upon sample injection, the solution was instantly mixed with the buffer solution via the rapidly rotating working electrode and an immediate increase in current was measured. The initial rate velocity, A. niger . Initial rate velocities were used to calculate the enzymatic activity kinetic parameters. Measured turnover (kcat) of the commercial GOx was 2.79×104 (µmol product/µmol active subunit/min) compared with 2.75×104 reported by the manufacturer. The Michaels-Menten constant, KM, was measured to be 14.6 mM. This value was not reported by Biozyme, but is within the range of 3.5–38 mM published for native GOx from A. niger under similar conditions Validation of the electrochemical initial rate assay was obtained by performing initial rate velocity measurements at several different glucose concentrations using commercially available GOx from S. cerevisiae culture medium. To ensure that potential interferants that may remain following sample concentration and purification do not contribute to the electrochemical assay response, a sample from a yeast culture in which GOx expression was induced was compared to an uninduced culture sample. While the induced culture sample  showed an immediate increase in current upon injection , the uninduced culture sample  showed no increase in current response throughout the course of the experiment . No increase in current was recorded upon injection of samples prepared from induced cultures in the absence of glucose  showing that galactose metabolism, as opposed to glucose metabolism, did not contribute a response during assay. Therefore, the current response measured from filtered, concentrated, and buffer exchanged culture media samples can be attributed solely to H2O2 produced from GOx turnover with no contribution from remaining components of the spent media.Enzymatic activity assays of the parental GOx strain and mutant GOx strains were performed using filtered, concentrated, and buffer exchanged spent A. niger were selected for either site-directed mutagenesis to mirror the comparable residues from Penicillium, or for saturation mutagenesis. An initial library of nearly 1200 mutants with single point mutations was created. Although the electrochemical assay proved more reliable for collecting kinetic rate data for mutants, the assay time was lengthy and could not be used for high-throughput screening. With the large number of mutants generated, it became necessary to use the more high-throughput colormetric assay as a pre-screen of mutant activity followed by electrochemical analysis of promising mutants. For this pre-screen the ABTS assay was utilized.As detailed in the Of the 200 mutants colorimetrically screened from the first round of saturation mutagenesis, 14% reported activity comparable to or greater than the parent enzyme, 25% reported activity less than the parental enzyme, and 61% showed no measurable activity. These results are similar to that reported by other directed evolution enzyme studies −1. This represents a 3-fold improvement in specific activity over the parental A. niger GOx activity of 112±10.5 s−1 (per monomer). The KM and specificity constant for this mutant and the parental strain are reported in Of the mutants assayed following the first round of saturation mutagenesis, the mutant with the greatest increase in specific activity, as determined by electrochemical assay, had a specific activity of 328±20.6 skcat (V42Y) showed a 4.5-fold improvement over the parental A. niger GOx and a slight improvement over GOx from P.amag. However, this mutant had the lowest substrate affinity, 21% lower than the parental stain. Interestingly, a double mutant containing both T132S and V42Y mutations was identified and showed a significant improvement in KM over the parental strain with a similar kcat to mutant T132S. An additional double mutant was identified (T132S/T56V) which showed the most improved KM, 38% better than the parental enzyme, but the lowest improvement in kcat.Ultimately, three additional unique mutants with significantly improved catalytic activity over the parent enzyme were identified. The kinetic parameters for these mutants are also reported in T70% at 50 °C of 10.4 hours was measured. This is similar to T70%  = 11 hours reported by Kalisz et al. under similar conditions P. amag. under the same conditions kcat, also showed the greatest improvement in thermal stability.As the goal of this study was to improve the specific activity of a relatively more stable form of GOx, it was important to ascertain how mutations affected protein stability. A thermal stability assay monitoring enzyme activity over time at 50 °C was validated using GOx from Biozyme. A A. niger GOx. Mutants were initially screened via a high throughput colorimetric activity assay and further characterized by an electrochemical initial rate assay allowing quantification of kinetic rate parameters. Four mutants with improved catalytic properties were identified out of a library of approximately 4800 mutants, of which only 1400 have so far been screened. These mutants  showed a 3 to 4 fold improvement in specificity constant over the parental strain. The variant with the highest kcat (V24Y) showed a 4.5-fold improvement over the parental A. niger GOx and slight improvement over GOx from P.amag. at the expense of a 21% lower substrate affinity. Also in common with all mutants, V42Y showed a modest improvement in thermal stability (T1/2 = 40.3 hours) compared to the parental strain (T1/2 = 28.8 hours) and the commercially available enzyme (T1/2 = 20.1 hours).Site-directed mutagenesis and directed evolution were used to improve the catalytic activity of recombinant 2 reduction 2 concentration in this mutant may clarify the mechanism responsible for its enhanced activity, and are planned for future research.A recent report It is less obvious why the modifications at V42Y and T56V have such large impacts upon enzymatic kinetics. As depicted in By combining increased catalytic efficiency with modest improvements in stability, the mutant GOx enzymes produced in this work may prove useful as alternatives to the wild-type enzyme in applications ranging from biosensing, the food and wine industry, to the development of high-power biofuel cells.


Cacna1g gene expression. In the present study, we combined bioinformatics and experimental approaches to find new β-catenin targets in the adult thalamus.LEF1/TCF transcription factors and their activator β-catenin are effectors of the canonical Wnt pathway. Although Wnt/β-catenin signaling has been implicated in neurodegenerative and psychiatric disorders, its possible role in the adult brain remains enigmatic. To address this issue, we sought to identify the genetic program activated by β-catenin in neurons. We recently showed that β-catenin accumulates specifically in thalamic neurons where it activates in vitro by introducing its negative regulator Axin2 reduced the expression of six of the nine genes. Immunoprecipitation of chromatin from the brain tissues confirmed the interaction between β-catenin and some of the predicted LEF1/TCF motifs. The results of these experiments validated four genes as authentic and direct targets of β-catenin: Gabra3 for the receptor of GABA neurotransmitter, Calb2 for the Ca2+-binding protein calretinin, and the Cacna1g and Kcna6 genes for voltage-gated ion channels. Two other genes from the latter cluster, Cacna2d2 and Kcnh8, appeared to be regulated by β-catenin, although the binding of β-catenin to the regulatory sequences of these genes could not be confirmed.We first selected the genes with at least two conserved LEF/TCF motifs within the regulatory elements. The resulting list of 428 putative LEF1/TCF targets was significantly enriched in known Wnt targets, validating our approach. Functional annotation of the presumed targets also revealed a group of 41 genes, heretofore not associated with Wnt pathway activity, that encode proteins involved in neuronal signal transmission. Using custom polymerase chain reaction arrays, we profiled the expression of these genes in the rat forebrain. We found that nine of the analyzed genes were highly expressed in the thalamus compared with the cortex and hippocampus. Removal of nuclear β-catenin from thalamic neurons In the thalamus, β-catenin regulates the expression of a novel group of genes that encode proteins involved in neuronal excitation. This implies that the transcriptional activity of β-catenin is necessary for the proper excitability of thalamic neurons, may influence activity in the thalamocortical circuit, and may contribute to thalamic pathologies. The cN-terminus of LEF1/TCF recruits β-catenin, which does not bind to DNA itself but has a strong transactivation domain. The remaining regulatory domains and alternatively spliced C-termini of the proteins vary between these members and provide a molecular basis for the diversity and context dependence of LEF1/TCFs function. Lef1/Tcf genes are highly expressed during mouse embryogenesis, but their transcripts are virtually undetectable postnatally [Lef1 and Tcf7l2 expression have been observed in the thalamus in the adult brain [The nuclear mediators of canonical Wnt signaling include the transcription factors LEF1, TCF7 , TCF7L1 , and TCF7L2 , which are members of the high mobility group (HMG) family -8. The Htnatally ,10, excetnatally ,12. Surplt brain -16.Growing evidence suggests the involvement of canonical Wnt signaling in the proper functioning of the adult central nervous system . AberranCacna1G, encodes voltage-gated Ca2+ channels [in silico and experimentally validated a novel group of β-catenin-LEF1/TCF targets in thalamic neurons, consisting of genes that encode proteins that are important for neuronal function, including voltage- and ligand-gated ion channels and the Ca2+-binding protein calretinin. This indicates a role for β-catenin and LEF1/TCF transcription factors in the maintenance of neuronal excitability.Most of the approximately 100 known β-catenin targets are involved in development and cancer. In differentiated neurons, only a few genes have been shown to be activated by β-catenin; several are already known Wnt target genes that are not neuron-specific -37. As wchannels . In thishttp://www.stanford.edu/group/nusselab/cgi-bin/wnt/target_genes . We relied on the fact that enhancers are enriched in proximity to TSSs , and thehttp://david.abcc.ncifcrf.gov; accessed January 11, 2012) to identify functionally related groups of genes. As expected, our list was highly enriched in genes involved in development, patterning, and cell proliferation (Table Gabarap). Several genes were also excluded because they were not brain-specific . The final collection contained 41 putative neuronal LEF1/TCF targets grouped into five categories: nine VGCCs, 11 neurotransmitter receptors, eight proteins involved in synaptic vesicle organization, 10 synaptic structural proteins, and three other proteins implicated in synaptic transmission (Table We performed a gene ontology (GO) analysis of the 428 putative targets using DAVID Functional Annotation tools .The expression of 53 of the 55 VGCC genes was detected in the forebrain; no signal was obtained for l Figure . Fisher’We next expanded the analysis and profiled the expression of all 41 genes listed as putative neuronal targets of LEF1/TCF Table  in the cHtr3b. In the thalamus, nine genes (22.5%) were expressed at least two-fold higher than in the cortex and hippocampus  were expressed at a higher level, and no single gene was highly expressed in the hippocampus , four genes that represent neurotransmitter receptors , and the Calb2 gene that encodes the Ca2+ buffer calretinin  assay with designed primers  was used, which precipitated < 0.02% of the input. The signals for all of the examined fragments were at background levels in the case of the cortex and hippocampus Figure . However% Figure , indicat% Figure  and acet% Figure .Finally, we examined the effect of nuclear β-catenin removal in primary thalamic cultures on the expression of the nine genes identified by gene profiling. The cultures contained both neurons and glia (approximately 1:1), which is vital for the survival of thalamic neurons .in vitro maintain the nuclear localization of β-catenin [Axin2, the product of which is a component of the β-catenin destruction complex and as such should reduce its cytoplasmic and nuclear pool. The control cultures were transduced with Gfp-expressing adenovirus. The percentage of β-catenin-positive neurons decreased from ~40% to 10% in cultures with ectopic Axin2 expression  40% of the neurons were β-catenin-positive in the control, and (ii) 12% of the neurons still maintained the nuclear localization of β-catenin after Axin2 adenovirus treatment , Cavα2δ2 (Cacna2d2), Kv1.6 (Kcna6), Kv12.1/ELK1 (Kcnh8), and GABAA receptor 3 (Gabra3) are all voltage- or ligand-gated ion channels [2+, K+, and Cl- (in the case of the GABA receptor) ions and directly propagate, inhibit or modify electric signals [2+-binding protein [The neuronal genes with conserved LEF1/TCF motifs that were highly expressed in the thalamus, the regulation of which by β-catenin was confirmed experimentally, encode proteins involved in neuronal excitability. Cav3.1  did not show high expression in the thalamus. However, they still might be regulated by β-catenin and LEF1/TCF factors in some subtypes of neurons or under specific physiological conditions because the regulation of gene expression by β-catenin is very much context-dependent ,44,68,69in vivo. If so, then we could speculate that the inappropriate activity of β-catenin might affect proper signal transmission in thalamocortical circuits. Thalamocortical desynchronization underlies absence epilepsy [2+ channels [2δ2 regulatory subunit of voltage-gated channels [A receptor 3 [Tcf7l2 have been recently shown to be a risk factor in schizophrenia [Gabra3-deficient mice display impairments in sensorimotor gating, which is a feature of this disorder [in vivo studies are required to elucidate this issue.We do not yet know whether variations in the nuclear level of β-catenin affect the expression of genes that encode VGCCs and neurotransmitter receptors and shape neuronal excitability epilepsy , and manchannels ). Specifchannels ,76, in achannels  and GABAceptor 3 . Schizopceptor 3 -84. Moreophrenia ,85, and ophrenia . Interesdisorder . These rWe identified a novel group of genes regulated by β-catenin-LEF1/TCF that encode proteins that underlie the transmission of nerve impulses. These results point to a novel role for β-catenin in the thalamus, in which β-catenin is constantly present in the cell nuclei. The identified and confirmed genes are directly involved in neuronal excitability, suggesting that β-catenin and LEF1/TCF transcription factors maintain the proper activity of thalamocortical circuits. The presented results may implicate the involvement of β-catenin transcriptional activity in thalamic pathologies, such as absence epilepsy and schizophrenia.http://www.stanford.edu/group/nusselab/cgi-bin/wnt/target_genes . The Wnt targets that had no CNSs, were indirect targets, or had confusing names were excluded from this analysis.For each human-rat orthologous gene pair in Ensembl (version 39), 20 kb flanks of the genomic sequence from −10 kb to +10 kb from TSSs in either species were aligned using the AVID global alignment algorithm. Conserved noncoding sequences, defined as sequence windows at least 100 bp long with at least 75% identity between human and rat, were selected as putative regulatory regions. The binding sites for LEF1/TCF were separately predicted using the position weight matrices in the V$LEFF family  with the MatInspector program for the human and rat sequences of each CNS pair. V$LEFF motifs with a nonzero number of instances in both the human and rat sequences of the same CNS pair  were considered conserved. To validate the method, the group of genes with at least one or at least two conserved V$LEFF motifs were crossed with known Wnt target genes listed at http://david.abcc.ncifcrf.gov; accessed January 11, 2011) were used to annotate the genes [p value, for the proportions of genes that fell into each GO term.The functional annotation tools of DAVID Bioinformatics Resources . The experimental procedures were approved by the Local Commission for the Ethics of Animal Experimentation no. 1 in Warsaw .g and briefly homogenized in ice-cold PBS supplemented with protease inhibitor cocktail using an Eppendorf-fitting pestle homogenizer. After two cycles of washing in PBS , the samples were lysed in 2.5 ml of lysis buffer  for 30 min at 4°C and then disrupted in a Potter-Elvehjem homogenizer (~100 strokes). The homogenates were centrifuged for 10 min at 2,400 × g to pellet the nuclei, which were then frozen at −80°. After thawing, the samples were diluted in digestion buffer from the ChIP-IT Express Enzymatic kit (Active Motif) and sonicated on ice for 10 × 20 s with 40 s breaks at a 50% duty cycle and 50% power using a Sonopuls Bandeline sonicator to disrupt the nuclei and preshear the chromatin. An enzymatic shearing protocol using the ChIP-IT Express Enzymatic kit was then followed. Shearing was conducted with 40 μl of Enzymatic Shearing Cocktail per one sample for 15 min at 37°C, and the reaction was stopped with EDTA. Finally, the sheared chromatin was separated from debris by centrifugation at 18,000 × g. This treatment yielded 125–500 bp DNA fragments. A subsequent immunoprecipitation procedure was performed on an equivalent 30 mg of tissue with 10 μg of anti-rabbit IgG (Sigma-Aldrich), 5 μg of anti-acetyl-histone H3 , or 40 μg of anti-β-catenin  antibodies, according to the Millipore protocol using salmon sperm DNA protein A-agarose (Millipore). Immunoprecipitated DNA was used as a template for RT-qPCR with SYBR Green chemistry. The obtained data were analyzed using the relative quantification method with the 2-ΔCT formula  and are presented as a percentage of the input. The primers used in the ChIP assay are listed in Table Three-month-old male Wistar rats were sacrificed by cervical dislocation, and the brains were removed and sectioned. Approximately 200 mg of cortical, hippocampal, and thalamic tissues obtained from two rats were chopped with a razor blade and fixed at room temperature for 30 min in 1 ml of fresh 3% formaldehyde in phosphate-buffered saline (PBS) supplemented with protease inhibitor cocktail (Roche) in an Eppendorf tube. Fixation was stopped by adding glycerol to a final concentration of 0.125 mM and incubation for a further 10 min to quench formaldehyde. The tissue was then spun down for 5 min at 1,000 × 5 cells per well of a 24-well plate in Minimal Essential Medium  supplemented with 10% fetal bovine serum and 0.2 mM glutamine (Sigma). The next day, the medium was replaced with Neurobasal (Gibco) supplemented with B27 (Gibco), 0.5 mM glutamine, 12.5 mM glutamate, and penicillin/streptomycin (Sigma) mixed in a 1:1 ratio with the cortical neuron conditioned medium.Dissociated primary thalamic cells were obtained from the brains of embryonic day 19 Wistar rat embryos as described previously . The celin vitro, purified virus suspensions at a multiplicity of infection of 100 were added to each well of a 24-well plate for 6 h at 37°C. The virus solution was then removed and replaced with fresh medium. Seventy-two hours later, the transduced cultures were collected for RNA isolation or fixation.The construction of recombinant adenoviral plasmids that expressed Gfp and Axin2 and the preparation and purification of the adenoviruses were described previously . On day The immunofluorescence analysis of β-catenin localization was performed as described previously . BrieflyTo isolate RNA from fresh brains, the brains from male, 2-month-old Wistar rats were sectioned, and different structures were homogenized separately using a Potter-Elvehjem homogenizer. RNA was extracted with the RNeasy Lipid Tissue Mini kit with additional DNase treatment (Qiagen). For neuronal cultures, the cells from one to three wells of a 24-well plate were pooled together and homogenized with Qiashredders (Qiagen), and RNA was isolated with the RNeasy Plus Mini kit (Qiagen). cDNA was synthesized by reverse transcription .-ΔCT formula . In thalamic cultures, gene expression levels were examined in individual qRT-PCR reactions. For Cacna1g, Cacna2d2, Kcna6, Knch8, Drd3, Glra1, Gabra3, Grid2, and Calb1, commercial primers were used (Qiagen). For Map2, commercial primers and FAM dye-labeled TAqMan probes (Applied Biosystems) were used. Lef1 and Gapdh expression was measured with the following primers: Lef1 , Gapdh . SYBR Green chemistry was used, with the exception of Map2, for which TaqMan was used (Applied Biosystems). The obtained data were quantified using the relative standard method.All RT-qPCR reactions were performed using the 7900HT system (Applied Biosystems). Gene profiling in the brain was performed with custom-designed TaqMan Low Density Arrays (Applied Biosystems), hereinafter referred to as RT-qPCR arrays. Approximately 1.5 μg of cDNA was loaded per array (384 assays). The obtained data were analyzed using the relative quantification method with the 2p values for the relationships between high expression in the thalamus and being a putative LEF1/TCF target , and compare the percentage of β-catenin-positive neurons in two types of thalamic cultures  or using relative values for the results obtained with individual RT-qPCRs .To calculate s Figure ; n = 6 op values in the ChIP experiment, in which we compared three brain regions at the same time, we used repeated measures ANOVA followed by Tukey’s post hoc test .To calculate t Figure ; n = 4.CNS: Conserved noncoding sequence; GABA: The γ-aminobutyric acid; GO: Gene ontology; RT-qPCR: Quantitative real-time polymerase chain reaction; TSS: Transcription start site; VGCC: Voltage-gated cation channel.The authors declare no conflicts of interest.in silico screening. KM performed immunofluorescence and microscopic analysis. AN and KM grew the primary cultures. JK discussed and reviewed the project during its implementation and participated in drafting the manuscript. All of the authors critically read and approved the manuscript.MBW conceived, designed, and coordinated the study, drafted the manuscript, and performed the GO analysis, ChIP assays, and gene expression analysis. AN designed the VGCC arrays, produced adenoviral vectors, and performed viral transduction. MD performed Excel file with a list of 428 rat genes with at least two LEF1/TCF motifs in the same CNS.Click here for file


In α-catenin conditional mutant optic cups, the major cell fates, including the optic fissure margin, neural retina and retinal pigmented epithelium, are specified normally, and the retinal progenitor cells proliferate normally. However, adherens junctions components, including N-cadherin, β-catenin and filamentous actin, fail to accumulate on the apical side of α-catenin mutant retinal progenitor cells, where adherens junctions are normally abundant, and the organization of the neural retina and the optic fissure margin is disrupted. Finally, the α-catenin mutant retina gradually degenerates in the adult mouse eye. Therefore, our results show that α-catenin-mediated cell adhesion and cell organization are important for the fissure closure in mice, and further suggest that genes that regulate cell adhesion may underlie certain coloboma cases in humans.Coloboma is a congenital disease that contributes significantly to childhood blindness. It results from the failure in closing the optic fissure, a transient opening on the ventral side of the developing eye. Although human and mouse genetic studies have identified a number of genes associated with coloboma, the detailed cellular mechanisms underlying the optic fissure closure and coloboma formation remain largely undefined. N-cadherin-mediated cell adhesion has been shown to be important for the optic fissure closure in zebrafish, but it remains to be determined experimentally how cell-cell adhesions are involved in the mammalian optic fissure closing process. α-catenin is required for cell adhesion mediated by all of the classic cadherin molecules, including N-cadherin. In this study, we used the Cre-mediated conditional knockout technique to specifically delete  Epithelial fusion is an important morphogenetic process frequently used to close originally open structures during embryogenesis and wound healing. It is also one of the key steps in vertebrate eye development. The development of the vertebrate eye begins with the bilateral evagination of the diencephalon in the early neurula, leading to the formation of the hollow tube-shaped structure, called the optic vesicle (OV). When it approaches the surface ectoderm, the OV begins to invaginate to form the optic cup (OC) Pax2, Vax1 and Vax2, produce the coloboma phenotype Shh, which promotes the proximal optic stalk fate but suppresses the distal retinal fate, have been found in coloboma patients, suggesting that the proper specification of the proximal-ventral retinal fate is critical for the proper OF closure β-catenin disrupts proper RPE differentiation, resulting in the coloboma phenotype Chd7 and Pitx2, which are expressed in mesenchymal cells and essential for their development, cause coloboma formation Phactr4, which encodes a negative regulator of cell proliferation, leads to the coloboma phenotype When the OF closure fails, a permanent opening remains on the ventral side of the eye, the congenital abnormality known as coloboma. Coloboma contributes significantly to childhood blindness, occurring in isolation or within syndromes pac, which encodes N-cadherin, causes tissue organization defects and the coloboma phenotype, providing direct evidence that adherens junctions (AJs) are important for eye patterning and morphogenesis α-catenin from the developing mouse retina to examine its role in retinal development. α-catenin is a filamentous actin (F-actin) binding and bundling protein, and is one of the key components of the AJ complex The OF closure is accompanied by cell morphological changes and rearrangement at the OF region. Cell-cell adhesion has been shown to be essential for cell morphological changes and rearrangement by providing structural connections between cells Six3-CrefxCtnna1All animal work was performed in compliance with the protocols approved by the Institutional Animal Care and Use Committee at the Stowers Institute for Medical Research (SIMR). The following mice are used in this study: Embryos were fixed overnight in 4% formaldehyde, cryo-preserved with 15% sucrose followed by 30% sucrose, and frozen using a freezing bath (Thermo Scientific) with isopentane. Because the OF is a ventral-specific structure of the OC, most specimens were sectioned para-sagittally. The nasal-temporal orientation of the OC was determined based on the brain structures around the eye according to The Atlas of Mouse Development For immunohistochemistry, tissue sections were heated in citrate buffer (pH 6.0) at 95°C for 10 minutes. They were then incubated with the primary antibodies at 4°C overnight, and then with Alexa 488- or Alexa 568-conjugated goat or donkey secondary antibodies (Invitrogen) for 2 hours at room temperature. Finally, tissue sections were counter-stained with DAPI for 5 minutes, washed and mounted. The following antibodies were used: Mouse anti-α-catenin (Invitrogen), rabbit anti-β-Catenin (Invitrogen), rabbit anti-Pax2 (Invitrogen), mouse anti-Pax6 , sheep anti-Vsx2 (Chemicon), mouse anti-Mitf (lab Vision), goat anti-pMLC (Santa Cruz), rabbit anti-N-cadherin (Santa Cruz), and mouse anti-Laminin (Sigma). Images were taken under either Leica SP2 or SP5 confocal microscope.The time-mated mice were injected intraperitoneally with BrdU at 0.1 mg/g body weight two hours before sacrifice. Embryos were fixed, sectioned and immuostained for BrdU (Amersham) as described above. For each para-sagittal section of the OC, about 60–100 retina progenitor cells (based on DAPI staining) in the central region of nasal and temporal retinas and retinal cells within a 5-cell diameter from the margin of the OF were counted. For each genotype, four eyes from four mice were quantified. For statistical analysis, Student's t-test was applied.α-catenin mutant embryos die at blastocyst stage Cre line, Six3-Creα-catenin conditional allele, fxCtnna1α-catenin specifically in the developing retina. Six3 is a homeobox-containing transcription factor that is expressed in all mouse retinal progenitor cells and the optic stalk (OS) from the optic vesicle stage Six3-Cre starts its expression in the OC and the OS from E9.5 α-catenin mutant embryos at E13.5 to examine whether there is any defect in the OF closure. At E13.5, the OF in the control mouse has already closed, which is evidenced by the appearance that the eye ball is completely enwrapped by an intact pigmented epithelium . As we showed earlier, the deletion of α-catenin is mosaic in the fx/fxSix3-Cre; Ctnna1 OCs, especially in the nasal retina  and Mitf are specifically expressed in the NR and RPE layers to control their cellular fate and development, respectively. At the control OFM, Vsx2 and Mitf are expressed in the inner and outer layers, respectively, though maybe slightly weaker than in the rest of the OC . Even thl retina . These rfx/fxSix3-Cre; Ctnna1 OCs are similar to those in the control OCs (fx/fxSix3-Cre; Ctnna1 OCs (Pax2 is essential for the OF closure in both humans and mice trol OCs . Howeverx/fx OCs . Taken tα-catenin also regulates cell proliferation in the retina. Two hours after BrdU was injected into pregnant female mice intraperitoneally, the embryos were harvested for detection of the BrdU label by fluorescent immunostaining. At E11.5, NR progenitors in nasal and temporal regions proliferate actively at similar rates, but OFM progenitors proliferate relatively slower than those in the other regions (fx/fxSix3-Cre; Ctnna1 retina, the progenitors in different regions show similar BrdU incorporation rates to their counterparts of the control (α-catenin not only regulates cell-cell adhesion, but also has been shown to regulate cell proliferation through modulating signaling pathways  regions . In the  control . These rfx/fxSix3-Cre; Ctnna1 eyeballs have a smaller pupil, which is shifted to the ventral side of the eye, suggesting that the mis-organized mutant retina may affect the development of a normal eye structure or the development of the iris (α-catenin mutant eye.The control adult eye balls show a perfect round pupil in the center . Histolothe iris . In the the iris . These rα-catenin from the developing OC results in coloboma formation in mice. Furthermore, we show that α-catenin mutant retinal and OFM progenitors fail to form AJs on the apical side, and exhibit abnormal cell morphologies and tissue organization, suggesting that α-catenin-mediated cell-cell adhesion is important for the successful closure of the OF.Although coloboma is a congenital birth defect that contributes significantly to childhood blindness, the cellular mechanisms underlying the defect are largely unclear. Although cadherin-mediated cell adhesion has been shown to be important for driving the OF closure in the fish, it remains unclear if it is also involved in the regulation of the OF closure in mammals. In this study, we show that a conditional deletion of α-catenin mutant retinal progenitors fail to form AJ foci on their apical side, which are likely responsible for their abnormal cell morphology, detachment from the basal membrane and the retina disorganization. In the process of the OF closing, the progenitor cells at the OF margin need to coordinate their cell shape changes to ensure that the two sides of the OF are aligned perfectly for the proper fissure closure. Such abnormal cell morphology and organization of the α-catenin mutant OFM progenitors likely causes the misalignment between two OF margins and thus the OF closing defect. Although α-catenin has been suggested to modulate different signaling pathways and thus cell fate and proliferation fx/fxSix3-Cre; Ctnna1 OCs, and that they also proliferate properly. Based on our experimental findings, we propose that α-catenin primarily regulates cadherin-mediated adhesion and possibly actin dynamics in OFM progenitors, which collectively contribute to the OF closing process.As an F-actin binding and bundling protein, α-catenin plays important roles in regulating the assembly and dynamics of actin-cytoskeleton network


The distinctive anisotropy of the spin splitting was observed while the temperature is below approximately 200 K. This anisotropy emerges from the combined effect of Dresselhaus spin-orbit coupling plus asymmetric potential gradients. We also exploit the temperature dependence of spin-splitting energy. Both the anisotropy of spin splitting and the in-plane effective g-factor decrease with increasing temperature.The in-plane spin splitting of conduction-band electron has been investigated in an asymmetric (001) GaAs/AlPACS: 78.47.jm, 71.70.Ej, 75.75.+a, 72.25.Fe, B > 0.The properties of spins in semiconductor materials have attracted much more attentions since the invention of spintronics and spin-based quantum information -3. In thB > 0 in semiconductor is described by a finite Zeeman splitting energy and characterized by the effective g-factor, which is necessary for the spin manipulation and spin-based qubit with an external electrical/magnetic field in semiconductor. So far, the effective g-factor has been intensively investigated in many literatures during past few decades )  and [1-10].In general, the spin splitting of electron or hole at  decades -13. For aterials . It is isymmetry . For exa//100]) -7,10. Fu0) -7,10.plitting . Up to nroposals  have bee in Ref. , can resh et al.  In this h et al. ,16 techn0.28Ga0.72As barrier layer, an 8-nm-wide GaAs quantum well, the other sloping barrier grown with content of Al changing from 4.28% to 28% on the length of approximately 9 nm, and the barrier layer of a width 50 nm. The upper part of the structure is covered with a 5-nm GaAs layer to avoid the oxidation of barrier. TRKR experiment was carried out in an Oxford magneto-optical cryostat supplied with a 7-T split-coil super-conducting magnet. The sample was excited near normal incidence with a degenerate pump and delayed probe pulses from a Coherent mode-locked Ti-sapphire laser . The center of the photon energy was tuned for the maximum Kerr rotation signal for each temperature setting. The laser beams were focused to a spot size of approximately 100 μm, and the pump and probe beams have an average power of 5.0 and 0.5 mW, respectively. The helicity of linearly polarized pump beam was modulated at 50 kHz by a photoelastic modulator for lock-in detection. The temporal evolution of the electron spins, which were generated by the circularly polarized pump pulse, was recorded by measuring Kerr rotation angle θK(Δt) of the linearly polarized probe pulse while sweeping Δt.The sample on investigation here is grown on (001) oriented semi-insulating GaAs substrate by molecular beam epitaxy. It contains a 50-nm-wide AlθK(Δt) measured at 1.5 K with an in-plane magnetic field of B = 2.0 T . The exgeometry . It is oS0 is the initial spin density, τs is spin lifetime, and ω the Larmor frequency. By this way, we obtain the exact value of the Larmor frequency ω and then the splitting energy ΔB//[110] E= 0.0263 meV and ΔB//[1-10] E= 0.0326 meV through the equation ΔE = ћω with in-plane magnetic field parallel to [110] and [1-10], respectively. Here, we use where B > 0. As calculated by Winkler, the spin-splitting energy writes as  and ζ = -1 for B//[110], and γ is the cubic Dresselhaus constant. The Zeeman-like term B > 0. As expected in Equation 2, the measured spin splitting is linearly dependent on the magnetic field with a prefactor B linear dependence will allow us to obtain the value of G accurately, which are G[110] = 0.0130 meV/T and B along [110] and [1-10]. The difference of two values results from the opposite sign of prefactor ζ. According to Equation 3, B > 0 in an asymmetric (001) GaAs/AlGaAs QW. However, the prefactor B > 0 in an asymmetric QW. Additionally, the Rashba term also gives rise to a nontrivial splitting in the presence of a magnetic field, but the splitting is isotropic  and [1-10], respectively. Both values decrease while the temperature is elevated. It is noted that the difference of spin splitting is maximum at low temperature of approximately 1.5 K and almost disappears when the temperature is up to 200 K. In order to clearly show the anisotropy of spin splitting, we have extracted precisely the values of B > 0. Let us recall the expression of prefactor g-factor can also be extracted from Equation 2. It is about g* = 0.25 at 1.5 K and very closed to that (g* = 0.26) in 10-nm-width well with the same Al fraction [g-factor. It decreases with increasing temperature. This trend is consistent with the former reports [Finally, we systematically investigate the anisotropy of in-plane spin splitting for the temperatures between 1.5 and 300 K keeping the fixed excitation power of approximately 5 mW and the fixed external magnetic field of approximately 2 T. Figure to 300 K . We beliB along 0 and [1- reports ,12,13.B > 0 combining the asymmetric confinement potential via the numerical comparison with the proper anisotropic Zeeman splitting.We observed the anisotropic in-plane spin splitting of the conduction-band electron in an asymmetric (001) GaAs/AlGaAs quantum well using TRKR technique with applied magnetic fields. It is confirmed that Dresselhaus spin-orbit coupling can significantly affect the in-plane spin splitting at BIA: bulk inversion asymmetry; DP: D'yakonov-Perel; QW: quantum well; SIA: structure inversion asymmetry; SOC: spin-orbit coupling; TRKR: time-resolved Kerr rotation.The authors declare that they have no competing interests.BL conceived and designed the experiments. HQ and CC carried out the experiments with contribution from GW and HM. HT and WX provided the sample. ZX contributed to the calculation. BL supervised the work. HQ and BL wrote the manuscript. All authors read and approved the final manuscript.This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.


IgG featuring terminal sialic acids were shown to induce less inflammatory response and increased serum half-life.Glycosylation is an important posttranslational modification of proteins influencing protein folding, stability and regulation of the biological activity. The sialyl mojety  is usually exposed at the terminal position of N-glycosylation and therefore, a major contributor to biological recognition and ligand function, The biosynthesis of sialyl conjugates is controlled by a set of sugar-active enzymes including sialyltransferases which are classified as ST3, ST6 and ST8 based on the hydroxyl position of the glycosyl acceptor the Neu5Ac is transferred to . The ST6Pichia pastoris, Spodoptera frugiperda and E. coli).To our knowledge, the access to recombinant ST6Gal-I for therapeutic applications is still limited due to low expression and/or poor activity in various hosts  by transient gene expression in HEK293 cells with yields >100 mg/L featuring distinct mono- (G2+1SA) as well as bi- (G2+2SA) sialylation activity.Two N-terminally truncated fragments of human ST6Gal-I  were designed for transient gene expression (TGE): Instead of the natural leader sequence and N-terminal residues, both ST6Gal-I coding regions harbor the Erythropoietin (EPO) signal sequence in order to ensure correct processing of the polypeptides by the secretion machinery. Following cloning into pM1MT, expression of the ST6Gal-I coding sequences is under control of a hCMV promoter followed by an intron A.Sialyltransferase assays: 1. Asialofetuin was used as acceptor and CMP-9F-NANA as donor substrate. Enzymatic activity was determined by measuring the transfer of 9F-NANA to asialofetuin. 2. Recombinant humanized IgG1 and IgG4 monoclonal antibodies (mabs), characterized as G2+0SA, as well as desialylated EPO were used as targets in sialylation experiments (30 μg enzyme/300 μg target protein). Both enzyme variants of ST6Gal-I (delta89 and delta108) were used under identical reaction conditions and the sialylation status was analyzed by mass spectrometry.In using the suspension-adapted human embryonic kidney (HEK) 293-F cell line, a modified serum-free FreeStyle™ medium platform plus transfection by the 293-Free™ reagent, we were able to install a TGE shaker fermentation process with product yields of up to 200 mg/L culture supernatant. Both variants delta89 and delta108 could be isolated to >98% purity by a simple 2-step purification protocol.To our surprise, both variants show a distinct and different sialylation activity as shown by sialylation kinetics of a IgG4 molecule Figure .Recently, the crystal structure of the delta89 variant could be determined as first human ST6Gal-I by SIRAS phasing using an iodide soak as derivative I : An elonDue to their high expression rates and easy purification, both recombinant variants (delta89 and delta108) of human ST6Gal-I are available in large quantities and high purity. Both variants are active with high molecular weight substrates like monoclonal antibodies. To our surprise, they show different performance in sialylation experiments using with bi-antennary glycans such as mabs as well as tetra-antennary glycans (data not shown) as substrate. Under identical reaction conditions, bi-sialylated glycans are obtained in using variant delta89, whereas delta108 yields mono-sialylated glycans.Our findings on variant delta108 are in contradiction to previous studies  claimingTo our knowledge, these human ST6Gal-I variants are the first enzymes available in large quantities and currently, recombinant alpha-2,3 sialyltransferase 1  and beta-1,4 galactosyltransferase 2  are developed in order to strengthen this enzyme portfolio. Together with the already available donor substrates (activated sugars), a complete set of reagents will be soon available for the commercial glycoengineering of proteins.


P=0.021), increased expressions of β-catenin (P=0.038) and its downstream targets; CyclinD1 (P<0.001) and FGF9 (P<0.001). In addition, using HEK293T cell model, we found that Sox7 was able to inhibit TCF/LEF-1-dependent luciferase activity induced by Wnt-1. This was further proved by that Sox7 could significantly suppress the expressions of Wnt targets; Cyclin D1 and C-myc in endometrial cells. Immuno-fluorescent microscopy revealed that Sox7 was co-localizaed with either mutant β-catenin or TCF4 protein in nucleus, while co-immunopreciptation assay demonstrated that Sox7 could physically interact with not only wild-type but also mutant β-catenin, as well as TCF4 proteins. Functionally, enforced expression of Sox7 could significantly inhibit endometrial or endometrioid ovarian cancer cells (OEA) harboring either wild-type or mutant β-catenin. These data suggest Sox7 is a negative regulator of Wnt/β-catenin signaling pathway through impeding the transcriptional machinery of β-catenin/TCF/LEF-1 transcriptional complex, and the loss of expression may be involved in the pathogenesis of endometrial cancer.Although the mortality rate of endometrial cancer is comparatively low in gynecologic malignancies, a rising trend of this cancer has been observed for the past decade. The understanding of the molecular mechanism will favor for the clinical management of this disease. Aberrant activation of Wnt/β-catenin signaling pathway plays a major role in the pathogenesis of endometrioid adenocarcinoma including this cancer type. In this study, we reported that Sox7, one of Sox transcriptional factors, was frequently underexpressed in endometrial cancer and importantly, it was associated with dysregulation of the Wnt/β-catenin signaling activity. Immunohistochemical and quantitative RT-PCR analyses showed that Sox7 was underexpressed and was associated with high-grade tumor ( Endometrial carcinoma is one of the common female malignancies in the western countries but it is getting common in Asian including Hong Kong Chinese. According to the Hong Kong Cancer Registry from 1999 to 2007, endometrial carcinoma has increased from the ninth to the fourth most common gynecologic malignancies for females overall in Hong Kong (Hong Kong Cancer Registry 2007). Although the mortality rate of this disease is keeping at approximately 1.0%, a significant increase for the incidence trend has been revealed from 1999 to 2007 (Hong Kong Cancer Registry 2007). This indicates endometrial cancer becomes more common in Hong Kong women nowadays. There are two primary types of endometrial cancer according to histopathology, cell biology and clinical course [β-catenin, APC and Axins are often associated with the upregulation of β-catenin and the pathogenesis of endometrioid-type of endometrial cancer and ovarian cancer [Wnt/β-catenin or canonical Wnt signal transduction pathway is a conserved molecular mechanism in metazoan animals. This signaling pathway regulates a remarkable variety of cellular process such as cell fate, cell proliferation, cell survival, cell behavior and migration . In thisn cancer -10. The n cancer , 12. Hown cancer -15. ThisIn this study, we identified Sox7, one of the Sox transcription factors family, was significantly down-regulated in high-grade endometrial cancer and inversely correlated with Wnt/β-catenin signaling activity. Enforced expression of Sox7 could remarkably suppress not only Wnt/β-catenin signaling and its downstream oncogenes, but also endometrial cancer cell growth. Importantly, we demonstrated that Sox7 inhibited Wnt/β-catenin signaling in endometrial or endometrioid ovarian cancer cells (OEA) harboring not only wild-type but also mutant β-catenin. Our findings manifest the significance of Sox7 in the pathogenesis and the regulatory mechanisms of aberrant activation of Wnt/β-catenin signaling activity in endometrial cancer. This new knowledge generated may reveal novel strategies for rational design of Wnt/β-catenin inhibiting agents to combat this cancer.2 in Dulbecco's modified Eagle medium (DMEM/F12)  with 10% fetal bovine serum (Gibco) and 1% Penicillin-Streptomycin (Gibco).Surgical resection of 43 tumor samples from primary endometrial cancer patients and 37 normal endometrial samples from benign diseases such as uterine fibroid after surgery were randomly chosen for Q-PCR analysis. The histology of all endometrial cancer tissue samples have been verified by surgical pathologists. The histological subtype and stage of the tumors were categorized according to International Federation of Gynaecology and Obstetrics (FIGO) classification. Written informed consent was taken and the use of these clinical samples was approved by Institutional Review Board of the University of Hong Kong/ Hospital Authority Hong Kong West Cluster (HKU/HA HKW IRB). Five endometrial cancer cell lines   and Ishikawa  were used for investigation of Sox7 functions. Human Embryonic Kidney 293 cells (HEK 293) (ATCC) was used for TOP/FOP luciferase reporter assay, and L Wnt3A cell line (CRL-2647) was used for Wnt3A conditioned medium (ATCC). The cell lines authentication was done by in-house STR DNA profiling analysis and were cultured at 37°C in 5% COThe Flag/Sox7 expressing plasmid  was for ectopic expression of Flag-tagged Sox7 fusion protein. The Sox7 cDNA was subcloned into pEGFP-C1  to generate GFP/Sox7 expressing plasmid. The HA/β-catenin expressing plasmid  and the GFP/mutant β-catenin S37A expressing plasmid  were used for expressing wild-type and mutant β-catenin respectively. The Myc/TCF4 expressing plasmid  was used for expressing Myc/TCF4 fusion protein. The pSuper8XTOPFlash and pSuper8XFOPFlash plasmids  were used for TOP/FOP luciferase reporter assay. The pLNCX-Wnt1 construct  was used for induction of Wnt/β-catenin activity. Lipofectamine™ LTX was used for cell transfection according to the manufacturer's instructions .Sox7, FGF9, SFN, Cyclin D1 and C-myc using Taqman® Gene expression Assays; human Sox7 (Assay ID: Hs00846731_s1), human FGF9 (Assay ID: Hs00181829_m1), human SFN (Assay ID: Hs00968567_s1), human Cyclin-D1 (Assay ID: Hs00765553_m1) and human C-myc (Assay ID: Hs00153408_m1), in an ABI PRISM™ 7500 system (Applied Biosystems). The human 18S rRNA (Assay ID: Hs99999901_m1) was used as an internal control.The total RNA was isolated from clinical samples and cell lines by TRIzol reagent (Invitrogen). The cDNA was synthesized using Reverse transcription reagent kit . Real time quantitative reverse transcriptase-PCR (Q-PCR) was used for evaluating the expressions of Cell lysate was prepared from cells using lysis buffer  containing protease inhibitor (Sigma) and Phenylmethylsulfonyl fluoride (PMSF) . The equal amount of protein samples was separated by 10% SDS-PAGE and electroblotted onto the Hybond-P membranes . Blots were blocked with 5% skim milk, followed by incubation with β-catenin  and C-Myc (N262) , Cyclin-D1 , anti-HA , anti-GFP (Santa Cruz), anti-Flag and β-actin (Sigma) overnight at 4°C. Blots were then incubated with anti-mouse or anti-rabbit (Amersham Pharmacia Biotechnology) secondary antibodies conjugate with horseradish peroxidase for 1 hour in room temperature and visualized using ECL™ Western Blotting Detection Reagent (Amersham).For co-immunoprecipitation assay, HEK293 cells were transiently co-transfected with Flag/Sox7 and HA/β-catenin, or GFP/mutant β-catenin, or Myc/TCF4 plasmids. The procedure of immunoprecipitation was performed as previously described , 17.For immunohistochemical analysis, a commercial ovarian cancer tissue array  was immunostained with primary rabbit polyclonal anti-Sox7  in 1:250 dilution, anti-β-catenin  in 1:400 dilution, and anti-FGF9 (Santa Cruz) in 1:200 dilution. The immunoreactivity of immuno-positive sample was determined by multiplying the intensity of the staining  and percentage of stained area (0-100%). The mean of immunoreactivity value of normal and borderline cases was used to normalize all cases. The examination and scoring of all tissues were done by two investigators independently.For immunofluorescent analysis, HEC1B cells were cultured on cover slips and transiently transfected with either RFP/Sox7 or GFP/mutant β-catenin S37A, or GFP/Sox7 and Flag/TCF4 and Myc/TCF4 expressing plasmids. After 24 hours, the transfected cells were fixed with 4% paraformaldehyde, treated with 0.1% Triton, incubated with anti-Flag (Sigma) and Alexa Fluor® Fluorescent Streptavidin Conjugates (Invitrogen), followed by counter-stained with DAPI  (Invitrogen), the fluorescent signals were examined and photographed by fluorescent microscopy (Leica Q550CW).Renilla luciferase activity. All experiments were repeated in 3 independent experiments.HEK293 cells were seeded in 24-well plates and transiently transfected with various amounts  of Flag/Sox7, 100 ng pLNCX-Wnt1, a pSuper8XTOPFlash or pSuper8XFOPFlash luciferase reporter constructs. All transfections were normalized with pcDNA vector and incubated for 24 hours. The luciferase activity was measured using the Dual-luciferase Reporter Assay System (Promega) and transfection efficiency was normalized with Cell viability was evaluated by Cell Proliferation Kit II (XTT) for 5 days according to the manufacturer's instructions (Roche). The experiment was repeated in three independent experiments.t test (for parametric data) and the Mann-Whitney test (for non-parametric data) were used. Statistical analyses on clinicopathological correlation were performed using the SPSS version 13.0 software . A p-value was considered significant when less than 0.05.Student's Sox7 in endometrial cancer was 4.2-fold less than normal endometrium (P = 0.005)  and Stratifin (SFN) [FGF9 and SFN which were 17.9-fold (P = 0.048) and 7.27-fold (P = 0.008), respectively, higher in endometrial cancer as compared with normal endometrium (see P = 0.021) and high level of β-catenin (P = 0.038). On the other hand, we found that 51.7% (62/120) endometrial cancer cases showed relatively higher in β-catenin expression (>2.7 folds) (Table P = 0.045), and two Wnt/β-catenin specific downstream targets; Cyclin D1 (P < 0.001) and FGF9 (P = 0.003) [Emerging reports have documented that Sox7 is down-regulated in several human cancers and may be a negative regulator of Wnt/β-catenin signaling activity -20. As a005) see . To invein (SFN) , 23 wererium see . To furtrium see . Clinico= 0.003) . This inSox7 in human endometrial cancer cell lines by Q-PCR analysis. Of 5 endometrial cell lines  (TOV112D)), HEC-1A, HEC-1B, Ishikawa and TOV112D which have previously showed high Wnt/β-catenin signaling activity [Sox7 but higher levels of CyclinD1, C-myc and FGF9   Fig. . Moreove01) Fig. . Intrigu01) Fig. . Taken tP < 0.02) and TOV112D (C5 and C6) (P < 0.01) exhibited significant reduction of cell proliferation rate (28 to 35%) as compared with their vector controls  and TOV112D (P < 0.001) further displayed suppressive effect on cell proliferation rate as compared with their vector controls , and a mutant β-catenin cell line TOV112D (C5 and C6). Western blotting showed that the ectopic expression of Sox7 was accompanied with the reduced expression of CyclinD1 and C-myc in all clones of both cell lines Fig. . When cools Fig. . Upon trols Fig. . These dGiven that Sox7 can negatively regulate Wnt/β-catenin signaling activity through reduction of its downstream targets, it may interrupt the transcriptional activity of this pathway. Therefore, we were interested in whether Sox7 would interact with transcriptional factor complex β-catenin/TCF/LEF. We firstly examined the localization of Sox7, β-catenin and TCF4 in endometrial cancer cells, HEC-1A. By immunofluorescent microscopy, we demonstrated that RFP/Sox7 or GFP/Sox7 was localized mainly in the nucleus Fig. . SimilarThis study showed that Sox7 was a potent negative regulator in the Wnt/β-catenin signaling pathway and was frequently underexpressed in endometrial cancer. The underexpressed Sox7 was associated with increased Wnt/β-catenin signaling activity and high-grade endometrial cancer. Importantly, we demonstrated that the enforced expression of Sox7 remarkably impeded Wnt/β-catenin signaling mediated cancer cell growth and such suppressive effect was independent on the presence of wild-type or mutant β-catenin in endometrial cancer or OEA cells.Previous studies documented that approximately 40% of endometrial cancer have aberrant activation of Wnt/β-catenin signaling pathway , 8. MostMounting evidences have showed that aberrant activation of the Wnt/β-catenin signaling has associated with human cancers including endometrial cancer and ovarian endometrioid adenocarcinoma (OEA) -29. TherAlthough further studies are needed to delineate how the interaction of Sox7 mediated inhibition of -catenin/TCF/LEF-1 complex transcriptional activity, our findings collectively highlight the suppressive roles of Sox7 in Wnt/β-catenin signaling and its tumorigenic capacities in endometrial cancer cells carrying either wild-type or mutant β-catenin.


The aim of this study was to prepare three types of multiwall carbon nanotubes (CNT)-based composite electrodes and to modify their surface by copper electrodeposition for nonenzymatic oxidation and determination of glucose from aqueous solution. Copper-decorated multiwall carbon nanotubes composite electrode (Cu/CNT-epoxy) exhibited the highest sensitivity to glucose determination. Nowadays, a large community of researchers is focusing on the development of different applications for carbon nanotubes (CNT) in the field of electrochemical sensing. The high sensitivity, chemical stability, excellent electrical conductivity, and modifiable surface that provides the possibility to fabricate multifunctional electrochemical sensors are only a few important properties which recommend them for sensing applications ,2. Using2. The ratios were chosen to reach 20% wt CNT, 20% wt SZ, and 20% wt NZ. The surface of prepared electrodes was then decorated with copper by electrodeposition at a potential of −0.5 V vs. Ag/AgCl for 20 s in the presence of 0.1 M CuSO4 solution. Scanning electron microscopy (SEM) was performed using an Inspect S PANalytical instrument  coupled with the energy-dispersive X-ray analysis detector (EDX). Voltammetric and amperometric measurements were carried out using an Autolab PGSTAT101  controlled with NOVA 1.6 software  and a three-electrode cell, with an Ag/AgCl reference electrode, a platinum counter electrode, and composite working electrodes.Multiwall CNT with an average diameter of 9.5 nm and average length of 1.5 μm were purchased from Nanocyl, Belgium . A synthetic A-type zeolite (SZ) was prepared using natural clinoptilolite as a silicon source and sodium aluminate as aluminum source, as we previously described . The comFigure −1 for Cu/CNT-epoxy, 5.718 mA∙mM−1 for Cu/SZCNT-epoxy and 2.826 mA∙mM−1 for Cu/NZCNT-epoxy, superior to the other values previous reported for catalyst-modified composite electrodes [A series of cyclic voltammograms recorded at the tested Cu decorated electrodes in 0.1 M NaOH and in the concentration range 0.02 to 0.12 mM glucose is illustrated in Figure −1, smaller than the one recorded by voltammetric techniques.Further amperometric tests were performed using the best performing electrode for the determination of glucose selected by cyclic voltammetry, i.e., Cu/CNT-epoxy. The chronoamperometry was employed in two variants, without and under stirring conditions , in comparison with the amperometric measurements under stationary conditions. This fact reflects an oxidation process of glucose control by a rate-determining step involving mass transfer and a surface-controlled reaction. Better responses were reached under the batch system analysis conditions of multiple-pulsed amperometry  of 3.5% shows a good reproducibility. A recovery test was also performed by analyzing three parallel glucose serum samples. This test was run for 0.1 mM glucose in 0.1 M NaOH supporting electrolyte, and a recovery of 98% was found with an RSD of 3.8% using multiple-pulsed amperometry.-1 and 0.2 μM glucose as limit of detection.The electrocatalytic activities of the copper-decorated CNT-epoxy, SZCNT-epoxy and NZCNT-epoxy electrodes towards the oxidation of glucose in an alkaline solution were demonstrated. All copper-decorated composite electrodes exhibited useful properties for the direct oxidation and simple nonenzymatic determination of glucose on tested electrodes surface. The differences between the electroanalytical performances of the electrodes are related to composite structure and morphology, which influenced copper particle size and distribution on the surface of the composite material. The best electroanalytical performances obtained for the detection of glucose by cyclic voltammetry were recorded with the copper-decorated CNT-epoxy electrode, i.e., electrode sensitivity of 8.45 mA∙mMCNT, Carbon nanotubes; CNT-epoxy, Carbon nanotubes-epoxy electrode; Cu/CNT-epoxy, Carbon nanotubes-epoxy copper decorated electrode; Cu/NZCNT-epoxy, Carbon nanotubes-natural zeolite-epoxy copper decorated electrode; Cu/SZCNT-epoxy, Carbon nanotubes-synthetic zeolite-epoxy copper decorated electrode; EDX, Energy-dispersive X-ray spectroscopy; NZ, Natural zeolite; NZCNT-epoxy, Carbon nanotubes-natural zeolite-epoxy electrode; SEM, Scanning electron microscopy; SZ, Synthetic A-type zeolite; SZCNT-epoxy, Carbon nanotubes-synthetic zeolite-epoxy electrode.The authors declare that they have no competing interests.AP carried out the electrodes preparation, electrochemical detection measurements and data interpretation. FM conceived the study and participated in data interpretation and coordination. CO performed SEM measurements and contributed at electrodes preparation. SM and EI contributed in the electrochemical characterization tests. NV and JS participated in paper design and coordination. All authors read and approved the final manuscript.AP and CO are postdoctoral research associates. FM is an associate professor of Electrochemistry Applied for Environment Remediation and Monitoring. NV is a professor of Electrochemistry. JS is a professor of Advanced Materials. SM and EI are PhD students.