Charge (electron) transfer in systems based on PANI/AuNPs(6nm)-GOx, PANI/AuNPs(AuCl4−)-GOx, Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs(AuCl4−)-GOx nanocomposites was investigated. Cyclic voltammetry (CV)-based investigations showed that the reported polymer nanocomposites are able to facilitate electron transfer from enzyme to the graphite rod (GR) electrode. Significantly higher anodic current and well-defined red-ox peaks were observed at a scan rate of 0.10 V s−1. Logarithmic function of anodic current (log Ipa), which was determined by CV-based experiments performed with glucose, was proportional to the logarithmic function of a scan rate (log v) in the range of 0.699–2.48 mV s−1, and it indicates that diffusion-controlled electrochemical processes were limiting the kinetics of the analytical signal. The most efficient nanocomposite structure for the design of the reported glucose biosensor was based on two-day formed Ppy/AuNPs(AuCl4−)-GOx nanocomposites. GR/Ppy/AuNPs(AuCl4−)-GOx was characterized by the linear dependence of the analytical signal on glucose concentration in the range from 0.1 to 0.70 mmol L−1, the sensitivity of 4.31 mA mM cm−2, the limit of detection of 0.10 mmol L−1 and the half-life period of 19 days.Nanocomposites based on two conducting polymers, polyaniline (PANI) and polypyrrole (Ppy), with embedded glucose oxidase (GOx) and 6 nm size gold nanoparticles (AuNPs The development of easy, fast and cheap technology for the determination of glucose is very important in clinical diagnostics, biotechnology, biofuel cells and food manufacturing ,7,8,9,10Through the achievements in nanotechnology and nanoscience, the amplification of the electrochemical signal of the sensor and efficiency of biofuel cells can be achieved ,22,23,24Electrodes modified by conducting polymers have been extensively developed during the last decades ,28,37,383+ cation is reduced and forms Au+ cation, which is related to the nuclei formation; then, Au+ cation is reduced to Au0 by the formation of gold clusters [+ cation is able to activate inert conjugated molecules  glucose biosensor, which was based on a GC electrode modified by GOx-poly(L-lysine)/reduced graphene oxide-ZrO2 composites and was operating in the absence of red-ox mediators. Another group of researchers developed a mediator-free fructose sensor based on a GC electrode modified by a composite consisting of 790 nm AuNPs, Ppy, chitosane and fructose dehydrogenase, with high biocatalytic current density of 2.45 mA cm−2 [−1cm−2 [Different polymer/AuNPs nanocomposites formation methods have been developed: (i) electrochemical synthesis of conducting polymers in the presence of AuNPs, which is based on monomer’s oxidation with following incorporation of metallic nanoparticles , (ii) inclusters ,30,40. As, etc., ) with thnd AuNPs ,43,44. Bnd AuNPs ,26,27, f[−2 mM−1 . The sam [−1cm−2 .4−) (PANI/AuNPs(6nm)-GOx, PANI/AuNPs(AuCl4−)-GOx, Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs(AuCl4−)-GOx) nanocomposites. Cyclic voltammetry (CV) was applied for the investigation of analytical characteristics of the glucose biosensor and for the evaluation of these modified electrodes in the determination of glucose in real serum samples in the presence/absence of some interfering materials.The main aim of the present study was to evaluate some characteristics of a glucose biosensor based on polyaniline (PANI) and polypyrrole (Ppy), with embedded GOx and 6 nm size AuNPs or gold-nanoclusters formed from chloroaurate ions  was purchased from Fluka , tetrachloroauric acid (HAuCl4·3H2O) – from Alfa Aesar GmbH & Co KG , sodium citrate – from Penta , and tannic acid, D-(+)-glucose, D(–)-fructose, D(+)-mannose, D(+)-galactose, D(+)-xylose and D(+)-saccharose – from Carl Roth GmbH + Co . The solutions of sugars were stored for 1 day before the measurements in order to establish equilibrium between α and β isomers of sugars. The buffer of sodium acetate  in the presence of 0.1 mol L−1 KCl was prepared from sodium acetate trihydrate and potassium chloride, which were obtained from Reanal  and Lachema , respectively. Aniline and sodium hydroxide were obtained from Merck KGaA , phenazine methosulfate (PMS), L-ascorbic acid (AA) and uric acid (UA) – from AppliChem GmbH , pyrrole – from Acros Organics , and hydrochloric acid – from Sigma-Aldrich . The reagents were characterized as analytically pure or the highest quality. Colored components of PANI and Ppy were removed by filtration through a filtration column (of 5 cm length) filled with α-Al2O3 with 0.3 µm diameter grains (Type N), which was purchased from Electron Microscopy Sciences . All working solutions were prepared using deionized water and were stored between investigations at 4 °C. 6 nm AuNPs of 2.3 × 1016 particles L−1 were synthesized according to a methodology presented previously [Glucose oxidase -GOx, PANI/AuNPs(AuCl4−)-GOx, Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs(AuCl4−)-GOx nanocomposites were formed by enzyme-assisted synthesis at room temperature (20 ± 2 °C) in the dark over 2 or 4 days. The formation of polymer nanocomposites (PNC) was performed in 0.05 mol L−1 SA buffer, pH 6.0, containing 0.05 mol L−1 of glucose, 0.75 mg mL−1 of GOx, 0.50 mol L−1 of aniline or pyrrole in the presence of 0.46 × 1016 particles L−1 of AuNPs(6 nm) or 0.6 mmol L−1 of HAuCl4. To separate enzyme-assisted synthesized nanocomposites from the polymerization solution, a centrifugation by centrifuge IEC CL31R Multispeed  during 8 min (14.6 × 103 g) was performed. Then, PNC were washed two times with 0.05 mol L−1 SA buffer, pH 6.0, and then collected by the centrifugation and were used for further measurements. PANI/GOx and Ppy/GOx nanocomposites were formed during a synthesis procedure lasting 2 days, according to the above-described protocol in the absence and presence of AuNPs.PANI/AuNPs2) served as a working electrode and was used for electrochemical investigations. To design the working electrode, GR was cut, polished firstly by fine emery paper and then by slurry α-Al2O3 powder. After that, GR was washed by deionized water, dried at room temperature and sealed within a silicone tube to avoid any contact of electrode sides with a solution during electrochemical investigations. Enzyme-assisted synthesized PNC were deposited on the surface of the GR electrode and damped by a drop of 0.05 mol L−1 SA buffer, pH 6.0. Then, the surface of the working electrode was covered with a polycarbonate membrane, to avoid the detachment of PNC.Graphite rod (surface area 0.071 cm(AuCl4−)-GOx and Ppy/AuNPs(AuCl4−)-GOx nanocomposites proceeded within 2 days. PNC were dispersed and homogenized in 50 μL of deionized water. Then, PNC were deposited on the surface of the Si substrate and the morphology and size of this structure were evaluated by a Hitachi SU-70  field emission scanning microscope (FE-SEM).Enzyme-assisted formation of PANI/AuNPsv) from 0.005 to 0.30 V s−1. All measurements were performed in three-electrode cells, with a working electrode—GR electrodes modified by PNC, a reference electrode—Ag/AgCl(3 mol L−1KCl) obtained from Metrohm , and a counter electrode—platinum spiral. Electrochemical experiments were carried out in 0.05 mol L−1 SA buffer, pH 6.0, with 0.1 mol L−1 KCl and with or without 6 mmol L−1 PMS, which shifted the potential from −0.70 to 0.80 V vs. Ag/AgCl(3 mol L−1KCl) at room temperature.Experiments were performed by computerized potentiostat/galvanostat PGSTAT 302/Autolab with GPES 4.9 software, which was purchased from EcoChemie , using cyclic voltammetry at scan rates -GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes were stored at 4 °C in a closed vessel hanging over the solution of SA buffer, followed by a wash with distilled water before each measurement.Electrochemical detection of the analytical response was performed at a concentration interval from 0.10 to 48.4 mmol LCyclic voltammograms of all measurements were plotted and the parameters of Michaelis-Menten kinetics and the limit of detection (LOD) were estimated using statistic software SigmaPlot version 12.5. Calibration curves of all investigations were obtained while measuring analytical responses three times. The calculated LOD represents the lowest concentration of glucose, which provides an analytical signal greater than the background value plus 3 δ.(6nm)-GOx, PANI/AuNPs(AuCl4−)-GOx, Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs(AuCl4−)-GOx nanocomposites followed by electrochemical evaluation of the nanocomposite-modified electrodes are presented in The principle schema of enzyme-assisted formation of PANI/AuNPs−1 SA, pH 6.0, and centrifuged at 14.6 × 103 g. All investigations were performed using the GR electrode modified by Ppy/AuNPs(AuCl4−)-GOx nanocomposites. The electrochemical measurements were done in 10 times diluted human serum with 10 mmol L−1 of glucose before and after the addition of 1 mmol L−1 fructose, mannose, galactose, xylose, or saccharose. To evaluate the influence of ascorbic and uric acids on the developed biosensor, CV measurements were applied in 10 times diluted human serum with 10 mmol L−1 of glucose, 10 mmol L−1 of glucose and 0.01, 0.05 or 0.1 mmol L−1 of ascorbic acid (AA), and 10 mmol L−1 of glucose and 0.01 or 0.05 mmol L−1 of uric acid (UA).The human serum sample was diluted (1:10) in 0.05 mol LNovel nanocomposites based on AuNPs and polymers are able to improve various useful characteristics of biosensors . The mai(6nm)-GOx, PANI/AuNPs(AuCl4−)-GOx, Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs(AuCl4−)-GOx nanocomposites, was investigated by CV in 0.05 mol L−1 SA buffer, pH 6.0, in the presence of a soluble red-ox mediator—PMS. The reduction of the pyrrole monomer and the oxidation of chloroaurate ions proceeded simultaneously with the formation of Ppy and Au0 [(6nm)-GOx, Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs(AuCl4−)-GOx nanocomposites, instead of PANI/AuNPs(AuCl4−)-GOx-based nanocomposites. After the deposition of the nanocomposite’s layer on the working electrode, the form of registered cyclic voltammograms changed and became much wider than that before the modification. This effect illustrates a significant increase of the electrical capacitance of the modified electrode, and this fact is in line with previously published research on the evaluation of Ppy layers of different thickness and/or morphology [Investigations of the shape of CV and the influence of scan rate on a shift of red-ox peaks provide some important information on the mechanism of electrochemical reaction and the rate of electron transfer from electrochemically active species . The per and Au0 . The infrphology .(6nm)-GOx, GR/PANI/AuNPs(AuCl4−)-GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes, Epa has been shifted to more positive values, from −0.002 to 0.23 V, from 0.096 to 0.26 V, from −0.009 to 0.24 V and from −0.007 to 0.21 V respectively, when the scan rate increased from 0.005 to 0.30 V s−1. These red-ox peaks are attributed to the oxidation of glucose to gluconolactone, as it was reported previously [It was determined that the diffusion barrier of GR electrodes, additionally modified by PANI or Ppy using enzyme-assisted polymerization, increased. It was observed that for GR/PANI/AuNPseviously , and ind(6nm)-GOx, GR/PANI/AuNPs(AuCl4−)-GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes, Epc has shifted towards more negative potentials, from −0.090 to −0.48 V, from −0.031 to −0.47 V, from −0.10 to −0.44 V and from −0.019 to −0.42 V respectively, by an increase of scan rate from 0.005 to 0.30 V s−1. The peak at potential of approximately −0.42 (−0.48) V for working electrodes may be observed due to the reduction of gold oxides, which occurs in potential of −0.40 V [−1, anodic and cathodic peaks shifted for GR/PANI/AuNPs(6nm)-GOx by 0.23 V (ΔEpa = Epa(0.30 V s−1) − Epa(0.005 V s−1)) and by −0.39 V (ΔEpc = Epc(0.30 V s−1) − Epc(0.005 V s−1)) respectively, for GR/PANI/AuNPs(AuCl4−)-GOx—by 0.16 V and by −0.44 V, for GR/Ppy/AuNPs(6nm)-GOx—by 0.25 V and by −0.34 V, and for GR/Ppy/AuNPs(AuCl4−)-GOx—by 0.22 V and by −0.40 V. It is seen that in the case of GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx, electrodes’ anodic peaks have shifted by 0.02 and 0.06 V towards more positive potential than that of GR/PANI/AuNPs(6nm)-GOx and GR/PANI/AuNPs(AuCl4−)-GOx electrodes, respectively. The form of anodic peaks of the GR/PANI/AuNPs(AuCl4−)-GOx electrode significantly differs from that of GR/PANI/AuNPs(6nm)-GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes. Cathodic peaks registered using GR/PANI/AuNPs(6nm)-GOx and GR/PANI/AuNPs(AuCl4−)-GOx electrodes have shifted by 0.05 and 0.04 V towards more negative potential than that of GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes  at 0.30 V s−1 for GR/PANI/AuNPs(6nm)-GOx was −0.13 V, for GR/PANI/AuNPs(AuCl4−)-GOx was −0.11 V, for GR/Ppy/AuNPs(6nm)-GOx was −0.10 V and for GR/Ppy/AuNPs(AuCl4−)-GOx was −0.11 V. It indicates that the GR electrode modified by PNC nanocomposites containing GOx and AuNPs was characterized by a rather high degree of the reversibility and sufficient charge transfer rate. It was determined that the scan rate in the range from 0.10 to 0.30 V s−1 was the most optimal for accurate determination of red-ox peaks in cyclic voltammograms.A diffusion-controlled, quasi-reversible electrochemical process was observed and the average value of the anodic and cathodic potential was calculated according to the methodology presented in Reference . The forIpa) is proportional to the applied scan rate and the relationship between log Ipa and log v is linear when the “classic” electrochemical system is under investigation [(6nm)-GOx, GR/PANI/AuNPs(AuCl4−)-GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes in the absence and in the presence of glucose is presented in Scan rate influences the electrochemical behavior of electrodes: a rather thick diffusion layer is formed when a slow scan rate is applied, and on the contrary, a thinner layer is formed when a fast scan rate is applied . Anodic tigation ,47,48. T−1 in the absence of glucose, the value of the anodic current obtained using GR/PANI/AuNPs(6nm)-GOx, GR/PANI/AuNPs(AuCl4−)-GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes increased by 6.08 times (from 0.141 to 0.857 mA), 4.51 times (from 0.0732 to 0.330 mA), 8.63 times (from 0.0562 to 0.485 mA) and 2.92 times (from 0.0703 to 0.205 mA), respectively (Ipa registered using GR/PANI/AuNPs(6nm)-GOx, GR/PANI/AuNPs(AuCl4−)-GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes increased by 7.41 times (from 0.147 to 1.09 mA), 4.75 times (from 0.0823 to 0.391 mA), 11.0 times (from 0.0788 to 0.870 mA) and 16.9 times (from 0.0343 to 0.578 mA) respectively, with the increase of scan rate from 0.005 to 0.30 V s−1 ((6nm)-GOx and GR/PANI/AuNPs(AuCl4−)-GOx electrodes (−1 scan rate in the presence of glucose was 1.27 and 1.18 times higher in comparison with the anodic current registered for the same electrodes in the absence of glucose. Anodic currents for GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes -GOx, GR/PANI/AuNPs(AuCl4−)-GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes in the presence of glucose is presented in v in the range of 0.699–2.48 mV s−1, which is in the agreement with data published in another paper [(6nm)-GOx, GR/PANI/AuNPs(AuCl4−)-GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes ((6nm)-GOx, GR/PANI/AuNPs(AuCl4−)-GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes is diffusion-controlled and, therefore, such electrode can be applied for electrochemical biosensing of glucose in a rather broad concentration range. Mathematically, the slope of line (a) is determined by the equation of linear dependence /(x2 − x1) = tan(a)). As is seen from (6nm)-GOx electrode (tan(a) = 0.754) is 1.25 times steeper than that calculated for the GR/PANI/AuNPs(AuCl4−)-GOx electrode (tan(a) = 0.601). The same difference of the slope was determined for GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes. As it is seen from (AuCl4−)-GOx electrode (tan(a) = 0.781) is 1.12 times steeper in comparison with the results calculated for the GR/Ppy/AuNPs(6nm)-GOx electrode (tan(a) = 0.700). The steeper slope presents the increase of electron rate on the surface of GR/PANI/AuNPs(6nm)-GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes. It is in agreement with a statement that AuNPs could enhance the sensitivity and stability of a glucose biosensor by catalyzing oxidation of H2O2, which is formed during enzymatic reaction, and effectively facilitate the electron transfer through a nanocomposite matrix due to good conductivity [The relationship between log ectrodes B achieveAuNPsAuCl−-GOx, GR−1 [(6nm)-GOx, PANI/AuNPs(AuCl4−)-GOx, Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs(AuCl4−)-GOx nanocomposites increased from 679 to 1128, from 570 to 659, from 512 to 594 and from 366 to 388 nm, respectively [(6nm)-GOx, GR/PANI/AuNPs(AuCl4−)-GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes will be decreased when too-long polymerization duration is applied. Gold nanoparticles promote the electron transfer between glucose oxidase and the working electrode [(6nm)-GOx, GR/PANI/AuNPs(AuCl4−)-GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes, the enzyme-assisted polymerization was performed for periods between 2 and 4 days in the dark at room temperature. To evaluate the anodic current of CV correctly, the difference of anodic peaks in the absence and the presence of 48 mmol L−1 glucose was calculated, and the obtained results are presented in During enzyme-assisted polymerization, low molecular weight polymers remain in aqueous solutions while large molecular weight polymers are adsorbed on the electrode surface . The eff−1 . However−1 . In our −1 . It was ectively . We predlectrode ,51. To e(6nm)-GOx, GR/PANI/AuNPs(AuCl4−)-GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes has decreased by 1.79 times (from 0.123 to 0.0687 mA), 1.72 times (from 0.0241 to 0.0140 mA), 7.69 times (from 0.233 to 0.0303 mA) and 11.7 times (from 0.263 to 0.0225 mA) respectively, with the increased duration of enzyme-assisted polymerization from 2 to 4 days. It led us to predict that a more sensitive glucose biosensor can be designed when enzyme-assisted polymerization lasting 2 days is applied for the formation of a nanocomposite-based layer.As it is seen from (6nm)-GOx, GR/PANI/AuNPs(AuCl4−)-GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes calculated from data presented in −2, respectively. It was determined that after 2 days of enzyme-assisted polymerization, the sensitivity of the GR/PANI/AuNPs(6nm)-GOx electrode was 5.08 times higher, if compared with the GR/PANI/AuNPs(AuCl4−)-GOx electrode. Meanwhile, the sensitivity of the GR/Ppy/AuNPs(6nm)-GOx electrode was 1.13 times lower than that obtained on the GR/Ppy/AuNPs(AuCl4−)-GOx electrode. The values of the sensitivity for GR/PANI/AuNPs(6nm)-GOx and GR/PANI/AuNPs(AuCl4−)-GOx electrodes after 2 days of PNC formation were 1.89 and 10.9 times lower than that obtained using GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes. To evaluate the role of AuNPs on the sensitivity in glucose sensing, the results obtained in the presence and absence of AuNPs in the structure of PANI and Ppy nanocomposites after 2 days of polymerization were compared. As can be seen from the results presented in −2) electrode was 1.37 times lower if compared with the GR/PANI/AuNPs(6nm)-GOx electrode. Meanwhile, the sensitivity of the GR/PANI/GOx electrode was 3.72 times higher in a comparison with the GR/PANI/AuNPs(AuCl4−)-GOx electrode, which were characterized as presented above. The sensitivity of the GR/Ppy/GOx (0.0314 mA mM cm−2) electrode was 2.18 and 2.46 times lower than that of GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes, respectively. It indicates the advantage of polymeric nanocomposites with AuNPs. In any case, 2 days of enzyme-assisted polymerization is the most suitable duration for the development of GR/PANI/AuNPs(6nm)-GOx, GR/PANI/AuNPs(AuCl4−)-GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes. The higher value of the anodic current calculated from differences of anodic peaks of CV in the presence and absence of glucose for GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes is based on the conducting nature and increased ‘active surface area’ of nanocomposites, which offers more freedom regarding the orientation of entrapped GOx [The sensitivity of GR/PANI/AuNPsv is the scan rate (0.1 V s−1) and A is the surface area of the working electrode (0.071 cm2), the constants R = 8.314 J K−1 mol−1, T = 294 K and F = 96485 C mol−1. The values of the surface coverage of GOx for GR/PANI/AuNPs(6nm)-GOx, GR/PANI/AuNPs(AuCl4−)-GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes were evaluated as 4.54 × 10−9, 8.91 × 10−10, 8.63 × 10−9 and 9.71 × 10−9 mol cm−2 respectively, which are higher than that (1.76 × 10−10 mol cm−2) obtained on the glassy carbon electrode modified by GOx/P-L-Arg/f-MWCNTs composites [(6 nm) and Ppy/AuNPs(AuCl4−) nanocomposites facilitate the high enzyme loading.The surface concentration of the electrochemically active enzyme on the electrode surface (Γ) can be calculated by the following equation :Ipa = n2(AuCl4−)-GOx and Ppy/AuNPs(AuCl4−)-GOx nanocomposites obtained after 2 days of enzyme-assisted synthesis were evaluated by field emission scanning microscope and are presented in The morphology and size of PANI/AuNPs(AuCl4−)-GOx and Ppy/AuNPs(AuCl4−)-GOx nanocomposites had spherical structures. The size of PANI/AuNPs(AuCl4−)-GOx and Ppy/AuNPs(AuCl4−)-GOx nanocomposites was in the range of 300–452 nm and 80–113 nm respectively, and was similar to that of Ppy structures formed in the absence of AuNPs [4 and toluene sulfonic acid [4− ions during the formation of polymeric nanocomposites increased the size of PANI/AuNPs(AuCl4−)-GOx and Ppy/AuNPs(AuCl4−)-GOx nanocomposites by 9 and 2 times if compared with the size of PANI/GOx and Ppy/GOx nanocomposites, which were synthesized in our previous research [As can be seen from of AuNPs . Huang enic acid . The pre(6nm)-GOx, PANI/AuNPs(AuCl4−)-GOx, Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs(AuCl4−)-GOx nanocomposites synthesized by the enzyme-assisted approach were investigated in 1.0 mol L−1 HCl by cyclic voltammetry performed in the potential range from −0.300 to 1.20 V. The cyclic voltammograms of bare GR electrodes and GR electrodes modified by PANI/AuNPs(6nm)-GOx, PANI/AuNPs(AuCl4−)-GOx, Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs(AuCl4−)-GOx nanocomposites are presented in The electrochemical characterization of PANI/AuNPs(6nm)-GOx electrode ((6nm)-GOx electrode ((6nm)-GOx electrode could be associated with the oxidation of emeraldine into pernigraniline form [(6nm)-GOx ((AuCl4−)-GOx -GOx B, line 4l4−)-GOx B, line 5−2) of GR/PANI/AuNPs(6nm)-GOx, GR/PANI/AuNPs(AuCl4−)-GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes was calculated from CVs presented in (6nm)-GOx and PANI/AuNPs(AuCl4−)-GOx ((6nm)-GOx and Ppy/AuNPs(AuCl4−)-GOx were characterized by the current of 0.105 and 0.069 mA at 0.425 and 0.545 V ((6nm)-GOx, GR/PANI/AuNPs(AuCl4−)-GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes was evaluated as 3.20, 5.78, 1.48 and 0.972 mA cm−2, and these values indicate rather high conductivity of formed structures, which increases the charge transfer ability of these PNC.The current density (mA cm(6nm)-GOx, Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs(AuCl4−)-GOx nanocomposites, which were formed by 2 days of enzyme-assisted polymerization, were evaluated. The application of PANI/AuNPs(AuCl4−)-GOx nanocomposites was refused due to the irregular form of cyclic voltammograms. The stability of the developed glucose biosensors was investigated within 6 or 22 days by the registration of CVs. GR electrodes modified by PANI/AuNPs(6nm)-GOx, Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs(AuCl4−)-GOx nanocomposites -GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes were equated to 100% and are presented in The next stage of investigations was the evaluation of the stability of glucose biosensors based on GR electrodes modified by the developed PNC. For this purpose, PANI/AuNPs(6nm)-GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes towards glucose after storage for 6 days decreased down to 16.3%, 10.1% and 78.1%, respectively, of their initial value. It means that the value of the anodic current obtained on GR/PANI/AuNPs(6nm)-GOx, GR/Ppy/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(AuCl4−)-GOx electrodes after 6 days was 6.13, 9.90 and 1.28 times lower than that immediately after the design of electrodes. It is seen that electrochemical biosensors based on the GR/Ppy/AuNPs(AuCl4−)-GOx electrode were 4.79 and 7.73 times more stable than those based on GR/PANI/AuNPs(6nm)-GOx and GR/Ppy/AuNPs(6nm)-GOx electrodes. The further investigation was performed with 2 days of enzyme-assisted synthesized Ppy/AuNPs(AuCl4−)-GOx nanocomposites due to their high stability and the simplicity of surface modification of the GR electrode. The τ1/2  for the developed GR/Ppy/AuNPs(AuCl4−)-GOx electrode was 19 days.The analytical response of GR/PANI/AuNPs(AuCl4−)-GOx nanocomposites formed on the surface of the GR electrode improved the efficiency of charge transfer from immobilized GOx and maintained their bio-catalytic activity. During electrochemical measurements, electrons are transferred toward the positively charged surface of the GR electrode and current is registered. In the presence of glucose and dissolved oxygen in the solution of SA buffer, immobilized on the GR electrode, GOx contained in polymeric nanocomposites generates hydrogen peroxide and gluconolactone, which is hydrolyzed to gluconic acid  directly to the GR electrode and (ii) through AuNPs [(AuCl4−)-GOx electrode, anodic current responses were measured by CV at the glucose concentration range from 0.10 to 48.4 mmol L−1. The anodic current of the GR/Ppy/AuNPs(AuCl4−)-GOx electrode increased due to the GOx-catalyzed reaction and the increased concentration of oxidizable products ((AuCl4−)-GOx electrode is also involved in the electrochemical reduction of O2 and H2O2 and it is in line with findings presented by some other researchers, which evaluated graphene/nano-Au/GOx systems immobilized on the GC electrode [(AuCl4−)-GOx electrode appeared at 0.138 V, while the cathodic peak appeared at −0.343 V. Ppy/AuNPsnic acid . PMS is gh AuNPs . AuNPs agh AuNPs ,51. To eproducts A. As it products A, the GRlectrode . The ano(AuCl4−)-GOx electrode can be used for glucose determination due to the increase of the anodic current of CVs (Imax) and the apparent Michaelis constant (KM(apparent)) were correspondingly a and b parameters of hyperbolic function y = ax/(b + x), which has been used for the approximation of results. The reported glucose biosensor based on the GR/Ppy/AuNPs(AuCl4−)-GOx electrode was characterized by ΔImax = 0.292 mA and KM(apparent) = 0.348 mmol L−1. A low value of KM(apparent) indicates high affinity of glucose towards immobilized glucose oxidase [The GR/Ppy/AuNPst of CVs A. The am oxidase .(AuCl4−)-GOx electrode display that the linear detection range for the determination of glucose was up to 0.70 mmol L−1, with the correlation coefficient of 0.9895 (−1 glucose was determined as 4.31 mA mM cm−2. The sensitivity of the GR/Ppy/AuNPs(AuCl4−)-GOx electrode at a low concentration of glucose was higher than that at a high concentration. Lower sensitivity (0.0772 mA mM cm−2) at high concentrations (48.4 mmol L−1) of glucose than that at low concentrations could be related to two factors: (i) the slower diffusion of glucose, because at higher glucose concentrations the viscosity of glucose solution increases, and (ii) adsorption/desorption dynamics of glucose at high concentrations [−2) [2 composites (11.65 μA mM cm−2) [−1 on the GR/Ppy/AuNPs(AuCl4−)-GOx electrode was 13.9%. The value of the LOD for the developed glucose biosensor was determined as 0.10 mmol L−1. The calculated LOD is in the same range as that determined by some other researchers for the GC electrode modified by GOx/P-L-Arg/f-MWCNTs composites (0.10 mmol L−1) [2 composites (0.13 mmol L−1) [Analytical measurements of the GR/Ppy/AuNPsf 0.9895 C. The setrations . The evaons [−2)  and of tmM cm−2) . The relmol L−1) . Accordimol L−1) , the inc(AuCl4−)-GOx nanocomposites, even if the linear range of the developed glucose biosensor was lower than the physiological level of glucose in biological samples, in the range from 3 to 8 mmol L−1 [−1).The high sensitivity and the low limit of detection are significant advantages of the GR electrode modified by Ppy/AuNPsmmol L−1 ,56 -GOx nanocomposites for practical application.The addition of the interfering species, such as 1.0 mmol L glucose A, while −1 of AA  and 0.1 −1 of UA ) in huma(AuCl4−)-GOx nanocomposites for glucose determination in the samples of human serum was performed. For this purpose, human serum 10 times diluted with 0.05 mol L−1 SA buffer solution, pH 6.0, with known initial glucose concentrations, was investigated. Then, various amounts of glucose were added in order to imitate samples with increasing glucose concentrations in the linear detection range of the analyte. Each sample of human blood serum was assessed at least three or four times, and the achieved results were expressed by the average values and are presented in The suitability of the developed biosensor based on the GR electrode modified by Ppy/AuNPs−1 SA buffer, pH 6.0, sample containing 6 mmol L−1 PMS.The response of CV was registered in human blood serum 10 times diluted with 0.05 mol L(AuCl4−)-GOx nanocomposites are: (i) the high sensitivity (4.31 mA mM cm−2) and rather good stability (19 days), (ii) the low limit of detection (0.10 mmol L−1), (iii) the applicability in multiple analyses, (iv) the low price of a single analysis, (v) the short duration of a single measurement (20 s) and (vi) the high resistance to interfering materials, which allows to apply modified GR electrodes for glucose biosensing in clinical practice. The development of a glucose biosensor without a red-ox mediator is expected to be the next step of our investigations in this research area.The advantages of the developed biosensor based on the graphite rod electrode modified by Ppy/AuNPsIpa and log v registered from CV anodic peaks in the presence of glucose was linear and the electrochemical reaction was controlled by the diffusion. The glucose biosensor based on GR modified by Ppy/AuNPs(AuCl4−)-GOx nanocomposites was characterized by high sensitivity (4.31 mA mM cm−2) and good stability . The developed glucose biosensor based on the GR electrode modified by Ppy/AuNPs(AuCl4−)-GOx nanocomposites was suitable for the determination of glucose in the sample of human blood serum in the presence of interfering species with the recoveries in the range of 93.6–94.8%. The presented bioelectrochemical system can be useful for the development of biosensors, biofuel cells and bioelectronics devices.PANI and Ppy nanocomposites with GOx and AuNPs were formed by enzyme-assisted synthesis of PANI and Ppy and were deposited on the surface of GR electrodes. The relationship between log


Aberrant signaling through β-catenin is an important determinant of tumorigenesis in rodents as well as in humans. In mice, xenobiotic activators of the constitutive androstane receptor (CAR), a chemo-sensing nuclear receptor, promote liver tumor growth by means of a non-genotoxic mechanism and, under certain conditions, select for hepatocellular tumors which contain activated β-catenin. In normal hepatocytes, interactions of β-catenin and CAR have been demonstrated with respect to the induction of proliferation and drug metabolism-related gene expression. The molecular details of these interactions are still not well understood. Recently it has been hypothesized that CAR might activate β-catenin signaling, thus providing a possible explanation for some of the observed phenomena. Nonetheless, many aspects of the molecular interplay of the two regulators have still not been elucidated. This review briefly summarizes our current knowledge about the interplay of CAR and β-catenin. By taking into account data and observations obtained with different mouse models and employing different experimental approaches, it is shown that published data also contain substantial evidence that xenobiotic activators of CAR do not activate, or do even inhibit signaling through the β-catenin pathway. The review highlights new aspects of possible ways of interaction between the two signaling cascades and will help to stimulate scientific discussion about the crosstalk of β-catenin signaling and the nuclear receptor CAR. Ctnnb1 gene  CAR activation in rodents selects for tumors with activated β-catenin, (2) both factors, CAR and β-catenin, are essential for this process, (3) CAR and β-catenin cooperate in the induction of proliferation and drug metabolism-related gene expression in non-transformed hepatocytes, thus suggesting synergistic effects of the activation of both pathways.MYC is observed after CAR as well as after β-catenin activation (see above). Several drug-metabolizing enzymes are also under the control of both, CAR and β-catenin, as e.g. documented for some glutathione S-tranferase and cytochrome P450 enzymes , a model target of β-catenin in the liver and physiologically expressed in a thin layer of hepatocytes around the central veins  , CAR, the pregnane-X-receptor (PXR), hepatocyte nuclear factor (HNF) 4α and the peroxisome proliferator-activated receptor (PPAR) α in livers of mice exposed to phenobarbital has been described , another nuclear receptor with known relevance in the regulation of drug and energy metabolism. In a transcriptome-wide analysis of phenobarbital effects in In vitro, phenobarbital has been shown to inhibit β-catenin-dependent signaling in mouse hepatoma cells (Groll et al. In summary, the exact molecular details how CAR allows hepatocytes with activated β-catenin to proliferate, remains to be elucidated. The studies published so far contain data that may favor a stimulatory role of CAR towards β-catenin as suggested by Shizu and Yoshinari , while a


The present study reveals a link between protein arginine methyltransferase 5 (PRMT5) and Homebox A9 (HoxA9) in the regulation of cardiomyocyte hypertrophy. In cardiomyocyte hypertrophy induced by β-adrenergic receptor agonist isoprenaline (ISO), PRMT5 expression was decreased while HoxA9 was upregulated. Silencing of PRMT5 or inhibition of PRMT5 by its pharmacological inhibitor EPZ augmented the expressions of cardiomyocyte hypertrophic genes brain natriuretic peptide (BNP) and β-Myosin Heavy Chain (β-MHC), whereas overexpression of PRMT5 inhibited ISO-induced cardiomyocyte hypertrophy, suggesting that PRMT5 ameliorates cardiomyocyte hypertrophy. On the contrary, HoxA9 promoted cardiomyocyte hypertrophy, as implied by the gain-of-function and loss-of-function experiments. HoxA9 was involved in the regulation of PRMT5 in cardiomyocyte hypertrophy, since HoxA9 knockdown prevented si-RPMT5-induced cardiomyocyte hypertrophy, and HoxA9 expression impaired the anti-hypertrophic effect of PRMT5. Co-immunoprecipitation experiments revealed that there were physical interactions between PRMT5 and HoxA9. The symmetric dimethylation level of HoxA9 was decreased by ISO or EPZ treatment, suggesting that HoxA9 is methylated by PRMT5. Additionally, PRMT5 repressed the expression of HoxA9. Chromatin immunoprecipitation (ChIP) assay demonstrated that HoxA9 could bind to the promoter of BNP, and that this binding affinity was further enhanced by ISO or EPZ. In conclusion, this study suggests that PRMT5 symmetric dimethylates HoxA9 and represses HoxA9 expression, thus impairing its binding to BNP promoter and ultimately protecting against cardiomyocyte hypertrophy. These findings provide a novel insight of the mechanism underlying the cardiac protective effect of PRMT5, and suggest potential therapeutic strategies of PRMT5 activation or HoxA9 inhibition in treatment of cardiac hypertrophy. During cardiovascular diseases such as hypertension and atherosclerosis, cardiomyocytes are exposed to continuous mechanical stress and neurohumoral stimulation, resulting to reactivation of fetal genes such as atrial natriuretic factor (ANF), brain natriuretic peptide (BNP) and β-Myosin Heavy Chain (β-MHC), protein synthesis, sarcomere assembly and ultimately enhancement of cell surface area . This prPRMT5, referring to protein arginine methyltransferase 5, belongs to the histone methyltransferase PRMTs family that specifically transfers methyl groups from the methyl donor S-adenosine methionine (SAM) to the nitrogen atoms on the guanidium of arginine residues . PRMT5 iThe present study revealed that PRMT5 was able to regulate Homebox (HOX) A9 in cardiac hypertrophy. HOX genes encode a series of highly conservative evolutionary transcription factors harboring a DNA-binding motif of 60 amino acids . All of Our preliminary studies showed that there were interactions between PRMT5 and HoxA9 in pathological cardiac hypertrophy and that HoxA9 was involved in the regulation of PRMT5 in cardiomyocyte hypertrophy. Thus, the present study was designed to investigate the underlying mechanisms.2 culture flasks for 1 h with 5% CO2 to separate cardiac fibroblasts and cardiomyocytes. Finally, cardiomyocytes were re-collected and seeded in culture dishes with 60–70% of confluent. To prevent potential contamination and fibroblasts proliferation, 1% penicillin-streptomycin and 0.1 mM 5-bromodeoxyuridine (5-BrdU) were necessary to added into the culture medium. 24 h later, culture medium was replaced by fresh one, and cells could be implied for subsequent experiments after another 12 h.Neonatal rat cardiac myocytes (NRCMs) were collected from hearts of Sprague-Dawley (SD) rats one to three days after birth according to previous protocol . Hearts Animal procedures used in this study were in line with institutional guidelines for the Care and Use of Laboratory Animals  and were approved by Laboratory Animal Center of the Sun Yat-sen University. 12 Sprague-Dawley (SD) male rats of 200–220 g in SPF grade were obtained from the Experimental Animal Center of Sun Yat-sen University . Briefly, SD rats were randomly allocated into two groups: the control (NS) group and isoprenaline (ISO) group. Rats in ISO group (n = 6) were intraperitoneally administrated with ISO , while rats in NS group received the same volume of saline. After 7-days-treatment, all rats were performed echocardiography by Vevo 2,100 ultrasound system , in order to evaluate the left ventricular (LV) function. Subsequently, rats were anesthetized and sacrificed by intraperitoneally injecting 7% chloral hydrate. Hearts were immediately removed in diastolic state and trimmed transversely and separated in two parts. One part was fixed with 4% paraformaldehyde and embedded in paraffin for hematoxylin-eosin staining (HE staining), the other part was utilized for Western blotting analysis or Real-time qPCR assay.The following primary antibodies were utilized in this study: Anti-HoxA9 (sc-81291), anti-αTubulin (sc-398103), anti-PRMT5 (sc-376937) and Anti-β-MHC (sc-53089) mouse monoclonal antibodies were purchased from Santa Cruz Biotechnology Inc. . Anti-BNP rabbit polyclonal antibody (PAA541Ra01) was purchased by Cloud-Clone Corp . Anti-mouse Anti-mouse IgG, HRP-linked secondary antibody (#7076P2), and Anti-mouse Anti-rabbit IgG, HRP-linked secondary antibody (#7074P2) were purchased from Cell Signaling Technology, Inc. .ISO was acquired from TargetMol . Rhodamine phalloidin and 4′6-diamidino-2-phenylindole (DAPI) were purchased from YeasenBiotechCo. Ltd. . EPZ015666 was bought from MedChem Express .Total protein of NRCMs or heart tissues was extracted by RIPA lysis buffer . The concentration of protein samples were determined by BCA Protein Assay Kit . Subsequently, equal amount of proteins from different samples, together with 5xSDS loading buffer  were loaded to 8∼12% sodium dodecyl sulfate-polyacrylamide gels and were subjected to electrophoresis (SDS-PAGE) under the condition of 70 V for 30 min and 110 V for 70 min. Then, proteins were transferred from gels to methanol-presoaked PVDF (polyvinylidene fluoride) membranes  via under the constant current of 230 mA for 100 min. After that, membranes were blocked with 5% non-fat milk for 1 h and washed by TBST buffer  for three times, 5 min each time. Membranes were incubated with primary antibodies at 4 °C overnight. After that, membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies  for another 1 h at ambient temperature. Blots were developed with enhanced chemiluminescence reagent  and detected by the LAS4000 imager . The intensities of the blots were quantified by the Quantity One (Bio-Rad) software. α-Tubulin was used as an internal control for total proteins.∆∆CT method by using β-actin as internal control. Results were presented as fold change to control group. Primers of target genes were shown in Total RNA from NRCMs or heart tissues were extracted by Trizol Reagent , according to manufacturer’s instructions. The concentration and purity of the RNA were measured by NanoDrop 2000 spectrophotometer from Thermo Fisher Scientific . 2 μg of total RNA was reversed and transcripted into first strand cDNA by using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) in RT-PCR system (Thermo Fisher Scientific). The mRNA expression levels were detected by loading cDNA, primer, buffer and THUNDERBIRD™ SYBR qPCR Mix  in the proportion of 1:1:3:5 into one well of 96-well white plate and conducted in triplicate in PikoReal Real-Time PCR System (Thermo Fisher Scientific) for 40-cycle-amplilfication. Cycle values that reached threshold were recorded. mRNA expression was calculated by −2Experiments were conducted according to protocols . NRCMs oTotally 500 μg protein was used for immunoprecipitation, while additional 10 μg protein was used as input. In general, protein lysate was separately incubated with anti-PRMT5 antibody , anti-HoxA9 antibody  or anti-mouse IgG antibody  overnight at 4 °C. Then 25 μl of protein A/G beads  was added to protein lysate and turned upside down at 4 °C. 4 h later, beads were washed, collected and boiled with 30 μl 2XSDS loading buffer, followed by SDS-PAGE immuno-blotting analysis.NRCMs cultured in 24-well plates were fixed with paraformaldehyde diluted in PBS buffer  for 15 min at ambient temperature, followed by 0.3% Triton-100 treatment for 10 min. Then NRCMs were washed by PBS buffer. After that, NRCMs were incubated with 0.1% (v/v) rhodamine-phalloidin in dark for 1 h, and were further stained with DAPI before rinsing NRCMs for another three times with PBS buffer. Images of the NRCMs were detected via High Content Screening system . The cell surface area from randomly selected fields (3 for each group) was determined using ImageJ analysis software. Data was presented as fold change to control group.Small interference RNAs (si-RNA) specific to PRMT5 (si-PRMT5) and HoxA9 (si-HoxA9), along with non-specific si-RNA (si-NC), were purchased from Guangzhou RiboBio Co., Ltd . Target sequences were shown in Wild type PRMT5 and mutant PRMT5 without methylation activity  were both cloned into pcDNA3.1(+) vector by Vigenebio . In general, 2 μg plasmid was transfected into cardiomyocytes in the same way of si-RNA transfection.Immunoprecipitated DNA was obtained by using SimpleChIP® Plus Enzymatic Chromatin IP Kit (#9005) from Cell Signaling Technology, Inc. Briefly, cells cultured in 100 mm plates with 80% of confluent were cross-linked with 1% of formaldehyde for 10 min, then cell pellets were sonicated and digested by 3 μl of micrococcal nuclease at 37 °C for 20 min to digest DNA to length of 150–900 bp. After that, cell pellets were immunoprecipitated with antibodies against PRMT5 or HoxA9, together with immunoglobulin G (IgG) antibody as negative control  overnight. Subsequently, each immunoprecipitation sample was incubated with ChIP Grade Protein G Magnetic Beads contained in SimpleChIP® Plus Enzymatic Chromatin IP Kit for 2 h at 4 °C with rotation. DNA sample were eluted from Magnetic beads by using magnetic separation rack and proteinase K. DNA fragments were purified by using spin columns of the kit. Finally, purified DNA samples were applied for subsequent PCR assay to investigate the level of PRMT5 or HoxA9 binding to target DNA regions.Bonferroni post hoc test for multiple comparisons using GraphPad Prism Software Version 5.01 . p < 0.05 was considered to be statistically significant.Data were presented as means ± SEM, and analyzed by two-tailed unpaired Student’s t-test between two groups and by one-way ANOVA followed by the in vivo cardiac hypertrophic model was successfully established. In this model, the protein expression of HoxA9 was found to be elevated, whereas PRMT5 was downregulated  HoxA9 deficiency reversed cardiomyocyte hypertrophy induced by PRMT5 silencing or inhibition; 2) the binding of HoxA9 to BNP promoter was enhanced by PRMT5 inhibitor EPZ.This study further investigates the mechanisms by which PRMT5 regulates HoxA9 in cardiomyocyte hypertrophy. Taken into account that PRMT5 is a symmetric dimethylase, and that PRMT5 interacted with HoxA9, it is most likely that PRMT5 directly symmetric dimethylates HoxA9. Indeed, the symmetric dimethylation level of HoxA9 was abrogated when PRMT5 was inhibited by EPZ or suppressed by ISO. These results are consistent with a previous study taken in vascular endothelial cells , supportIntriguingly, PRMT5 affects the expression of HoxA9 at the transcriptional level. This is based on the observations that si-PRMT5 augmented HoxA9 expression while PRMT5 overexpression decreased HoxA9 protein level, and that knockdown or inhibition of PRMT5 increased the mRNA level of HoxA9. These results imply that PRMT5 suppresses HoxA9 through histone methylation as an epigenetic modification mechanism, since PRMT5 is a vital histone methyltransferase catalyzing symmetric dimethylation in histones H4R3 and H3R8 . By methIn conclusions, PRMT5 exerts potent anti-hypertrophic effect by symmetric dimethylating HoxA9 and repressing HoxA9 expression. These findings suggest that activation of PRMT5 or inhibition of HoxA9 might serve as potential therapeutic strategies in treatment of cardiac hypertrophy.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Institutional Animal Care and Use Committee (IACUC), Sun Yat-Sen University.SC and RL designed and performed research, analyzed data and wrote the paper; PW and JL contributed to new reagents, analytic tools and experimental protocols; TX contributed to data collections. MW and YC contributed to cell culture and preparations for sample inspection. PL and ZL helped provide constructive informations to the framework of this manuscript and contributed to refine the main idea of this article.This work was supported by grants from the National Natural Science Foundation of China , Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01Y093), National Major Special Projects for the Creation and Manufacture of New Drugs (2019ZX09301104), Special Program for Applied Science and Technology of Guangdong Province (2015B020232009), National Engineering and Technology Research Center for New drug Druggability Evaluation , Guangzhou Science and Technology Program Project , Yang Fan Project of Guangdong Province (Grant no. 2014 YT02S044), and Guangdong Provincial Key Laboratory of Construction Foundation (No. 2017B030314030).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Transcriptional regulation of growth, maturation, and cell turnover in adrenal cortex during postnatal development has been significantly less studied than in embryonic period, while elucidation of factors mediating its normal postnatal morphogenesis could clarify mechanisms of tumorigenesis in adrenal cortex. Expression of transcription factors Hex, β-catenin, and Wnt signaling in the adrenal cortex of male pubertal and postpubertal Wistar rats were examined. Adrenal cortex morphology and hormone production during postnatal development were also studied. Adrenocortical zones demonstrated similar reduction of Ki-67-expressing cells, but different patterns of morphological and functional changes. Age-dependent decrease in percentage of cells with membrane localization of β-catenin and stable rate of cells with nuclear β-catenin, indicative of Wnt signaling activation, were revealed in each cortical zone. Nuclear β-catenin was not observed in immature areas of zona fasciculata. No association between Wnt signaling activation and rates of proliferation as well as changes in secretion of adrenocortical hormones was observed in postnatal development of rat adrenal cortex. Hex, known as antiproliferative factor, showed up-regulation of expression after puberty. Strong inverse correlations between ratio of Hex-positive cells and proliferating cells were found in zona glomerulosa and zona fasciculata. Zona reticularis demonstrated moderate correlation. Thus, these findings suggest a role for Hex in proliferation control during postnatal development of the rat adrenal cortex and possible implication of Hex down-regulation in adrenocortical dysplasia and neoplasia, which requires further study. Evaluation of Hex expression may also be considered a potent tool in assessment of cell proliferation in rat adrenal cortex. Adrenal cortex, postnatal development, transcription factors, Hex, β-catenin, Wnt signaling, proliferation, hormones, morphology. Recent data suggest that embryonic transcription factors can have various effects on tissue and organ development before and after birth . Lack ofEstablishment of functional zonation and regulation of growth is actively studied for last decades, but most research focuses on fetal and perinatal periods ,6,7. TheSince activation of canonical Wnt/β-catenin signaling is associated with acceleration of proliferation and even tumorigenesis in the adrenal cortex ,19,20,2122.1nd and 70th day of age at 9–10.00 AM. 42nd day of postnatal development (PND42) is pubertal age after adrenarche and prior to gonadarche, when adrenals actively grow, and the 70th day postnatal development (PND70) is a time of maximal development of adrenal cortex and termination of growth [Male Wistar rats (n = 40) were obtained from Scientific Center of Biomedical Technologies of Federal Medical and Biological Agency of Russia. The animals were housed at +22–23 °C with a 12/12-hr light-dark cycle and given a pelleted standard chow ad libitum. The rats were sacrificed by zoletil overdosage at 42f growth . The blo2.22, and diameter of capillaries were measured.The adrenal glands were fixed in Bouen solution. After standard histological processing the tissue samples were embedded in paraffin. Equatorial sections of the adrenals were stained with hematoxylin and eosin. Histological examination included light microscopy and computer morphometry of ZG, ZF, and ZR, performed with “Leica DM2500” light microscope and “ImageScope” software . Surface area, size of adrenocortical cells, number of the cells in 1μm2.3Immunohistochemical evaluation of β-catenin, Hex, and Ki-67 was performed on paraffin-embedded tissues. After antigen retrieval by boiling with 10mM sodium citrate (pH 6.0) endogenous peroxidase was blocked with Hydrogen Peroxide Block and then endogenous immunoglobulins was blocked Protein Block  according to manufacturer's protocol. The slides were incubated with primary antibodies to β-catenin , Hex , and Ki-67  overnight at 8 °C. The reaction was visualized with anti-mouse/antirabbit “UltraVision LP Detection System” reagent kit  according to manufacturer's recommendations. The sections were counterstained with Mayer's hematoxylin. After dehydration the sections were mounted in polymer media. Sections processed without primary antibodies were used as negative control for secondary antibodies. No nonspecific reactions were registered.2.42 of each zone.Immunohistochemical reactions were evaluated in ZG, ZF, and ZR. Hex expression was assessed as a percent of cells with immunopositive (brown and dark brown) nuclei. The number of cells with the membrane, cytoplasmic, and nuclear localization of β-catenin was calculated and expressed as the percent of the total number of cell in each zone of the adrenal cortex. Cells with immunopositive membranes and negative cytoplasm and nucleus were considered as cells with membrane localization of β-catenin. Cells with immunopositive cytoplasm and immunonegative nuclei were referred to cells with cytoplasmic localization. Cells with immunopositive nuclei with presence/absence of β-catenin in membranes and cytoplasm were considered as cells with nuclear localization. Translocation of β-catenin into nucleus was considered as a marker of activation of canonical Wnt signaling ,35. Rate2.5Collected blood samples were incubated at room temperature for 30 min. Clotted blood was centrifuged at 2700 rounds per minute for 15 min. Serum was transferred into polypropylene tubes. Mineralocorticoid hormone aldosterone, glucocorticoid hormone corticosterone, and adrenal sex steroids androstendione and estrone were measured for assessment of ZG, ZF, and ZR function, subsequently. Hormone concentrations in serum were determined by enzyme-linked immunosorbent assay according to manufacturer's protocols  with “Anthos 2010” microplate reader at 450 nm.2.62. Associations of the total number of Ki-67- and Hex-positive and Ki-67- and nuclear β-catenin-positive cells in each term of the investigation were analyzed using Pearson correlations test. Differences were considered statistically significant at p < 0.05.The statistical analyses were carried out using the software package Statistica 7.0 . The central tendency and dispersion of quantitative traits with approximately normal distribution were presented as the mean and standard error of the mean (М±m). Quantitative comparisons of independent groups were performed using Student's t test, taking into account the values of Levene's test for the equality of variances, and χ33.1nd-days old rats exhibited well developed adrenal cortex with three distinguishable concentric layers: ZG, the outermost layer, ZF, and ZR, the innermost layer. Some areas in outer ZF, representing immature developing sites, had not typical bundle structure. Interrupted zona intermedia was observed between ZG and ZF. Intermedia cells demonstrated smaller size and pleomorphic nuclei. Histological examination on the 70th day of postnatal development revealed some changes in structure of the cortex. Diminished area of ZG and enlarged ZF were observed .This work was supported by the The authors do not have permission to share data.The authors declare no conflict of interest.No additional information is available for this paper.


The growing demand for patient-compliance therapies in recent years has led to the development of transdermal drug delivery, which possesses several advantages compared with conventional methods. Delivering protein through the skin by transdermal patches is extremely difficult due to the presence of the stratum corneum which restricts the application to lipophilic drugs with relatively low molecular weight. To overcome these limitations, microneedle (MN) patches, consisting of micro/miniature-sized needles, are a promising tool to perforate the stratum corneum and to release drugs and proteins into the dermis following a non-invasive route. This review investigates the fabrication methods, protein delivery, and translational considerations for the industrial scaling-up of polymeric MNs for dermal protein delivery. Proteins, as drugs, play key roles in the treatment of several diseases, such as inflammation or neurodegenerative disorders, cancer, vaccinations, genetic diseases, and so on. They possess several functions in the body, e.g., as enzymes, immunostimulators, cellular regulators, or molecular transporters . CurrentTo overcome these limitations, micrometric length microneedles (MNs) have emerged. They are capable of penetrating the epidermis layer at a fixed depth, avoiding the stimulation of the nerve endings; in this way, the macromolecules can be rapidly absorbed in the capillaries and lymphatic networks without producing pain for the patients . They alIn addition, MNs are able to encapsulate proteins with high efficiency and store them in a bioactive state without requesting further expedients (cold-chain), hence minimizing the cost of transportation . MoreoveDespite these restrictions, the progress in MN fabrication and protein encapsulation make microneedles a promising platform for the dermal delivery of drugs. In the following chapters, fabrication methods, protein delivery, and translational considerations for the industrial scaling-up of polymeric MNs are discussed.Micromolding is the most commonly used methodology for the fabrication of microneedles. It involves the fabrication of a master with the desired microstructures from which molds can be obtained. Then, after mold fabrication, microneedles can be replicated on it. MN masters are usually produced by techniques such as photolithography using deep X-ray lithography of Lithographie Galvanoformung Abformung ,16 and uMicromolding includes hot embossing, injection, and solvent casting a. In hotA polymer MN fabrication technique based on a drawing lithography process  has been3); under an appropriate thermal stimulus applied to the crystal, an electric field is generated, which drives the microneedle drawing process. During the shaping process, the liquid cone solidifies following the fast solvent evaporation, in this way fixing the microneedle’s desired shape. The technology is free from a high process temperature and UV radiation, with the biopolymers processed in solution form at temperatures between 20 and 40 °C. Recently, this process has been improved in terms of shape by removing the presence of a pedestal below the microcones and in terms of parallelization which now needs to be automated in order to provide an industrial scalable process ,4039,40] process .Among mold-free technologies, additive manufacturing, or 3D printing, has been shown to be an emerging field in MN structure fabrication. In fact, employing Computer Aided Design (CAD) software allows the design of MNs with costumed density, length, and geometry, which can be printed with high reproducibility through the consecutive deposition of layers . AdditivAdditive manufacturing is often combined with other technologies. For example, fused deposition modeling (FDM) was recently introduced to fabricate biodegradable polymer MNs f and wasAdditive manufacturing has been shown to be a promising and challenging technology to fabricate MNs and MN integrated devices. However, some limitations need to be overcome. First of all, there is a low availability of 3D printable biomaterials (with specific properties and viscosities at certain temperatures or photo-sensitivities) which are biocompatible and which have the appropriate biodegradation rate in the case of drug-containing materials. Additionally, the material needs to have good mechanical performance to allow MNs to pierce the skin. Lastly, the nature of the protein has to be considered: proteins can degrade when exposed to high temperatures or UV radiation.2O3), calcium sulfate dehydrate, calcium phosphate dehydrate, and Ormocer can be used to manufacture MNs [alt-maleic anhydride) (Gantrez AN-139) or poly (Gantrez S-97®) have also been employed [Silicon, metal, silica glass, ceramics, and polymers have been employed to prepare MNs. The first study regarding silicon MNs to enhance drug delivery through the skin was published in 1998 . The useture MNs ,53. Carbture MNs ,55. Theyture MNs ,57,58,59ture MNs , polymetture MNs , poly and shear strength tests are essential to provide a comprehensive profile of microneedle behavior during their applications, because skin surface irregularity often causes the imperfect penetration of MN arrays, causing MN transverse bending [The  bending . In this bending ,70. A li bending . On the other hand, the main challenge in using MNs is to transport biomolecules into the skin in an efficient and reproducible way. A lot of improved techniques in combination with MNs have been investigated to enhance drug delivery after MN insertion. For instance, Wu et al.  studied MNs can be classified into solid MNs and hollow, coated, dissolvable, degradable, and swellable systems. Recently, some responsive polymers were embedded into MNs in order to achieve an on-demand release . Solid MFor example, prolonged release can be achieved by encapsulating the drug in polymeric microparticles/nanoparticles and hydrogel-forming structures, while burst release can be performed using dissolvable matrices. Furthermore, MNs, as intelligent carriers, are responsive to internal and external stimuli and have been designed to have a smart drug delivery system. Hollow MNs emerged to inject liquids  and suspIn a recent study, four types of nanoparticles (NPs) including PLGA, liposomes, mesoporous silica, and gelatin NPs delivered by hollow MNs were used to encapsulate OVA and its adjuvant in order to evaluate the different physiochemical properties of the NPs. PLGA NPs, particularly cationic liposomes, induced the highest immune responses, arising from the strong interaction between the antigen/adjuvant and the nanoparticle matrix. Gelatin and mesoporous silica have faster release, which may be due to the weak electrostatic interaction between the antigen/adjuvant on the surface of NPs. Antigen co-encapsulated with adjuvant induced antibody responses in a more effective way compared with the free OVA/adjuvant. This demonstrated that hollow MNs accompanied by the applicator are a promising tool for intradermal vaccination . Using tIn this kind of patch, drugs are adhered directly onto the surface of solid or polymeric MNs. Since the coating layer reduces the mechanical strength and sharpness of the needles, the drug loading on the needle surface is limited to a low amount. As a consequence, coated MNs are only usable for some specific applications in which a low dose is needed ,81. HumaDissolving MNs were created to encapsulate drugs within a water-soluble polymer matrix, and they become completely dissolved once the MNs are inserted into the skin, with a dissolving time from minutes to hours ,104. SulAdditionally, for influenza vaccination, materials such as trehalose  and carbIn the direction of burst release, HA-MNs were loaded with an amyloid-β 42-amino acid peptide antigen to develop a vaccine for Alzheimer’s disease. The patients showed an efficient immune response after MN application . The inc1/O/W2 emulsion method and successfully introduced into the microneedle patches [In order to achieve sustained drug release, MNs with biodegradable polymer particles were designed. The drug payload is gradually released by simple diffusion and hydrolysis of the polymer . PLGA is patches . The res patches . In thisMuch effort has been made to solve the deactivation of protein structures during the fabrication steps. In this context, ovalbumin (OVA), as an antigen model, was loaded in PLGA microparticles by the self-healing method. This method guarantees the stability of the antigen structure since microparticles are incubated with the antigen solution, preventing the antigen from exposure to the mechanical stresses present during the preparation process . Long teIn the context of the prolonged release of the vaccine, MNs composed of a silk tip supported on a poly(acrylic acid) PAA base have been designed. PAA bases dissolve quickly to deliver the antigen while also implanting silk hydrogel depots for sustained cutaneous release over 1–2 weeks. Microneedle arrays containing OVA loaded in the silk needle tips or OVA in the PAA pedestals allow for bolus and sustained release . The useSwellable MNs, also known as cross-linked hydrogels, are solid systems that swell after the uptake of interstitial fluid (IF), after which they deliver the payload, and are removed intact from the skin ,120. TheSome limitations regarding swellable MNs are related to the need to keep the patch on the skin for all release timeframes. Additionally, particular attention has to be given to the choice of cross-linkage reaction, which needs to be biocompatible. Bio-responsive materials have the outstanding characteristic of sensing physiological or pathological signals and regulating on-demand release based on the presence and intensity of a specific stimulus . One of Applying another strategy, Juiang et. al. created polymeric MNs loaded with insulin containing glucose-responsive mesoporous bioactive glass nanoparticles (BGNs). The silica NPs were coated by a pH-responsive material and glucose oxidase (GOx), which made them link BGLs as the pH changed, consequently giving the ability of glucose level sensing. When the pH drops because of the high concentration of glucose, this system can sense pH alteration through the pH-responsive material and lead to insulin release . In anotGlucose control can also be achieved by live (cell-based) and synthetic glucose-responsive systems, for instance, glucose-responsive MNs based on cross-linked hyaluronic acid with β-cells encapsulated in microgels on the tip of MNs . AmplifiBio-responsive MNs are the state-of-the-art MNs, but again, their complexity needs to be justified. Additionally, they need to be kept on the skin for the release timeframe, but this is not a limitation in applications, such as insulin delivery, where the use of a reservoir is needed anyway to guarantee the amount of the molecule being delivered. Mechanical strength and indentation ability are strongly dependent on the amount of water residual in the polymeric MNs. For this reason, here, we take a brief look at the characterization of MNs. For instance, the addition of sucrose to the dissolvable MNs can change the mechanical properties. Also, the addition of sucrose results in the faster release of sucrose/dissolvable MN formulations compared with MN formulations without sucrose . Unlike An encapsulated biomolecule does not have the same stability and sensitivity to temperature, humidity, and shelf life in solvent as a free biomolecule. For example, proteins, antibodies, and viruses are very susceptible to degradation in comparison to organic molecules; in particular, proteins are vulnerable if they are exposed to some organic solvents, such as dichloromethane, ethyl acetate, and dimethylcarbonate (DMC), which are used during PLGA microparticle production by the double emulsion technique. Since the liquid and commercially-available lyophilized drugs lose their activity after preservation at room temperature, the thermostability of MNs is great of interest. The common fabrication methods employed for protein-loaded MNs involve the use of high temperatures, a vacuum, or exposure to an ultraviolet source, which may be harmful to protein activity . AnotherRecently, Yang et al.  developeCandida albicans and Pseudomonas aeruginosa pathogens through cell membranes is lower in comparison with the use of a 21G hypodermic needle [Staphylococcus aureus. Sterilization, packaging, and storage should be taken into account for scaling up MNs before being commercialized. In contrast to conventional transdermal delivery platforms, MNs are not applied to the skin surface; indeed, MNs pierce the outermost protective skin layer, penetrating into the epidermis and dermis, which are normally sterile parts of the body . Therefoc needle . Togethec needle  reportedHowever, the best solution is to always subject the MNs to a complete sterilization process. One of the possibilities can be to use gamma irradiation after the complete fabrication of MNs. Several studies about hydrogel MN arrays showed that no measurable bioburden was detected in any gamma-sterilized devices, and endotoxin levels were below the Food and Drug Administration (FDA) limits (20 endotoxin units/device). Moreover, some types of MNs, e.g., hydrogel-forming MNs, are not affected by gamma irradiation (25 KiloGray (kGy)) in terms of their physical properties and drug bioactivity . AnotherHowever, the sterilization methods should be carefully chosen to avoid changing the product and increasing the manufacturing costs. For instance, aseptic manufacturing could be expensive, and heat or microwave heating could damage the MNs or their cargo. Alternatively, GMP productions would be needed to skip sterilization. Regarding storage, since MNs are highly sensitive to temperature and mechanical stress, it is necessary to keep the patches in a humidity-free state, e.g., in a desiccator. However, stability studies of long-term patches are needed. In this regard, Hiraishi et al.  reportedThe clinical development of proteins is faster than that of small-molecules or peptides , althougUnfortunately, they also have limitations, for example, MNs are still missing many requirements  to be defined as “smart devices” for biomedical applications. Moreover, great consideration should be given to reduce the skin allergies, redness, and irritation associated with MN treatments. Despite these restrictions, the growth in MN fabrication and biomolecule encapsulation processes make them a favorable platform for the dermal delivery of drugs.Microneedles can be considered emerging devices in the field of drug delivery compared with current techniques. In fact, MNs have achieved excellent results, especially in the cosmetic field, undergoing rapid growth in the last decade. For example, HA-MN patches are currently sold around the world for cosmetic purposes by the Raphas company . Moreove


Recent investigations have also suggested the regulatory effects of the Wnt/β-catenin signaling pathway in response to chemotherapeutic agents in CRC. Here, we particularly focus on reviewing the evidences suggesting the mechanisms of Wnt/β-catenin signaling in the chemoresistance modulation of colorectal cancer.Colorectal cancer (CRC) is a common malignancy with high morbidity and mortality worldwide. To date, chemotherapy plays an important role in the treatment of CRC patients. Multidrug resistance (MDR) is one of the major hurdles in chemotherapy for CRC, and the underlying mechanisms need to be explored. Studies have demonstrated that Wnt/ Colorectal cancer (CRC), one of the most prevalent malignancies, ranked third in cancer incidence in both genders and remains the second cause of cancer-related deaths in the world . CurrentMultidrug resistance is a major impediment to successful treatment of CRC, and overcoming MDR becomes a great challenge in fighting against CRC . Chemothβ-catenin signaling cascade [β-catenin signaling pathway, essential for maintaining cell homeostasis and embryonic development, was demonstrated to be associated with tumor cell proliferation, apoptosis, invasion, stemness, and chemotherapy resistance [β-catenin signaling pathway as a key pathway involved in various processes of CRC [β-catenin is associated with poor prognosis of CRC patients, and studies were performed to explore the roles and mechanisms of Wnt/β-catenin signaling in regulating cell apoptosis, stimulating angiogenesis, and maintaining highly resistant CSCs [The most common genetic changes accompanying CRC progression are mutations that deregulate the Wnt/ cascade . Wnt/β-csistance   transcription factors, which control a cluster of target genes including MDR1, c-myc, Met, MMP-7, c-Jun, and cyclin D1. In this pathway, the cysteine-rich and lipid-modified Wnt proteins could induce the activation of canonical and noncanonical Wnt signaling pathways after binding to Frizzled (Frz) and low-density lipoprotein receptor-related 5/6 (LRP5/6). In physiological conditions lacking the Wnt signal, β-catenin is degraded by the complex composed of glycogen synthase kinase 3β (GSK3β), Axin, adenomatous polyposis coli (APC), and casein kinase 1 (CK1). As a key scaffolding protein of the destruction complex, Axin becomes degraded and stops facilitating the destruction of β-catenin after ribosylation by Poly (ADP ribose) polymerases (PARPs). Followed by proteasomal degradation and ubiquitination, β-catenin is phosphorylated by GSK3β and CK1. Downregulated cytoplasmic β-catenin recruits the corepressor Groucho to TCF/LEF, ensuring transcriptional repression and blocking the target genes from being activated. Given the tight association between Wnt/β-catenin signaling and cancer apoptosis, EMT, stemness, and tumor microenvironment, the Wnt/β-catenin signaling pathway is thought to be associated with cancer chemoresistance. We then mainly discuss the underlying mechanisms by which the Wnt/β-catenin signaling cascade influences the drug resistance of colorectal cancer in the following sections.Wnt/c target . β-Catenβ-catenin signaling cascade, nuclear β-catenin could preferentially recruit CBP to the promoter region of MDR1 gene, and MDR1 has been proven to be one of the target genes of TCF/LEF [β-catenin transcriptional complex [β-catenin by RNA interference could significantly reduce the transcription and expression of the MDR1 gene, resulting in a reversal of its encoded P-gp efflux and restoration of sensitivity to drug-induced apoptosis. Zhou et al. [β-catenin signaling pathway by suppressing MDR1/P-gp expression. Moreover, RARg overexpression was shown to contribute to the multidrug resistance of CRC cells by upregulating MDR1 through activating the Wnt/β-catenin signaling pathway [β-catenin signaling pathway. The above studies suggested that the Wnt/β-catenin signaling cascade contributed to enhanced resistance of various chemotherapeutic agents in CRC through upregulating MDR1.One of the most typical mechanisms of MDR is that ABC membrane transporters pump various chemotherapeutic agents out of cells to reduce intracellular drug accumulation and attenuate drug-induced cytotoxicity , 12. Mos TCF/LEF , 17. The complex . Depletiu et al.  showed t pathway . Wang et pathway  also demβ-catenin signaling cascade is a prosurvival signaling pathway that has an intimate crosstalk with other prosurvival signaling pathways such as signal transducer and activator of transcription (STAT), mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3K)/Akt signaling pathways.Apoptosis, one of the major signs of effective chemotherapy, is characterized by DNA fragmentation, condensation of the nucleus, and specific protein degradation. Apoptosis resistance, a hallmark of tumors, acts as a crucial obstacle to anticancer therapy. As a critical mode of cell death induced by chemotherapy, apoptosis participates in chemotherapy resistance and plays a crucial role in regulating tissue homeostasis. Tolerance to DNA damage could be enhanced in chemoresistant cells through elevating the toxicity threshold by upregulating prosurvival signaling and downregulating apoptotic signaling pathways. The Wnt/β-catenin signaling pathway has been increasingly established to be associated with apoptosis. Previous evidence showed that inhibition of Wnt/β-catenin signaling by the inhibitor XAV939 could significantly increase the apoptosis induced by 5-FU in colon cancer cells [β-catenin signaling pathway, β-catenin could promote the expression of its target gene survivin, which inhibits apoptosis in colon cancer [β-catenin signaling pathway, including proteins Wnt, GSK3β, and APC, also participate in the process of apoptosis in CRC. As a member of Wnt proteins initiating the canonical Wnt signaling pathway, Wnt1 inhibits apoptosis of colorectal cancer cells through blocking the caspase-9 activation induced by chemotherapeutic drugs, and this sensitivity to apoptotic stimuli could be blocked by inhibiting β-catenin/TCF transcription [β could constitutively phosphorylate β-catenin and serve as a negative regulator of the Wnt/β-catenin signaling pathway [β could increase the apoptosis of CRC cells. Adenomatous polyposis coli (APC), another crucial component of the canonical Wnt signaling cascade, could downregulate β-catenin. As a tumor suppressor protein, APC could induce cell death of CRC through apoptosis in CRC [β-catenin signaling cascade have been reported to modulate drug resistance through regulating apoptosis. For instance, MMP-7 could increase the oxaliplatin resistance of colon cancer cells by decreasing the Fas receptor that promotes cell apoptosis [β-catenin signaling. Li et al. [β-catenin signaling pathway. Similar results were also observed in Chinese medicine including luteolin and Sanguisorba officinalis which could induce apoptosis by enhancing the Bax and caspase-3 expression and suppressing Bcl-2 expression through Wnt/β-catenin signaling in CRC cell lines [β-catenin signaling pathway [β-catenin signaling in apoptosis give it a status in the chemoresistance of CRC, and the underlying mechanisms need further exploration.Wnt/er cells . As the n cancer . Other ccription . As a se pathway . Dewi et pathway  showed ts in CRC . Moreovepoptosis . Lastly,poptosis  reportedi et al.  demonstrll lines , 32. MAS pathway . Thus, tCancer stem cells (CSCs) are a small subpopulation of cells that are endowed with the ability to self-renew, and differentiate into heterogeneous cell lineages that constitute the tumor , 35. CSCβ-catenin signaling modulates the expression of CSC marker genes and plays a role in the self-renewal ability and undifferentiated status of CSCs [β-catenin signaling pathway, which regulates normal stem cell differentiation and proliferation, is important in maintaining cancer stem cell properties [β-catenin signaling inhibitor IC-2 reduced the expression levels of CSC marker proteins and increased the cytotoxicity of 5-FU in CRC cells. As a negative feedback regulator of Wnt/β-catenin signaling, Axin2 could control Wnt-induced transcriptional responses. Suppression of Axin2 by miR-103/107 was demonstrated to enhance CRC chemoresistance by promoting cell stemness via Wnt/β-catenin signaling [β-catenin enhances both the chemoresistance and radioresistance of locally advanced rectal cancer through regulating EMT/CSC properties, and nuclear β-catenin in pretreatment-biopsied samples is promising in predicting the efficacy of chemoradiotherapy in rectal cancer patients [β-catenin cascade. It was also observed that zerumbone could suppress the stemness properties of CRC by inhibiting the β-catenin signaling pathway [β-catenin signaling pathway plays crucial roles in fostering chemoresistance of CRC through stemness.It has been proven that Wnt/ of CSCs –45. Accuoperties , 47. Uruoperties  demonstrignaling . Moreovepatients . Liu et patients  showed t pathway . These sRecently, accumulating evidence suggests molecular and phenotypic associations between epithelial–mesenchymal transition (EMT) phenotype and tumor chemoresistance –55. EMT β-catenin, Notch, TGF-β, and Hedgehog pathways while the underlying mechanisms have not been clearly defined [β-catenin signaling converging on activation of transcription factors such as ZEB and Snail was triggered to induce the expression of mesenchymal genes and repress E-cadherin expression [β-Catenin and E-cadherin form a complex in the area of cell–cell junction, providing the basis for cell–cell association [β-catenin signaling pathway and has been characterized as a trait of EMT cells [β-catenin and disassociation of the E-cadherin/β-catenin complex activate β-catenin–TCF transcription, decrease E-cadherin levels, and increase Snail expression. CRC metastasis could be also promoted by inducing EMT through a β-catenin–dependent pathway [β-catenin, downregulation of epithelial markers, and overexpression of mesenchymal markers, in both cellular and animal models of colorectal cancer. Collectively, Wnt/β-catenin signaling may modulate the chemosensitivity of CRC through EMT.Since studies have reported that residual resistant cells following chemotherapy are associated with an EMT phenotype in animal models as well as in clinical settings, EMT has now emerged as the focus of research into the cause of chemoresistance in several tumor types –63. The  defined , 65. As pression , 66. β-Cociation . Previouociation –70. In CMT cells . Chen etMT cells  showed t pathway . Another pathway  demonstrAlthough the investigation of chemoresistance in CRC has been focused on mechanisms intrinsic to tumor cells, alternative views propose a role for the tumor microenvironment (TME) in promoting chemoresistance. The tumor microenvironment, regarded as the tumor bed, contains components including extracellular matrix (ECM) proteins, aberrant vasculature, and cancer-associated cells . TME alsβ-catenin activity and thereby affect stemness features which are associated with chemoresistance in colorectal cancer cells [β-catenin signaling pathway via acting as a competing endogenous RNA sponge [β-catenin signaling pathway [β-catenin signaling cascade [β-catenin signaling infiltrates immune effector cells into a tolerant state and inhibits the DC-dependent cross-sensitization of antitumor CTLs [β-catenin promote resistance to immunotherapy with anti-PD-1, which impairs T-cell activity and involves the deficient recruitment of DCs [β-catenin signaling duration in HCT116 cells [α and TGF-β2 to promote chemoresistance in colorectal cancer. Nevertheless, the underlying mechanism was not related to the β-catenin signaling pathway [In stromal cells, it is reported that hepatocyte growth factor (HGF) secreted by myofibroblasts could activate nuclear er cells . As the er cells . In coloA sponge . lncRNA  pathway . lncRNA  cascade . In immumor CTLs . Active t of DCs . As a cr16 cells . Hypoxia pathway , 81.β-catenin cascade [β-catenin signaling pathway in In recent years, an increasing number of studies have indicated that regulation of gene expression by various noncoding RNAs (ncRNAs) such as microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) were involved in the acquisition of chemoresistance after treatment . miRNAs  cascade , 87. Theβ-catenin signaling in CRC has been shown to be associated with a variety of mechanisms including decreased intracellular drug accumulation, apoptosis inhibition, presence of highly resistant CSCs, EMT, tumor microenvironment, and some ncRNAs. The Wnt/β-catenin signaling pathway could be further investigated as a promising target in the development of new drugs to alleviate chemoresistance. Nowadays, preclinical studies and clinical trials showing patients' responses to therapy with inhibitors of Wnt/β-catenin signaling pathway are undergoing. Some experimental evidence from preclinical studies already suggests a beneficial consequence of β-catenin signaling pathway blockade. For example, as a soy-derived isoflavone, which could be used as an inhibitor of the Wnt pathway by inactivating β-catenin signaling through overexpressing GSK3β and E-cadherin, Genistein has been reported to play a role in reversing resistance to fluoropyrimidine compounds and platinum [β-catenin signaling pathway need to be further explored in the future. Finally, more progresses in molecular biology enabling clinicians to reverse drug resistance of CRC are eagerly anticipated.Chemoresistance remains a considerable challenge preventing better cure rates after treatment initiation in CRC nowadays. Unraveling the molecular mechanisms driving the chemoresistance of CRC would be beneficial for identifying invaluable therapeutic targets for clinical applications. As mentioned in the present review, chemoresistance related to platinum . Moreove


CTNNB1 and SUFU mutations. To the best of our knowledge, this is the first report of panfollicular carcinosarcoma and of the presence of a CTNNB1 mutation in trichoblastic carcinosarcoma.We present a case of trichoblastic carcinosarcoma with panfollicular differentiation. An 80‐year‐old man presented with a lesion on the left ear, which had been present for several months. Histopathology revealed a well‐demarcated neoplasm in the dermis composed of intimately intermingled malignant epithelial and mesenchymal cells. The epithelial component showed multilineage follicular differentiation toward all of the elements of a normal hair follicle. Molecular analysis revealed identical molecular aberrations in both epithelial and mesenchymal components including  Only eight cases of trichoblastic carcinosarcoma have been reported in the literature.2An 80‐year‐old man with no relevant past medical history presented with a lesion on the left ear, which had been present for a few months. Histopathology revealed a well‐demarcated neoplasm in the dermis without subcutaneous involvement or connection to the epidermis Figure . The lesImmunohistochemically, the epithelial component was positive for cytokeratin CKAE1/AE3), BerEp4 , and p63. Aberrant expression of p53 was demonstrated in both components. Interestingly, both the epithelial and mesenchymal components revealed focal strong nuclear beta‐catenin staining, mainly in inner root cells, matrical cells, and spindle cells. Ki‐67 proliferation index was high (30%) in both components and accentuated the nuclear polymorphism in both components  and truncating mutations in both CDKN2A (encoding p16/p14arf) and SUFU   of cancer‐associated genes and single‐nucleotide polymorphism (SNP) analysis for copy number alterations was performed using two different gene panels . Separat3Trichoblastic carcinosarcoma is an exceedingly rare tumor. To date, only eight cases have been reported in the literature.CTNNB1 mutation . This specific mutation in Serine 45 has not been reported earlier, although Serine 45 is a mutational hotspot in other tumor types and different mutations in this residue have been shown to be oncogenic/activating. Mutations in exon 3 are associated with translocation of the beta‐catenin protein from the membrane to the nucleus and activation of Wnt/beta‐catenin signaling. Aberrant expression of beta‐catenin has been reported in tumors with matrical differentiation, such as pilomatricoma, pilomatrix carcinoma, and basal cell carcinoma with matrical differentiation. Furthermore, Kazakov et al described one case of a trichoblastoma harboring a CTNNB1 mutation. To our knowledge, this is the first report of a trichoblastic carcinosarcoma harboring a CTNNB1 mutation.Wnt activation leads to binding of beta‐catenin to transcription factors resulting in pluripotent gene regulation.Wnt signaling leads to dysregulated follicular differentiation.Interestingly, using immunohistochemistry, we found nuclear beta‐catenin staining in parts of both the epithelial and mesenchymal components. Indeed, additional molecular analysis confirmed a SUFU protein acts as a negative regulator of the Hedgehog signaling pathway by binding to and inhibiting Gli1 protein. Inactivating mutations in SUFU can result in upregulation of Gli transcription factors leading to aberrant activation of the Hedgehog pathway. Furthermore, Hedgehog signaling regulates the growth and morphogenesis of hair follicle epithelium during the anagen portion of the hair cycle.SUFU mutation in both components of the tumor. Mutations in SUFU gene have been associated with Gorlin syndrome, multiple hereditary infundibulocystic basal cell carcinoma syndrome, familial meningioma, and medulloblastoma.SUFU mutations.The Hedgehog signaling pathway is essential for embryonic development and plays a crucial role in tumorigenesis. The According to the literature, trichoblastic carcinosarcoma are more frequently seen in older individuals, with male predominance, and are mainly located in the head and neck region, as in our case. Trichoblastic carcinosarcoma has not been reported in young adults or children.No guidelines regarding treatment and follow‐up of trichoblastic carcinosarcoma have been established because of the rarity of these tumors. In our patient, re‐excision with Mohs surgery was performed since the tumor was found close to the resection margin in the first excision. Although the follow‐up period is limited, there has been no local recurrence over a period of 6 months. There were no lymph node metastases.SUFU mutations, in the epithelial and mesenchymal components, suggesting that trichoblastic carcinosarcoma might originate from a single progenitor cell . This finding may shed light on the underlying biology of this tumor type. However, further investigation including further molecular studies is needed for a better understanding of trichoblastic carcinosarcoma.In conclusion, we describe the first case of trichoblastic carcinosarcoma with panfollicular differentiation and identify identical molecular changes, including beta‐catenin and AppendixS1: Supporting informationClick here for additional data file.