The physical and chemical characteristics of samples were evaluated with different analyses. The obtained results demonstrated higher surface area for PTL/Ar and negligible change in crystal structure, compared to NL. It was found that the synergistic effect between ozone and PTL/Ar nanocatalyst was led to highest PhP degradation efficiency. The kinetic study confirmed the pseudo-first-order reaction for the PhP degradation processes included adsorption, peroxone and ozonation, catalytic ozonation with NL and PTL/Ar. Long term application (6 cycles) confirmed the high stability of the PTL/Ar. Moreover, different organic and inorganic salts as well as the dissolved ozone concentration demonstrated the predominant role of hydroxyl radicals and superoxide radicals in PhP degradation by catalytic Ozonation using PTL/Ar. The main produced intermediates during PhP oxidation by PTL/Ar catalytic ozonation were identified using LC–(+ESI)–MS technique. Finally, the negligible iron leaching, higher mineralization rate, lower electrical energy consumption and excellent catalytic activity of PTL/Ar samples demonstrate the superior application of non-thermal plasma for treatment of NL.The present study has focused on the degradation of phenazopyridine (PhP) as an emerging contaminant through catalytic ozonation by novel plasma treated natural limonite (FeOOH·xH Furthermore, physical processes like adsorption, coagulation or membrane processes are not favorable because pollutants only transfer from one phase to another that needs more treatment6. In the recent decay, advanced oxidation processes (AOPs) are of great interest due to their potential to degrade any resistant contaminants by in-situ generation of active oxidizing agents, good mineralization of pollutants and no extra waste9. Among different types of AOPs, much attention has been paid to the ozone based processes because of its accessibility, feasibility, high reactivity and purification of final effluent12. Ozone with 2.07 V redox potential destroys organic contaminants through direct oxidation of pollutants and/or indirect mechanisms by generation free hydroxyl radical  contains fine crystal grains from multiphase mixture with porous structure and large specific surface area as well as a high surface energy which results in a stronger capability. The salient property of NL that enhances its catalytic activity is the presence of superficial hydroxyl groups on its surface which participates in the production of hydroxyl radicals22. The modification of natural minerals is an effective way to increase their application, which is done using both physical and/or chemical processes. Mechanical modification is a simple, inexpensive and efficient method to reduce the size of minerals from micrometers to nanometers. Furthermore, chemical activation is effective to enhance surface hydroxyl groups as well as specific surface area23. From different types of modification methods, non-thermal plasma technique is one of the newest methods for surface modification. The main feature of plasma technology is environmental-friendly, cost-effective, quickness, simplicity and ability of various nanostructures production24. Phenazopyridine hydrochloride (PhP) is a heterocyclic aromatic azo-compound that is used to help with the irritation, pain or urgency caused by urinary tract infections25. Due to the low water solubility (0.202 g/L) and low biodegradability, PhP accumulates in the sewages and therefore, treatment of PhP contaminated waters are very important26.The pharmaceutical wastewaters contain extensive amounts of bio-resistant contaminants that are not effectively remediated through conventional biological methods such as activated sludge, sequencing batch reactor, biofilm reactors, etc.pzc and EDX confirmed the physical and chemical characteristics of synthesized samples. Moreover, in this research, the influence of the eco-friendly modified NL was evaluated by degradation of PhP antibiotic through catalytic ozonation process. For this purpose, the effect of main process parameters such as catalyst dosage, solution pH, initial contaminant and ozone concentration were investigated on the performance of process. In the following sections, the catalyst stability and reusability of as-prepared catalysts, Fe ions release, mineralization and also assessment of electrical energy consumption were also investigated. By evaluating the effect of organic and inorganic salts and also, the dissolved ozone concentration the PhP degradation mechanism was proposed. In addition the LC–( ESI)–MS/MS analysis used to recognize the produced intermediates and by-products during the PhP degradation by catalytic ozonation using PTL/Ar nanocatalysts. Only few reports have been worked on PhP oxidation intermediates in ozone based AOPs.To the best of our knowledge, there is no comprehensive research on non-thermal glow discharge plasma method for treatment of NL in order to improve the structural properties and catalytic activity of this iron mineral. Different characterization analysis such as: FESEM, XRD, AAS, TEM, FTIR, BET-BJH, pH2S2O3.5H2O) as ozonation reaction quenching, sodium hydroxide (NaOH) and sulfuric acid (H2SO4) for pH adjustment, potassium hydrogen phthalate  for TOC calibration and, scavenging agents such as t-butanol (C4H10O), benzoquinone (BQ), sodium dihydrogen phosphate (NaH2PO4), sodium carbonate (Na2CO3) and sodium nitrate (NaNO3) were provided from Merck Co. (Germany). Ultrapure water used for all experimental tests. All reagent used in the AOPs were pure and used as received without any purification.NL ore was provided from Tekab iron mine . The PhP was used as pharmaceutical contaminant which characteristics are given in Table 12. High-voltage direct current (DC) supplied by a DC power source was connected by the aluminum bonnets to generate glow discharge plasma. Argon gas with flowrate of 3 cm3/s was pumped within the reactor to bring the pressure to 50 Pa. The NL plasma-treated under Ar was denoted as PTL/Ar.The limonite nanostructures were produced from NL by non-thermal glow discharge plasma under argon atmosphere. The treatment procedure is schematically presented in Fig. The crystalline structure of NL and PTL/Ar samples were evaluated by X-ray diffraction (XRD)  armed with Cu-Kα . The size and morphology of the NL and PTL/Ar samples were investigated using Field Emission.BET), porosity, pore diameter and volume of NL and PTL/Ar samples were calculated by physical nitrogen adsorption and desorption experiment  using Brunauer Emmett Teller (BET) analysis . The Microstructure Distance Measurement software was used to evaluate particle size distribution of the nanoparticles . The atomic absorption spectroscopy (AAS) analyses measured the concentration of Fe ions released in the aquatic medium . The point of zero charge (pHPZC) was performed by a procedure proposed by Mustafa et al.27. In this method, ten glass bottles containing 40 mL NaNO3 (0.1 M) in ultrapure water with different pH values between 2 and 11 were prepared, each bottles containing 0.2 g of NL or PTL/Ar. The suspensions stirred for 48 h at 25 ℃. The pHPZC is defined by plotting the final pH versus initial pH.Scanning Electron Microscopy equipped with an EDX microanalysis  and Transmission Electron Microscopy . Fourier Transform Infrared spectra (FTIR) were conducted in Bruker Tensor 27 (400–4000 1/cm). The specific surface area (S2S2O3 (0.01 M). Then, filtered by a 0.45 µm PTFE filter for further analysis.Semi-batch experiments of ozonation based processes were performed in a tubular Pyrex reactor with 4 cm internal diameter and 35 cm in height, as illustrated in Fig. 28.The concentration of PhP at different times of reaction was evaluated at 270 nm through a UV–Vis spectrophotometer . A VCSH TOC analyzer  was conducted to measure the Total organic carbon (TOC) of samples. The residual ozone concentration in the solution was evaluated by Indigo colorimetric procedure0 (mg/L) and Mt (mg/L) are the concentration/TOC at initial and t (min) time of concentration, respectively.The degradation percent of PhP and TOC removal were calculated using Eq.  where M00 and C (mg/L) are the initial and final concentration of PhP, respectively. The time of reaction and the apparent first-order rate constant are represented by k (1/min) and t (min), respectively.The kinetic rates of reaction were evaluated using pseudo first-order kinetics (Eq. ):2\docum3CN, LC–MS grade) and H2O (30:70) were used as the mobile phase. The LC–MS/MS was scanned in the mass region from 50 to 1000 m/z with sampling period of 0.1 s. The CDL and heating module temperature were both set as 350 °C and 450 °C, respectively.The liquid chromatography–mass spectrometry (LC-(+ ESI)-MS) technology was utilized to monitor the produced intermediates and by-products and propose the PhP degradation pathways in catalytic ozonation by PTL/Ar. The LC–MS/MS analyses were performed using mass spectrum (EMS) scan with positive mode on a 3200 QTrap mass spectrometer . The measurements were carried out at 254 nm in a C18 column . Pure acetonitrile  with a hysteresis cycle, revealing the porous structure with meso–micro cavities. The obtained results also indicated that the closed pores converted to interconnected open cavities. Also, the H4 type hysteresis can be considered for limonite samples, indicating narrow gap cavities. The textural properties of NL and PTL/Ar including total pore volume, pore diameter and specific surface area are listed in Table 2/g which increased to 49.16 m2/g after plasma treatment. The pore volume of PTL/Ar samples was obtained about 0.039 cm3/g which improved significantly compared to NL samples (0.023 cm3/g) as a consequence of plasma treatment. This increase in specific surface area results in the generation of more active sites on the surface of the catalyst and consequently, enhances the performance of catalytic process.BET-BJH analysis was performed for determination of the specific surface area, pore diameter and volume of the raw and treated samples. Figure PZC was determined by plotting the final pH versus the initial pH. As depicted in Fig. PZC of the NL and PTL/Ar were measured 6.4 and 6.7, respectively, indicating successful hydroxylation of limonite after Ar-plasma treatment. The net surface charge of limonite is zero at pHPZC. Furthermore, the surface of limonite positively charged when pHsolution < pHPZC and negatively charged when pHsolution > pHPZC32. According to Eq. , ozone concentration (11 g/L), pH value (6.7) but at different initial PhP concentrations of 30, 40, 50 and 60 mg/L. The degradation of PhP was decreased from 98 to 71% by increasing its initial concentration from 30 to 60 mg/L, respectively. The degradation efficiency of PhP depends on the rate of radical generation. Whiles the oxidant concentration is constant, a competition occurred between the generated intermediates and byproducts with parent pollutant in reacting with radicals at high initial pollutant concentration36. Therefore, the possibility of ROSs reaction with PhP molecules decreases, which leads to reduction in degradation efficiency of oxidation process. A similar phenomena was observed by Khataee et al.37 that the degradation of textile dye in the sono-Fenton/pyrite process was decreased as initial dye concentration increased.Figure a of PhP (5.05) and pHPZC of nanocatalysts (6.7), so in the acidic condition both PhP repelled from the surface. In such condition the surface charge density of the nanocatalyst become positive while the PhP molecules are in cationic form thus an electrostatic repulsion forces occurred. On the other hand, in the alkaline condition the negative charge density of surface and anionic form of PhP molecules hinder the contaminant adsorption and consequent reactions. In the moderate pH as Fig. The initial pH of solution is one of the significant parameters in the heterogeneous processes. It was stated in the literature that the oxidation process conducted mainly near or on the surface of catalyst due to the short life-time of free radicals. Therefore, it is desirable that the contaminant not repelled but attracted to the surface. Considering the pK9. However, no significant difference was observed in the degradation efficiency of PhP between catalyst dosages of 0.75 and 1 g/L. Therefore, 0.75 g/L was selected as the optimum dosage of PTL/Ar nanocatalysts for further experiments. In the similar study conducted by Liu et al.38, no noticeable difference was observed for degradation of p-nitrophenol when limonite dosage changed from 2 to 4 g/L.The influence of catalyst loading on the performance of oxidation process is demonstrated in Fig. 3/L at constant pH, PhP concentration and catalyst dosage of 6.7, 30 mg/L and 0.75 g/L, respectively and the results are presented in Fig. 33. It should be noted that the final PhP degradation was the same in the experiments containing 11, 13 and 15 mgO3/L. Therefore, 11 mg/L ozone concentration was selected as optimum value for further experiments.The effect of initial ozone concentration was evaluated over the range of 7–13 mgO3 + NL and O3 + PTL/Ar processes, respectively. Therefore, the plasma treatment significantly decreased the amount of Fe release to the reaction medium. The improvement in catalyst stability after plasma treatment also was reported by researchers37. In order to investigate the reusability of the catalyst, the performance of PTL/Ar after 6 runs was evaluated. The results of reusability study were illustrated in Fig. 39.The stability of catalyst plays an important role in the long-term operation. For this purpose, the Fe content released in the solution was analyzed before and after oxidation process by AAS analysis. The results of Fig. 3, O3 + NL, O3 + H2O2 and O3 + PTL/Ar. The results showed that 37, 50, 60 and 71% of the TOC was removed in the O3, O3 + NL, O3 + H2O2 and O3 + PTL/Ar processes, respectively. As can be seen the highest mineralization of PhP was observed in O3 + PTL/Ar process. Comparing the ozonation process in the presence of NL and PTL/Ar revealed that the plasma treatment of NL highly affect the mineralization of PhP.The mineralization of PhP oxidation process was evaluated by measuring total organic carbon (TOC). As illustrated in Fig. EO) of the above processes was calculated using Eq. (40:0 (mg/L) and TOCf (mg/L) are the initial and final total organic carbons, respectively. The EEO values for degradation of PhP according to optimum condition were calculated and the results are shown in Fig. 3 + PTL/Ar process had the lowest energy consumption as well as highest TOC removal compared to O3, O3 + NL and O3 + H2O2 processes. Mehrjouei and co-workers41 compared the economic aspects of photocatalytic oxidation, photo and catalytic ozonation processes for degradation of dichloroacetic acid and oxalic acid and reported that photocatalytic ozonation represented the highest cost-effectiveness between the investigated processes.In this research, the electrical energy per order  on the performance of O3 + PTL/Ar process. TBA as free radical scavenger reacts with hydroxyl radical at a second-order-rate constant 8 1/M.s). Also, BQ was used as superoxide radical scavenger. The concentrations of scavengers were too high in order to sufficiently quench the generated radicals instead of reacting with PhP. From Fig. 9:Figure Therefore, the rate of radical production was decreased due to the presence of the salts and, carbonate had the greatest effect.42. The release of inorganic ions (NO3− and NO2−) from N heteroatoms as a consequence of PhP bond cleavage in sole and catalytic ozonation is presented in Fig. 3− and NO2− ions were accumulated in the medium gradually from the beginning of processes. More nitrate ions were created in both processes compared to nitrite ions. Nitrite ion concentration increased at beginning of processes and followed by a gradual decrease which can be due to conversion to nitrate. Nitrate ions were formed in a more extent in comparison with nitrite and the concentration was increased over time of reaction. Furthermore, the amount of NO3− and NO2− ions release was higher in O3 + PTL/Ar process than sole O3. This means ozonation process in the presence of PTL/Ar nanocatalysts had more degradation efficiency compared to ozonation process alone. These findings are in consistence with the other researcher coutcomes43.The concentration of dissolved ozone was evaluated during ozonation and catalytic ozonation (with PTL/Ar) processes and shown in Fig. ·OH attack to N atom of amine group of PhP. 2,3,6-Triaminopyridine (A) degraded to 3-hydroxy-2,6-diaminopyridine with m/z of 126 which can furthered degraded to 2,6-diaminopyridine (m/z = 110). The intermediate with m/z of 124 (“B) was identified as a result of N=N bond cleavage of “A structure. Phenol compound (m/z = 95) was generated due to hydroxylation of “B bonds. Finally, multiple hydroxylation reactions and bond cleavage occurred and further reaction of ·OH and other ROSs with different structures led to production of end-products such as H2O, CO2, and N ions.The produced intermediates and by-products PhP degradation during catalytic ozonation by PTL/Ar was evaluated by LC–MS analysis. The detected oxidation intermediates are summarized in Fig. 3, peroxone and O3 + NL processes, the O3 + PTL/Ar process expressed the highest mineralization and lowest electrical energy consumption under the optimized conditions. The PhP degradation mechanism was explored by different inorganic and organic scavengers as well as O3 concentration evaluation which proved that the indirect ozone attack was the dominant degradation pathway. The main intermediates during PhP oxidation by catalytic ozonation processes using PTL/Ar were identified using LC–MS technique. Considering the supreme activity and stability of the plasma treated nanocatalyst, it can be concluded that PTL/Ar has potential for further practical applications.In the present research, the beneficial limonite nanocatalysts were prepared by non-thermal glow discharge plasma under Ar atmosphere. Based on the characterization analyses, the small particle size, highly porous and rough nanostructure of plasma treated samples will improve the PhP degradation. A comparison between different degradation processes demonstrated that catalytic ozonation with PTL/Ar due to the higher specific surface area and total pore volume led to 96.4% degradation and 74% mineralization during 40 and 120 min of process, respectively. The excellent properties of nanostructures which were achieved by plasma treatment enhanced the penetration of pollutant molecules and thus improved the catalytic activity of PTL/Ar. The optimum amount of pH value, catalyst loading, initial ozone and contaminant concentration were observed to be 6.7, 0.75 m/L, 11 mg/L and 30 mg/L, respectively. Lower Fe ion release in addition to high reusability and stability during the catalytic ozonation process with PTL/Ar can be due to strengthening of the crystalline structure after plasma treatment in comparison with raw samples. The mineralization and electrical energy consumption results revealed that, in contrast to the O


Cotton production is reaching a global limit, leading to a growing demand for bio-based textile fibers produced by other means. Textile fibers based on regenerated cellulose from wood holds great potential, but in order to produce fibers, the components need to be dissolved in suitable solvents. Furthermore, the dissolution process of cellulose is not yet fully understood. In this study, we investigated the dissolution state of microcrystalline cellulose in aqueous NaOH by using primarily scattering methods. Contrary to previous findings, this study indicated that cellulose concentrations of up to 2 wt % are completely molecularly dissolved in 8 wt % NaOH. Scattering data furthermore revealed the presence of semi-flexible cylinders with stiff segments. In order to improve the dissolution capability of NaOH, the effects of different additives have been of interest. In this study, scattering data indicated that the addition of ZnO decreased the formation of aggregates, while the addition of PEG did not improve the dissolution properties significantly, although preliminary NMR data did suggest a weak attraction between PEG and cellulose. Overall, this study sheds further light on the dissolution of cellulose in NaOH and highlights the use of scattering methods to assess solvent quality. Cotton fibers are eminent as textile fibers, but cotton production is reaching a global limit, mainly due to environmental considerations . Since tSubstantial effort has been put toward identifying suitable solvents. One of the most well-studied of these is NaOH, which from an industrial perspective is both cheap and environmentally friendly. One of the challenges of using aqueous NaOH as a solvent for cellulose is that cellulose is only dissolved within a narrow range of NaOH concentrations (1.5–2.5 M NaOH) and at temperatures below 0 ℃ . FurtherIn order to improve the dissolution capability of NaOH, the effects of adding different additives to aqueous NaOH have been of interest. Amongst others, ZnO, urea and PEG have in the literature been reported to positively affect the dissolution properties of NaOH . In this2O, 99.5% D and D2O was obtained from Sigma-Aldrich, Merck KGaA, Darmstadt, Germany.Microcrystalline cellulose (MCC) was obtained from Sigma-Aldrich, Merck KGaA, Darmstadt, Germany  aqueous NaOH by using a freeze-thaw method: MCC was added to NaOH(aq) at 0 ℃ while stirring vigorously for 15–20 min (samples kept in an ice bath). The samples were subsequently left in the freezer at –18 ℃ overnight and next day thawed in an ice bath while being stirred for approximately 20 min. If applicable, additives were added to NaOH(aq) before the addition of MCC. Since the solubility of ZnO is strongly dependent on pH, ZnO was solubilized at higher NaOH concentrations (18–20 wt %), and afterwards the solutions were diluted to obtain 8 wt % NaOH. NMR samples were prepared similarly, but by using NaOD and Dq is defined by n, the wavelength Static light scattering experiments were performed using a 3D LS Spectrometer  equipped with a laser producing a photon beam with a wavelength of 660 nm. The data were obtained over an angular range of 60–140° with a step size of 1°. Exposure time was 10 × 30 s per step. The temperature was kept at 25 ℃, controlled by a circulating water bath . The scattering intensity of the neat 8 wt % NaOH solution was subtracted as background, and the scattering data were converted to absolute scale, i.e., the excess Rayleigh ratio rding to :(1)ΔR(q)A and B are sample-specific coefficients determined by fitting the equation to the measured refractive indices.Refractive indices were determined by using an Abbe 60 Refractometer . The temperature was kept at 25 ℃, controlled by a circulating water bath . The refractive indices for different concentrations of MCC were determined at three different wavelengths; 435.8, 546.1 and 579.0 nm. The refractive indices at 660 nm were obtained through Cauchy’s empirical equation:I is given as a function of the scattering vector q-range from approximately 0.004 to 3 1/Å. Data were brought to absolute scale using the SAXSGui software provided by the instrument. In order to assess the scattering intensity of MCC, Small-angle X-ray scattering measurements were performed using the laboratory-based SAXSLab Ganesha 300XL instrument (SAXSLAB ApS (Xenocs), Skovlunde, Denmark) equipped with an X-ray source producing a photon beam with a wavelength of 1.54 Å. The scattering patterns were recorded with a two-dimensional (2D) 300 K Pilatus pixel area detector . The samples were measured at a temperature of 25 ℃, which was controlled by an external recirculating water bath . The 2D scattering data did not show any angular dependency, and were thus azimuthally averaged; normalized by the incident radiation intensity, the sample exposure time and transmission; and corrected for background and detector inhomogeneities. The radially averaged intensity www.sasview.org [The respective volume fractions were calculated from the weight fractions by using the density of MCC, view.org ).G of 960 G 1/cm. Measurements were done at 25 ℃ using dry air as the temperature control gas. Pulsed gradient stimulated echo with a field gradient pulse of G 1/cm was used to measure water diffusion in samples of 2M NaOD and 0.1 or 1 wt % PEG-200 at different concentrations of MCC. The diffusion of PEG in the 0.1 wt % PEG samples was obtained in the same way, but with a varying gradient strength of G 1/cm. Non-stimulated pulsed spin echo was employed for the 1 wt % PEG samples in order to detect the sharp PEG peak and not the overlapping broad peak from cellulose (otherwise with the same parameters). The diffusion coefficients D were calculated using the software Topspin 4.0.6 by fitting an exponentially decaying function to the measured echo amplitude INMR diffusion experiments were performed on a Bruker Avance DMX200 spectrometer  operating at 200.13 MHz on the ic ratio ,20.q, the scattering intensity is similar for all four MCC concentrations. At low q, the scattering intensity of 1 and 2 wt % MCC seems to level off at approximately q can be ascribed to the presence of aggregates/branched cylinders in the solution.Different concentrations of MCC dissolved in NaOH were studied by using SAXS. A comparison of the SAXS curves for MCC concentrations of 1, 2, 3 and 4 wt % is shown in q-range, which indicates that cellulose is completely dissolved at these concentrations. This is in contrast to previous observations reported by Hagman et al. [q. Slight changes in the sample preparation protocol  are most likely the cause of the observed difference in dissolution and highlight the sensitivity of the cellulose-NaOH-system with respect to these parameters.The scattering patterns obtained for 1 and 2 wt % MCC are similar in the whole n et al. , where tAs already discussed by Behrens et al. , for MCCq-values in the range of approximately q can be expressed asK is the optical constant depending on the type of scattering technique, c is the concentration of the solute, To further investigate the state of dissolution at low wt % MCC, we focused on the X-ray scattering pattern for 2 wt % MCC in 8 wt % NaOH . The slor and a total contour length L, consisting of stiff segments with lengths given by a persistence length Due to the slope of length lp. The perK given byThe parameters applied in the displayed model where: 218.2±6.9, which cThe model fit depicted in ormation .q is significantly lower than the scattering intensity observed by Hagman et al. [q for 2 wt % MCC in NaOH is quite comparable to the reported scattered intensity at low q for 2 wt % MCC dissolved in TBAH, which was found to be close to a As discussed above, the scattering intensity at low n et al. , where tn et al.  the avern et al. , but seeq-values above At q, a static light scattering study was performed on different concentrations of MCC in 8 wt % NaOH , which is in good agreement with the SAXS observations, indicating that MCC is completely dissolved at these concentrations. At MCC concentrations above 2 wt %, the increased scattering intensity observed by using SLS  by scaling with the optical constants for SAXS and SLS, Equation . The optr et al. , the levr et al. .L, and the persistence length, A quick estimation of the observed effective relation (10)Rg2=Similarly, an estimate of the observed weight-averaged molecular weight  in TBAH .q, which supports the finding that MCC is fully dissolved at this concentration  compared to MCC in NaOH without ZnO (black curve). This indicates that the addition of ZnO reduces the formation of aggregates, which fits well with previously reported observations that the addition of ZnO improves the dissolution by acting as a stabilizer against gelation [The dissolution properties of MCC in NaOH have been reported to improve when adding 0.5–2 wt % ZnO [gelation ,29. The q for the 1 and 0.7 wt % ZnO samples is similar to the absolute intensity obtained for completely dissolved MCC in neat NaOH  compared to samples containing no additives (black curve), which suggests a slight decrease in the formation of aggregates. At a higher PEG content, the scattering intensity at low q is increased as compared to 4 wt % MCC in neat NaOH, indicating that the formation of aggregates is increased. Thus, only a low content of PEG-200 seems to slightly improve the dissolution properties of MCC in NaOH. However, the observed difference is so small that it is questionable whether this decrease in aggregation results in any noticeable difference of the properties on the macro-scale.The addition of PEG with an average molecular weight of 2000 g/mol has been reported to improve the dissolution properties of MCC in NaOH . In the 2O and PEG, respectively, were measured at different concentrations of MCC and divided by the diffusion in the neat solvent, To investigate possible interactions between PEG and cellulose, a NMR diffusion study was performed on PEG in neat NaOH versus in NaOH containing dissolved MCC. If PEG binds to MCC, the diffusion coefficient of PEG is expected to decrease, as MCC diffuses much slower than the small PEG molecules. The diffusion coefficients vent, D0 . The obtvent, D0 . The difvent, D0 . The fraAccording to the obtained data, this implies that approximately 20% of PEG was bound to MCC at a concentration of 4 wt %. The fact that the Previous studies reported in the literature seem to indicate that there is no interaction between PEG and cellulose in an aqueous state, although PEG has been found to associate with cellulose through hydrogen bonding if no water is present . The detNative cellulose is insoluble in water because of the very stable crystalline phase, often referred to as cellulose I. From the point of view of thermodynamics, this means that the transfer of cellulose molecules from the crystal state to water is associated with an increase in the Gibbs’ free energy, 2.5–13.5 . When th+ –> Nan can be a very large number. This results in a decreased cellulose solubility with increasing NaOH concentration. Thus, the solubility experiences two instabilities. At low pH it is unstable relative to native cellulose I, while at higher pH it is unstable with respect to the sodium salt, Na+ fits well into the dense crystalline lattice of cellulose, resulting in a stable crystalline phase. However, if we replace Na+ with a more bulky organic cation, such as tetrabutyl ammonium, TBA+, which does not fit into the crystalline lattice, then the salt crystal becomes unstable and large amounts of cellulose can be solubilized [However, increasing the NaOH concentration above 2 M does not lead to an increased solubility of cellulose. Instead, another instability sets in. The sodium salt of (deprotonated) cellulose also forms very stable crystalline phases with low solubility. Here, we need to consider the precipitation reaction celluloseubilized .+ is to destabilize the salt crystal state. Successful additives, instead, need to stabilize the solution phase. Many additives have been tested over the years, and in several cases improved solubility has been reported, although no dramatic improvement has been detected [That organic bases are superior relative to NaOH in solubilizing cellulose has also been known for a long time. Still, however, they have not been shown to be commercially viable. Instead, a lot of research have been invested over the years into finding suitable additives to improve the solubility of cellulose in NaOH . As discdetected ,28,29,32+ counter-ions, have the potential to stabilize the solution phase. Binding the Na+-ions in solution can be practically difficult because of the high concentration (2 M), although one can of course imagine the action of complexing agents. Concerning the cellulose chain, Lindman and co-workers have in a number of recent papers pointed out its amphiphilic nature [The question is then: What can be the action of an additive that leads to a decrease of cellulose’s chemical potential in solution and help “bind” it there? Additives that experience attractive interactions with the cellulose chains, alternatively with the Nac nature ,7, whichc nature . In addic nature .In conclusion, the current study sheds further light on the dissolution properties of cellulose in aqueous NaOH. SAXS and SLS were employed in order to assess and compare different concentrations of MCC and the effects of additives. Scattering data revealed that it was possible to molecularly dissolve up to 2 wt % MCC in 8 wt % NaOH, which is contrary to previous findings. The obtained data suggest that the dissolved cellulose molecules can be characterized as semi-flexible cylinders with relatively long, stiff segments. This indicates that the chains are sterically hindered, most likely due to either solvent interactions or folded chain conformations.SAXS data obtained in the current study furthermore revealed that the addition of ZnO reduced the aggregation level compared to cellulose in neat NaOH. Based on the observed levels of aggregation, the current study indicated that an optimal ZnO concentration for 4 wt % MCC in 8 wt % NaOH is approximately 0.7 wt % ZnO. The addition of PEG-200 did not improve the dissolution properties significantly, although the included diffusion NMR study did indicate a weak attraction between cellulose and PEG. The observed aggregation levels were slightly reduced for samples containing 0.1 wt % PEG, but the aggregation was increased for samples with a higher PEG content. Thus, the current study suggests that PEG most likely has no significant positive effect on the dissolution properties of cellulose in NaOH, at least not when following the sample preparation protocol employed in this study.


Carbohydrates are the most important energy source for monogastric animals, including humans, and dysregulation of carbohydrate metabolism has been associated with metabolic syndromes, such as type 2 diabetes mellitus (T2DM), hypertension, and obesity. Starch is the major carbohydrate source, consisting of amylose and amylopectin. This study investigated the effects of dietary starch structure on growth performance, serum glucose–insulin response, and intestinal health in weaned piglets, which may contribute to the principles of carbohydrate nutrition and facilitate the utilization of dietary starches.p > 0.05). Diets with higher amylose ratios  led to a significant reduction of the serum glucose concentration at 3 h post-prandium (p < 0.01), while high amylopectin diets (AR 0.31 and 0.14) significantly elevated The expression of gene s at this time point (p < 0.01). High amylopectin diets also increased the apparent digestibility of crude protein (CP), ether extract (EE), dry matter (DM), gross energy (GE), and crude ash (p < 0.001). Interestingly, diet rich in amylose (AR 2.90) significantly elevated the butyric acid content (p < 0.05) and decreased the pH value (p < 0.05) in the cecal digesta. In contrast, diet rich in amylopectin  significantly elevated the total bacteria populations in the cecal digesta (p < 0.001). Moreover, a high amylopectin diet (AR 0.14) tended to elevate the mRNA level of fatty acid synthase , but significantly decreased the mRNA level of sodium-dependent glucose transporter 1  in the duodenal and jejunal mucosa, respectively. These results suggested that blood glucose and insulin concentrations were improved in high AR diets, and the diet also helped to maintain the intestinal health.To investigate the effects of dietary starch structure  on serum glucose absorption metabolism and intestinal health, a total of ninety weaned piglets (Duroc × (Yorkshire × Landrace)) were randomly assigned to 5 dietary treatments and fed with a diet containing different AR . The trial lasted for 21 d. In this study, the growth performance was not affected by the dietary starch structure ( FAS) and acetyl CoA carboxylase (ACC), in liver and adipose tissue. However, ingestion of a high AR diet may lead to mild blood glucose and insulin responses . A. A13]. A2SO4 solution was added to 100 g of each fresh fecal sample to fix excreta nitrogen [Experimental diets were sampled and stored at −20 °C for chemical analysis of dry matter (DM), ether extract (EE), crude protein (CP), crude ash, gross energy (GE), total starch, and ratio of amylose to amylopectin. From days 18 to 21, fresh fecal samples were collected and placed in individual plastic bags and then 10 mL of 5% Hnitrogen .g for 15 min at room temperature, and serum was subsequently separated and stored at −20 °C for further analysis. At the end of the study, the selected piglets were killed by intravenous injection of sodium pentobarbital  [After the trial, one medium-weight piglet from each pen was selected and venous blood samples were collected at 07.00. Piglets were fed at 08.00 and then postprandial blood samples were taken once an hour, from 09.00 to 13.50 h. Blood samples were centrifuged at 3000× Feces from the last 4 days of one medium-weight piglet from each pen were mixed thoroughly and dried at 65 °C for 72 h, after which they were ground to pass through a 40-mesh screen. The Apparent total tract digestibility (ATTD) was evaluated by using acid insoluble ash (AIA) as the digestibility indicator. The AIA in diet and fecal samples were determined by a method described by Mccarthy et al.  with modof feces .Total starch and AR were determined by assay kits .g for 10 min. Then, 1 mL supernatant was transferred to a new sterile tube and then mixed with 0.2 mL 25% metaphosphoric acid and 23.3 μL 210 mmol/L crotonic acid simultaneously, then left at room temperature for 30 min. After 30 min, the sterile tubes were centrifuged at 15,000× g again for 10 min, then placed in sterile tubes in an ice bath for 30 min. Next, 500 μL supernatant was transferred to another sterile tube, mixed with 500 μL methanol, and homogenized for 10 min. After this, the mixture was centrifuged at 4 °C. Finally, the supernatant was injected into a gas chromatographic system  to separate and quantify the VFA.Cecal digesta samples were used to determine the concentration of VFA by gas chromatography according to Chen et al. . BrieflyThe serum glucose concentration was measured by corresponding assay kits  according to the manufacturer’s instructions. The serum insulin concentration was measured by enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer’s instructions.Total RNA was isolated from duodenal and jejunal mucous using TRIzol  and further purified by RNeasy Mini Kit . All the procedures were carried out as per the manufacturer’s protocol. The concentration of RNA was determined using spectrophotometry based on the optical density (OD) ratio OD260/OD280, which ranged from 1.8 to 2.0 in all samples, while integrity was monitored using an Agilent 2100 bioanalyzer . The reverse transcription reaction was done using Real-time reagent kits with gDNA Eraser (TaKaRa), following the manufacturer’s instructions.β-Actin was used as the reference gene. Real-time PCR for four target genes and the house keeping gene was performed using Applied Biosystems  Power SYBR Green PCR Master Mix in a Bio-Rad iCycler with minor modifications . Fluorescein was added at a final concentration of 10 nM as the reference dye. Cycling conditions were as follows: 95 °C for 5 min, forty-five cycles of 95 °C for 30 s, appropriate annealing temperature  to assay four differentially expressed genes . Here, βperature  for 30 sE. coli, Lactobacillus, Bifidobacterium, and Bacillus were obtained from the Qi et al. [E. coli, Lactobacillus, Bifidobacterium, and Bacillus were quantified by quantitative PCR with the method adapted from Diao et al. [Microbial genomic DNA was isolated from the cecal digesta samples by using E.Z.N.A Stool DNA Kit , according to the offered manual. Primers and probes  for totai et al.  and Fieri et al. , which wo et al. . Copies o et al. . Standarp < 0.05 and p < 0.10, respectively. Regression analysis was used to estimate the linear and quadratic relation between dietary starch structure and dependent variables with the use of regression curve estimation.Pen was the experimental unit for ADFI, ADG, and F: G. Gene expression data from replicate samples were averaged and analyzed using the Pfaffl  method tp > 0.05) in average daily gain or feed intake between the high AR and low AR groups during the 21 d experimental period.The growth parameters are reported in p < 0.01) and linearly increased at 4 h post-prandium (p < 0.05). Furthermore, the variation in insulin level did not respond correspondently to the postprandial blood glucose levels. The fasting insulin concentration quadratically increased in AR 2.90, 1.46, 0.68, and 0.31 groups, compared with piglets consuming the AR 0.14 diet (p < 0.01). Compared with the AR 0.31 and 0.14 groups, the insulin level (p < 0.01) at 1 h post-prandium was quadratically increased in AR 2.90, 1.46, and 0.68 groups. However, piglets in AR 0.14 group quadratically increased their insulin concentrations at 2, 3, 4, and 5 h post-prandium (p < 0.01). Moreover, the serum insulin levels in the five dietary treatment groups were increased to the peak at 2 h post-pranidum, and then decreased gradually over time. Overall, the serum insulin concentration in the AR 0.14 group showed greater changes than that of AR 2.90 group at 2, 3, 4, and 5 h post-prandium (p < 0.05).Fasting and postprandial glucose and insulin contents in serum are presented in p < 0.001) the digestibility of DM, EE, GE, CP, and ash compared with the other four groups (p < 0.05) the digestibility of DM, EE, GE, CP, and ash compared with piglets consuming the high amylose diet (AR 2.90). Piglets fed the high amylopectin diet (AR 0.14) significantly increased (r groups . Pigletsp < 0.05) the butyric acid concentration. Piglets that consumed the high amylose diet (AR 2.90) tend to have greater cecal concentration of acetic acid (p = 0.070) and propionic acid  than piglets consuming other four diets. In addition, increasing dietary amylopectin content linearly increased the cecal digesta pH value (p < 0.05)  .FAS tended to increase (p = 0.083) in the duodenum when piglets were fed with low AR (AR 0.14) diet. However, the expression of SGLT1 in the jejunum increased (p < 0.01) in high AR (AR 2.90) diet.The effects of DAR on intestinal gene expression are presented in Bacillus, Bifidobacterium, and Lactobacillus showed no differences (p > 0.05) in piglets fed diets with different AR. However, piglets fed the high amylopectin diet (AR 0.14) had quadratically increased (p < 0.001) total bacteria populations compared with piglets consuming the other 4 diets. Increasing dietary amylose had a linear tend to decrease (p = 0.075) Escherichia coli populations compared with piglets consuming the low AR diets.Cecal microflora data are shown in Starch is the main carbohydrate available for humans and monogastric animals . It has Previous studies showed that young pigs fed with a high amylose diet significantly decreased growth performance and feed efficiency . In the FAS in piglets of the low AR (AR 0.31 and 0.14) groups were higher than those of the high AR groups. The present results, however, agree well with previous findings showing that ingestion of a low AR diet significantly elevated the lipogenic gene expression in growing–finishing pigs [The present results showed that increasing dietary amylopectin content was associated with increased nutrient digestibility, which increased the postprandial glucose–insulin response. In addition, previous studies have demonstrated that ingestion of RDS leads to rapid increase in blood glucose and triggers insulin secretion from β-cells of the endocrine pancreas ,27. Typi AR AR 0. and 0.14ing pigs ,33.Bacillus, Lactobacillus, and Bifidobacterium, were observed. However, the result is consistent with the previous report showing that ingestion of a high amylose diet did not increase several beneficial bacteria populations, such as Bifidobacterium spp. and Firmicutes, in the cecum of growing pigs [E. coli in weaned pigs [To further understand the effects of DAR on intestinal health, cecal digesta samples were collected to measure SCFAs and microbes. We found that diets with high levels of amylose linearly decreased the digesta pH value, which was probably due to microbial fermentation. It is a well-known fact that SCFAs are the major products of indigestible starch fermentation in the large intestine and only a few SCFAs are excreted in feces ,35,36,37ing pigs . Additioned pigs .In conclusion, the present results suggested that the metabolic responses of weaned piglets fed with different DAR may vary widely depending on their composition, and ingestion of a low AR diet leads to a stronger insulinemic response and up-regulation the expression of lipogenic genes. In contrast, ingestion of a high AR diet is helpful for maintaining the intestinal microbiota and health.


Most of the derivatives showed significantly better stability than idebenone over 6 hours (p < 0.001). By extending the side-chain of SCQs, increased stability for some compounds was observed. Metabolic conversion from the derivative 3 to 5 and reduced idebenone metabolism in the presence of 5 were also observed. These results highlight the therapeutic potential of naphthoquinone-based SCQs and provide essential insights for future drug design, prodrug therapy and polytherapy, respectively.Short-chain quinones (SCQs) have been identified as potential drug candidates against mitochondrial dysfunction, which is largely dependent on their reversible redox characteristics of the active quinone core. We recently synthesized a SCQ library of > 148 naphthoquinone derivatives and identified 16 compounds with enhanced cytoprotection compared to the clinically used benzoquinone idebenone. One of the major drawbacks of idebenone is its high metabolic conversion in the liver, which significantly restricts its therapeutic activity. Therefore, this study assessed the metabolic stability of the 16 identified naphthoquinone derivatives  Mitochondrial dysfunction has been linked to a vast number of disorders ranging from primary mitochondrial disorders such as Leber’s Hereditary Optical Neuropathy (LHON) to common diseases associated with mitochondrial dysfunction such as diabetes ,2,3. Des1-16, p < 0.033) under conditions of mitochondrial dysfunction , wh, wh1 andL-phenylalaninol derivative 3 to the L-phenylalanine derivative 5 appeared to reach a plateau at ~10% conversion from t = 2 h and persisted for the entire test period until t = 6 h. Guided by the superior stability of 5, we hypothesized that 5 might be a metabolic enzyme inhibitor. A concentration series of the unstable reference SCQ idebenone was tested for metabolic stability for 6 h with or without 40 μM 5 (5 was observed from 20 μM idebenone onward. Not surprisingly, 5 was found to be stable, as described in combination with all concentrations of idebenone (5 inhibited metabolic enzymes and could be used in combination with idebenone as a polytherapy alternative to overcome its poor stability reported in vivo [The conversion from the  40 μM 5 . A signidebenone . These r in vivo ,18. FutuA library of novel short-chain naphthoquinones was designed to support the discovery of drug candidates to protect against mitochondrial dysfunction . From th


OCLN), claudin-3 and -7 (CLDN3 and CLDN7), desmoglein 3 (DSG3) and thymic stromal lymphopoietin (TSLP) in AR patients (n = 30) and non-allergic controls (n = 30). Nasal epithelial cells of non-allergic controls and AR patients were collected to examine their mRNA expression levels, and to correlate with clinico-demographical and environmental parameters. We demonstrated that the expression of OCLN (p = 0.009), CLDN3 (p = 0.032) or CLDN7 (p = 0.004) transcript was significantly lower in AR patients compared with non-allergic controls. No significant difference was observed in the expression of DSG3 (p = 0.750) or TSLP (p = 0.991) transcript in AR patients compared with non-allergic controls. A significant association between urban locations and lower OCLN expression (p = 0.010), or exposure to second-hand smoke with lower CLDN7 expression (p = 0.042) was found in AR patients. Interestingly, none of the TJs expression was significantly associated with having pets, frequency of changing bedsheet and housekeeping. These results suggest that defective nasal epithelial barrier in AR patients is attributable to reduced expression of OCLN and CLDN7 associated with urban locations and exposure to second-hand smoke, supporting recent findings that air pollution represents one of the causes of AR.The breakdown of nasal epithelial barrier occurs in allergic rhinitis (AR) patients. Impairment of cell junction molecules including tight junctions (TJs) and desmosomes plays causative roles in the pathogenesis of AR. In this study, we investigated the transcript expression levels of TJs including occludin ( Breakdown of nasal epithelial barrier integrity is attributable to reduced expression of tight junction (TJ) molecules which is observed in patients with allergic diseases such as allergic rhinitis (AR). TJ disruption-inducing factors include air pollutants ..The MX3005P qPCR thermal cycler was used in the qPCR reaction . The mixture for one qPCR reaction was prepared with 10 μL of iTaq Universal SYBR Green Supermix (2x) at final concentration of 1x, 2 μL (400 nM) each for forward and reverse primer, variable amount of cDNA template at final concentration of 50 ng and nuclease-free water was added into the mixture for a final volume of 20 μL. qPCR reaction was then performed according to the following thermal profile: (1) Polymerase activation step at 95 °C for 25 s; (2) Denaturation step at 95 °C for 5 s; (3) Annealing/extension step at 60 °C for 20 s. All three steps were repeated for 40 cycles. The primers were reconstituted to yield a 10 × concentration by resuspending in 950 µL of nuclease-free water followed by centrifugation at 2,500 rpm for 10 s before use. The relative transcripts quantity of every target gene in every sample was determined using the 2p-values were two-tailed and values < 0.05 were considered statistically significant.Data were analyzed using Student’s t-test  and Mann–Whitney U test  to determine the difference of gene expression between AR and non-allergic control groups. The distribution of clinico-demographical and environmental parameters in AR patients or non-allergic controls were compared in terms of each gene’s expression (median cut-off) using the χ2-test or Fisher’s exact test as appropriate . All Supplementary Video.Supplementary Information.


Psm ES4326/AvrRpt2 (AvrRpt2) was widely used as the reaction system of hypersensitive response (HR) in Arabidopsis. The study showed that in npr1 (GFP-ATG8a), AvrRpt2 was more effective at inducing the production of autophagosome and autophagy flux than that in GFP-ATG8a. The mRNA expression of ATG1, ATG6 and ATG8a were more in npr1 during the early HR. Based on transcriptome data analysis, enhanced disease susceptibility 1 (EDS1) was up-regulated in wild-type (WT) but was not induced in atg4a4b (ATG4 deletion mutant) during AvrRpt2 infection. Compared with WT, atg4a4b had higher expression of salicylic acid glucosyltransferase 1 (SGT1) and isochorismate synthase 1 (ICS1); but less salicylic acid (SA) in normal condition and the same level of free SA during AvrRpt2 infection. These results suggested that the consumption of free SA should be occurred in atg4a4b. AvrRpt2 may trigger the activation of Toll/Interleukin-1 receptor (TIR)-nucleotide binding site (NB)-leucine rich repeat (LRR)—TIR-NB-LRR—to induce autophagy via EDS1, which was inhibited by nonexpressor of PR genes 1 (NPR1). Moreover, high expression of NPR3 in atg4a4b may accelerate the degradation of NPR1 during AvrRpt2 infection. Psm ES4326/AvrRpt2 (AvrRpt2) infection [Arabidopsis R proteins resistant to Pseudomonas syringae 2 (RPS2) [P. syringae 4 (RPS4) under AvrRps4 infection; non-race specific disease resistance (NDR1) is required for a different R proteins called coiled-coil (CC)-NB-LRR (CC-NB-LRR), which was mediated by RPS2 under AvrRpt2 infection, and triggers HR independent of autophagy [AvrRpt2 during the initial of HR and the new functions of ATG4, related to EDS1.Autophagy is a highly conserved intracellular degradation and recycling procession, which exists in yeast, plants and mammals. It controls cellular homeostasis, stress adaptation, and programmed cell death (PCD) in eukaryotes . Autopha2 (RPS2) . Geneticutophagy . In thisP. syringae) stresses significantly induce the formation of free SA and SA glucose conjugates in Arabidopsis. Consistent with this induced response, SA glucosyltransferase (SGT) is induced by SA and appropriate biotic and abiotic stress, which catalyzes the conversion of SA into a conjugated form [Salicylic acid (SA) plays an important role in plant immunity against biotic and abiotic stress . Previouted form .AvrRpt2 infection still need further study.The components of the SA mediated immunity pathway are the nonexpressor of pathogenesis-related genes (NPR) proteins that include NPR1–NPR4 four close isoforms. NPR1 functions as a transcriptional activator, whereas NPR3 and NPR4 are transcriptional repressors. They all work independently and harmoniously to regulate the expression of downstream genes . NPR1 isAvrRpt2-induced autophagy and that ATG4 inhibits the consumption of free SA and alleviates the degradation of NPR1, providing a new insight into the plant autophagy.Here, we report that EDS1 is involved in the GFP-ATG8a and npr1 (GFP- ATG8a) with concanamycin A (ConA) and wortmannin (WM) by confocal microscopy under AvrRpt2 infection  cells. Then we used 8.95 μM WM to block autophagy effectively in Arabidopsis [GFP-ATG8a and npr1 (GFP-ATG8a) under AvrRpt2 infection as most of the fluorescence remaining diffuse within the cytosol (GFP-ATG8a (GFP-ATG8a and npr1 (GFP-ATG8a) increased with the time of AvrRpt2 infection and both reached the maximum at 6 h, but decreased significantly at 12 h. The level of free GFP in each npr1 (GFP-ATG8a) group was higher than that in wild-type (WT) group  group B. These ATG1, ATG6 and ATG8a in WT and npr1 were examined by qRT-PCR  are essential for autophagosome formation . The exp qRT-PCR C. ComparAvrRpt2 and new function of ATG4, we performed transcriptome sequencing and analyzed on BMKCloud (www.biocloud.net) in WT and atg4a4b (ATG4 deletion mutant) under AvrRpt2 infection for 12 h. The number R2, which represent that means the square of the Pearson coefficient, was larger than 0.85 for both the tested samples  (Log2FC 1.6187) and heat shock protein 90 (HSP90) (Log2FC 1.0307) that associated with plant resistance showed red, which meant the expression was up-regulated. Surprisingly, the downstream factor EDS1 (Log2FC 1.2281) in response to AvrRps4 that triggered TIR-NB-LRR to induce autophagy also showed red. However, in atg4a4b, all the responding genes had no change under AvrRpt2 infection  shrunk. At 2 d.p.i of AvrRpt2 infection, the leaves of rps2 and atg4a4b were only yellowed, while the leaves of npr1 and atg8a were especially degraded. Numbers are ratios of leaves with HR phenotype from by repeating the experiment independently  in npr1, atg4a4b and atg8a were examined  [atg4a4b. With 12 h of AvrRpt2 infection, the NPR1 protein expression level increased significantly in WT, rps2, atg5 and atg8a, while decreased significantly in atg4a4b  . We unex atg4a4b D. ICS1 in atg4a4b increased, while total SA content decreased and free SA content unchanged when compared with WT at 12 h of AvrRpt2 infection  that detects conserved pathogen-associated molecular patterns (PAMPs) and initiates plant immune response by the name of PAMP-triggered immunity (PTI) . AnotherAvrRpt2 recognized by the CC-NB-LRR type R protein RPS2 and was considered to trigger hypersensitive cell death that was strictly NDR1-dependent but autophagy-independent [AvrRpt2 induced the production of autophagosomes and autophagy flux, which was inhibited by NPR1 ; these are very interesting. Previous work showed that AvrRpt2-induced the expression of several primary jasmonic acid (JA)-responsive genes and JA is a positive regulator of RPS2-mediated ETI [AvrRpt2-induced autophagy maybe involve in other key hormone signals (for example JA) besides SA signal, which needed further study.ependent ,23. For  by NPR1 A,B. In aes of HR C. These ated ETI . We specatg4a4b. We found that the expression of EDS1, SGT1 and HSP90 was up-regulated in WT, while there was no change in atg4a4b  and 82% relative humidity for 2–4 weeks. The Arabidopsis thaliana mutants (in ecotype Col-0), rps2, ATG8a-lacking mutant (atg8a) and NPR1-lacking mutant (npr1) were provided by Dr. Xinnian Dong . Transgenic GFP-ATG8a (Col-0 background) was donated by Dr. Kohki Yoshimoto of the Plant Science Center of Japan. npr1 (GFP-ATG8a) was produced by crossing npr1 and GFP-ATG8a. The atg4a4b was produced by crossing atg4a (SALK_085300) and atg4b (SALK_056994). atg8a (SALK_045344) was obtained from the Arabidopsis Biological Resource Center. SA was purchased from Sigma-Aldrich, China . Seeds were cultivated in soil culture in growth cabinets at 22 °C (day) and 18 °C (night), with a 16 h light period .The bacterial used in this study was Following the previous procedure ,31, 4-weArabidopsis leaves at indicated times after the treatment of TRI reagent according to the manufacturer’s instruction . The first-strand complementary DNA was synthesized from total RNA using a Reverse-iT first-strand synthesis kit . qPCR was performed using the ChamQ SYBR qPCR Master Mix  on ABI Life QuantStudio 6. The thermal cycles of qPCR were initial denaturation at 95 °C for 30 s, followed by 40 cycles by 40 cycles at 95 °C for 5 s and 60 °C for 34 s. Ubiquitin 5 and AtACTIN2 were used as internal control. The primers used are listed in Total RNAs were extracted from w/v) Triton X-100, 0.2% (w/v) Nonidet P-40 and 1% (w/v) phenylmethanesulfonyl fluoride (PMSF). To extract NPR1, add 40 µM MG115, 1% β-ME, 500× protease inhibitor cocktail (PMSF not included) and 5000× phosphatase inhibitor cocktail to the protein extraction buffer. The extracts were centrifuged and the protein concentration of the supernatant was determined with a Bio-Rad protein assay.Amounts of 0.4 g leaves were ground in liquid nitrogen and the powder was resuspended in 1 mL ice-cold extraction buffer comprised of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.2%  seedlings (7 days old) were treated with MgCl2 or 1 μm concanamycin A (ConA)  or ConA + Psm ES4326/AvrRpt2 or ConA + 8.95 μm wortmannin (WM)  + Psm ES4326/AvrRpt2 for 3 h. Root epidermal cells below the cotyledon were imaged using a Zeiss LSM880 confocal laser scanning microscope. GFP fluorescence was excited by a 488 nm argon laser and detected at 505–550 nm by a photomultiplier detector. At least 10 sets of images were obtained for quantification analysis. GFP was counted per section according to 10 sets of images field of vision.rps2, npr1, atg4a4b and atg8a plants were infiltrated with Psm ES4326/AvrRpt2, and 6 leaf discs (8 mm diameter) were removed rapidly following infection and washed in 50 mL ddH2O (twice). After 10 min, we removed the wash water and replaced it with 15 mL of ddH2O. Ion leakage was then measured over time.Ion leakage assay was performed as previously described . The leaatg4a4b were infected by Psm ES4326/AvrRpt2 for 12 h, and then their samples were collected by taking 8–10 real leaves from three different culture pots as three biological replicates. Total RNA was extracted by TRIzol Reagent . RNA quality was determined using Agilent 2100 Bioanalyzer . RNA libraries were constructed from 2 lg of total RNA and subjected to deep sequencing at an Illumina Hiseq 2500 platform .WT and


A promotional role for androgen receptor (AR) signaling in hepatocellular carcinogenesis is emerging. In pre-clinical models, including diethylnitrosamine- (DEN-) induced hepatocellular carcinoma (HCC), anti-androgen therapies delay hepatocarcinogenesis. However, pharmacologic anti-androgen therapy in advanced HCC patients fails, suggesting that AR plays a role in HCC onset. This study aims to characterize AR expression and function throughout DEN-induced liver inflammation and carcinogenesis and evaluate the efficacy of prophylactic AR antagonism to prevent hepatocarcinogenesis. We demonstrate that pharmacologic AR antagonism with enzalutamide inhibits hepatocellular carcinogenesis. With enzalutamide treatment, we observe decreased CYP2E1 expression, reducing DEN-induced hepatocyte death and DNA ethyl-adducts. AR protein expression analyses show that DEN causes an initial upregulation of AR in portal fibroblasts and leukocytes, but not hepatocytes, suggesting that hepatocyte-autonomous AR signaling is not essential for DEN-induced carcinogenesis. Ablating androgen signaling by surgical castration reduced pre-carcinogen Kupffer cell populations but did not alter DEN-mediated immune cell recruitment nor AR expression. In this study, we identified that anti-androgen interventions modulate mutagenic DNA adducts, tumour initiation, and immune cell composition. Additionally, we find that AR expression in hepatocytes is not present during nor required for early DEN-mediated carcinogenesis. HCC emerges in the presence of chronic liver disease and the sexual dimorphism is evident regardless of the underlying aetiology including viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, and chronic aflatoxicosis. Intriguingly, the sexual dimorphism is consistent across species as multiple rodent models of HCC also exhibit a striking male predisposition4. Molecular evidence further supports a pro-carcinogenic role for the AR-signaling in HCC. Increased AR expression in both human and rodent HCC tumours has been reported9. More recent analyses implicate AR signaling may activate a variety of critical cancer-associated pathways in the neoplastic liver, including suppression of DNA repair, enhancing cancer cell metabolic activity and division, activation of known oncogenes, and promotion of liver cancer cell immortalization13.The androgen receptor (AR) is an enticing therapeutic target in hepatocellular carcinoma (HCC). This belief is founded upon the recognition of a marked sexual dimorphism in HCC risk, with men three to eight times more likely than women to develop or die from the disease15. The success of anti-androgen therapies in pre-clinical carcinogenesis models has yet to translate into humans as pharmacologic targeting of the AR-signaling axis consistently fails when studied in late-stage HCC17. These findings support a hypothesis that inhibition of the liver carcinogenesis cascade may require early and prolonged intervention of the AR-signaling axis. In both carcinogen- and viral transgene-induced models, surgical castration as a therapeutic intervention demonstrates a precipitous decline in anti-carcinogenic activity with delayed orchiectomy4. Thus, disruption of androgen signaling early in the carcinogenic cascade, perhaps with a chemoprevention strategy, warrants consideration.In rodent models, anti-androgen interventions including surgical castration, heritable mutations of the AR, and liver-specific AR-knockout reliably inhibit hepatocarcinogenesis, implicating a neoplasia-promoting capacity for AR signalingOur understanding of AR-signaling in early hepatocarcinogenesis—where its tumour-inhibiting outcomes are maximized in HCC animal models—remain poorly understood. Given the current body of evidence, we hypothesized that targeted pharmacologic intervention at the AR signaling axis would be most effective early in hepatocarcinogenesis. To test this hypothesis—and to interrogate mechanisms of action potentially explaining the efficacy of early antagonism of the AR signaling axis in HCC—we treated  adult rats with enzalutamide (ENZ), a potent AR antagonist, in a diethylnitrosamine (DEN)-induced model of hepatocellular carcinogenesis.18. ENZ dosages were based on a systemic exposure-matched therapeutic equivalent for patients receiving treatment for castrate resistant prostate cancer (100 mg/kg) and half-log equivalents thereof, 30 mg/kg and 10 mg/kg19. To ensure maximal AR antagonism, both DEN challenges were timed to occur at approximately within-dose maximal ENZ plasma concentrations (Tmax). Vehicle-treated SHAM animals receiving DEN/PH alone served as untreated controls while animals orchiectomized (ORX) three weeks prior to carcinogen challenge served as anti-androgen phenotype positive controls. We observed mortality at the time of carcinogen challenge within the 100 mg/kg group that left only two animals surviving to the end of study; this treatment group was thus excluded from follow-up analysis.To test the hypothesis that competitive antagonism of AR signaling inhibits hepatocellular carcinogenesis, we administered ENZ orally once daily to male rats in three separate dose groups Fig. a. To ens19. Surgical castration resulted in marked atrophy of the accessory sex glands.To ensure that low-dose ENZ as administered exhibits anti-androgen efficacy in the rat, the androgen-dependent tissues of the lower urogenital tract  were harvested and examined Fig. b. Consis19. To ensure therapeutic tolerability in an experimental design with considerable physiologic challenge to the liver, we assessed study morbidity, mortality, gross pathology and liver damage biomarkers. All rats on low dose  ENZ tolerated carcinogenic challenge and survived to end of study. Livers collected at necropsy were enlarged compared to age-standardized controls, a phenotype consistent with both the addition of DEN carcinogen and long-term ENZ administration20. There was no appreciable difference in gross appearance of the livers collected at sacrifice, and there was no impedance of hepatic regeneration with anti-androgen therapies as determined by liver weights normalized to bodyweight , regucalcin (Rgn), and CYP7A1 , we hypothesized that our preventive anti-androgen therapies may be differentially influencing hepatic Wnt/β-catenin-mediated metabolic zonation in the face of DEN challenge. To test this hypothesis, we evaluated downstream targets of Wnt/β-catenin activity using a candidate gene approach, namely: CYP1A2, CYP2E1, CYP7A1, and Glul  to evaluate DEN-induced hepatocyte death, and quantified DEN-adduct O2-ethyl-thymidine in DNA extracted from liver samples collected at PH. This particular ethyl-adduct was chosen for its documented persistence and mutagenicity29. Consistent with this hypothesis, preventive ENZ reduced the number of CCIII positive hepatocytes  altered the distribution of AR expression within the liver. In each case, the distribution pattern of hepatic AR expression was limited to the periportal region of the hepatic lobule, and there was no overlap in expression pattern when compared to the multifocal, scattered hepatocellular distribution of pGST.Contrary to our hypothesis and the reports of others, AR was undetectable in the broader parenchyma, including hepatocytes, in IHC analyses Fig. b9. InsteGiven the absence of the AR from the parenchymal and pre-neoplastic hepatocyte in the rat model, we were curious to evaluate AR expression in DEN-induced HCC lesions. To this end, intact male rats were subjected to the DEN/PH challenge, allowed to recover without intervention for four weeks, and then administered weekly IP injections of DEN up to 18 weeks post-PH to accelerate carcinogenesis  for activated portal fibroblasts  cells, we hypothesized that AR-positive immune cell recruitment could contribute to the increased AR expression described and observed in DEN carcinogenesis. To test this, we challenged SHAM and ORX mice with VEH or 50 mg/kg IP DEN and evaluated their hepatic immune cell populations by flow cytometry one-week after DEN exposure  was slightly increased in ORX mice in the absence of DEN challenge  and monocyte-derived liver macrophages in SHAM or ORX mice absent of DEN challenge and 2 and 7 days following DEN challenge Fig. . Liver mlls Fig. a.Figure Despite these baseline differences established by ORX, the proportions of immune cell subsets in the liver by 2 and 7 days after DEN challenge were essentially unaffected by castration Fig. b. In botearly interventions, including castration and genetic knockout of AR, inhibit cancer onset and burden35, whereas later stage interventions exhibit significantly less anti-cancer activity14. The evidence collectively suggests that the optimal strategy for AR-targeted intervention is early in the hepatocellular carcinogenesis cascade.Given the male predominance in disease incidence and mortality, building evidence of AR expression and activity, and an increasing number of available pharmacologic anti-androgens, the AR is a promising chemopreventive and therapeutic target in HCC. Pre-clinical models consistently indicate that What little is known about AR expression, activity, and suspected pro-carcinogenic mechanisms is limited to late-stage disease where targeted therapies are ineffective. The aim of our study was to determine the efficacy of early pharmacologic AR inhibition, interrogate mechanistic hypotheses potentially explaining how chemopreventive anti-androgen therapies inhibit hepatic carcinogenesis, and characterize AR expression and distribution in early carcinogenesis. To that end, we demonstrate: (1) Intervention with ENZ at 30 mg/kg indeed inhibits carcinogen-induced liver carcinogenesis; (2) Androgen deprivation alters hepatic metabolic zonation, reducing CYP2E1 expression and preventing activated DEN-induced toxicities including cell injury, death, and DNA-alkylation; and (3) Hepatic AR expression is upregulated during early DEN carcinogenesis and is largely due to AR expression within hepatic immune cells and fibroblasts with a minor contribution from hepatocytes (preneoplastic or otherwise).15. Delayed interventions exhibit a time-dependent decrease in therapeutic efficacy4. Based on these studies, we hypothesized that chemopreventive intervention would improve the likelihood of observing a benefit to anti-androgen therapy in HCC. Our results support this hypothesis by identifying a statistically significant decrease in the number of pGST positive, pre-neoplastic hepatocytes following preventive ENZ treatment  and cellular distribution (nuclear) consistent with human disease36. It is worth noting that AR-positive tumors represented a minority of those present. In total, these findings implicate an evolving role for the AR in hepatocellular carcinogenesis: an early, indirect role highlighted by the apparent upregulation in portal fibroblasts and leukocytes followed by an autonomous upregulation within the neoplastic hepatocyte. In agreement with our findings, an indirect role for AR in DEN carcinogenesis was also elegantly established by Kemp et al. who used Tfm mosaic neonatal mice to show that HCC is AR-dependent, yet autonomous hepatocyte AR expression is not a requirement for disease induction15. The same group later established that the sex hormonal effects on DEN carcinogenesis are dependent on wild-type levels of growth hormone37, which is well characterized to require intact hepatic androgen signaling38. Our discovery of non-hepatocyte, AR-positive cells raises the possibility for an intrahepatic, local and inflammatory influence of the AR. Furthermore, our finding that the pre-neoplastic hepatocyte is essentially devoid of AR expression suggests that genetic approaches ablating AR expression specifically in the hepatocyte may represent an inappropriate test of AR function in hepatocellular carcinogenesis that underestimates AR’s contribution9.Given our focus on early carcinogenesis and undetectable AR expression within hepatocytes, we wanted to confirm the presence of AR in DEN-induced rodent tumors. In our opinion, the only other study reporting similar results likely misinterprets normal hepatic structures  as abnormal, AR-positive dysplastic foci, and adequate tissue assessment is obscured by abundant, non-specific immunoperoxidase staining artefact40, and HCC is considered the end-stage event of chronic hepatic inflammatory processes. AR’s presence within immune cells suggests hormone signaling impacts inflammation associated liver carcinogenesis and may account for sexual dimorphism. Upon evaluation of AR+ cells within immune cell subsets from ORX and SHAM mice  at baseline, strongly suggesting an androgen-dependent effect on resident Kupffer cell populations  protocol (2015A00000079) approved by the IACUC at The Ohio State University  in compliance with their Animal Care and Use Program. Six-week old, male Sprague Dawley rats  were stratified by weight into treatment groups (n = 6) upon reception. Animals were surgically (ORX) or sham (SHAM) castrated (age = 7 wks) based on group assignment one week prior to initiating treatment. Treatment consisted of SHAM rats receiving 10, 30, or 100 mg/kg oral ENZ  diluted in 300 g/mol polyethylene glycol 300  once per day. Treatment started two weeks prior to carcinogen challenge (age = 8 weeks) and lasted to sacrifice (SAC). PEG300 vehicle (VEH) was delivered to control groups with ORX rats serving as a low-testosterone control and SHAM animals serving as negative controls. We initiated carcinogenesis with 50 mg/kg intraperitoneal (IP) DEN  (age = 10 wks) followed 24 h later by two-thirds PH followed again by IP DEN of the same dose . Animals recovered for 4-weeks then were given weekly 50 mg/kg IP DEN injections for 6, 10 or 14 weeks to accelerate tumorigenesis  were ORX or SHAM castrated two weeks prior to challenge with VEH or 50 mg/kg IP DEN (n = 8/group). DEN-treated mice from ORX and SHAM treatment groups were sacrificed at 48 h and 1 week following DEN injection. VEH mice were sacrificed 1 week following VEH injection  and IHC staining were performed on 4-µm thick FFPE tissue sections. Hepatic lesions were classified according to INHAND Hepatobiliary guidelinesMicroarray analysis was performed at The James Comprehensive Cancer Center’s Genomics Shared Resource. RNA was extracted from liver tissue with the Animal Tissue RNA Purification Kit . Briefly, RNA was hybridized to GeneChip Rat Gene 2.0 ST Arrays. Probe signal was background corrected, normalized and non-changing and/or undetected probes removed. Differentially expressed genes (DEGs) were determined (limma) using an FDR-corrected p-value of < 0.2. Significantly different genes were visualized using pheatmap, using Manhatten clustering, and the treatment groups indicated.O2-ethyl-thymidine was quantified using methods similar to those previously published53. Briefly, approximately 5 µg of extracted DNA was hydrolysed by sequential enzymatic reactions with nuclease P1 , phosphodiesterase I , and alkaline phosphatase . Hydrolysed O2-ethyl-thymidine was purified and concentrated using an Oasis HLB cartridge  and then quantified via LC–MS/MS tandem mass spectrometry using Waters Quattro Premier equipped with UPLC liquid chromatography and tandem mass spectrometry. LC conditions included mobile phases of A (10 mM ammonium formate/0.1% formic acid) and B (methanol/0.1% formic acid) with a gradient of 10–100% of mobile phase B which specifically 0.0 min (90% A and 10% B), 5.0 min (65% A and 35% B), 10.0 min (85% B), 18 min (100% B), 20 min (90% A and 10% B) at a constant flow rate of 200 µl/min. Analytes were separated using a C8(2) column . The mass transition of the analyte following precursor/product ion pair of m/z was 271.2–155.0 for O2-ethyl-thymidine. The mass spectrometer was tuned to its optimal sensitivity by direct infusion of pure O2-ethyl-thymidine. The data acquisition and peak integration were performed using Mass Lynx software (Waters).DNA was extracted from rat livers using the DNeasy Blood and Tissue Kit . DNA was processed and the adduct See Supplementary Methods online for mouse liver preparation for flow cytometry. All data was acquired on a BD LSRFortessa within one week of sample collection and fixation. Bead-based compensation was performed for each experimental run. See Supplementary Fig. 54.Statistical analyses were performed with Prism 8 . Figure legends contain corresponding test-specific analyses. Microarray analyses was undertaken using R platform for statistical computing and implementing various libraries (version 3.6.2)Supplementary Information.


Transcriptomic analyses revealed that low dox downregulated cell cycle genes while high dox upregulated DNA damage response genes. In chromatin immunoprecipitation (ChIP) assays with low dox, AR binding to sARE-containing enhancers increased, whereas AR was lost from cAREs. Further, ChIP-seq analysis revealed a subset of genes for which AR binding in low dox increased at pre-existing sites that included sites for prostate-specific factors such as FOXA1. AR dependence on cofactors at sAREs may be the basis for differential modulation by dox that preserves expression of genes for survival but not cancer progression. Repurposing of dox may provide unique opportunities for PCa treatment.Androgen receptor (AR) action is a hallmark of prostate cancer (PCa) with androgen deprivation being standard therapy. Yet, resistance arises and aberrant AR signaling promotes disease. We sought compounds that inhibited genes driving cancer but not normal growth and hypothesized that genes with consensus androgen response elements (cAREs) drive proliferation but genes with selective elements (sAREs) promote differentiation. In a high-throughput promoter-dependent drug screen, doxorubicin (dox) exhibited this ability, acting on DNA rather than AR. This dox effect was observed at low doses for multiple AR target genes in multiple PCa cell lines and also occurred  The progression of prostate cancer (PCa) depends on androgens acting via the androgen receptor (AR), and therefore androgen deprivation slows disease ,2. HowevThe consensus androgen response element (cARE) is a 6 bp inverted repeat, 5′-AGAACA-3′, with 3 bps intervening . cAREs din vitro of AR binding to cARE than sARE DNA. In PCa cells, dox at low dose increased expression of endogenous sARE-driven genes but repressed cARE-driven genes, and AR was differentially recruited to the chromatin of these genes. Since few genes with sAREs have been characterized, we considered genes sARE-like if their expression increased rather than decreased with low dose dox. The differential effect of low dose dox on gene expression was detectable in xenograft tumors. Dox dosage determined global differentiative versus proliferative patterns of gene expression by RNA-seq analysis and distinct landscapes of AR binding in ChIP-seq. The dose-dependent response to dox underscores that AR distinguishes selective from consensus response elements, the importance of these elements in driving functionally distinct patterns of gene expression, and an ability to differentially modulate AR activity on these elements. These results provide a basis to develop new PCa therapies that modulate rather than block AR activity, thus delaying resistance and producing fewer side effects.We developed a high-throughput screen for compounds that elicit differential AR regulation from distinct cARE or sARE elements, similar to a screen for differential modulators of GR . SurprisScreens were performed in the University of Michigan Center for Chemical Genomics (CCG). HeLa-A6 cells (a gift from E.M. Wilson ) served CV-1, HEK-293T, LNCaP, C42B and LAPC-4 cell lines were from American Type Culture Collection  and were validated, grown and transfected as previously described ,25. For 6 LAPC4 cells in matrigel were injected into both flanks of the mice , or in the case of mice with no palpable tumors, 3 weeks after grafting. Dox was prepared in 0.9% sterile saline and administered twice weekly with 100 μl intraperitoneal injections. The highest dose, similar to what is used in man, proved toxic. Mice were euthanized, and tumors harvested when the tumors reached about 2 cm3 in size, or 3 weeks after treatment initiation. At euthanasia, tumors were resected and portions stored in RNAlater Solution (Invitrogen), 10% formalin or snap frozen in liquid nitrogen for future analysis.CB17 scid mice from an in-house colony at the University of Michigan were surgically castrated and concurrently implanted with silastic tubing containing 25 mg testosterone for sustained release. After 1–1.5 weeks of allowing circulating testosterone levels to equilibrate to approximate human hormone levels, 3 × 10the mice . Tumors later. Solution was removed from −80°C and thawed, and RNA was isolated using the miRNAeasy kit, including the optional DNAse digestion . cDNA was synthesized from 1000 ng total RNA using Maxima First Strand cDNA Synthesis III Kit for RT-qPCR (Thermo Fisher Scientific), and/or the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Isolated RNA was quantified and checked for integrity and purity (260:280 ratio) using NANOdrop Spectrophotometer (Thermo Scientific). Quantitative real-time PCR was performed in triplicate using standard SYBR green reagents and protocols on a StepOnePlus Real-Time PCR system (Applied Biosystems). The target mRNA expression was quantified using the ΔΔCt method and normalized to β-actin expression. All primers (http://frodo.wi.mit.edu/primer3/) and synthesized by Integrated DNA Technologies .Total RNA was isolated from cells or homogenized xenograft tissue stored in RNA primers  were des2 fold change (FC) value  under R1881-treated conditions, were formulated as linear models and fit using Limma  and then treated 24 hrs with vehicle, 1 nM R1881, or R1881 plus 0.1, 0.4 or 0.7 μM dox. RNA-seq was performed on RNA extracted as above, using the Illumina HiSeq 2000 in paired end mode, as previously described . For eacC) value . These rC) value ,30. Signng Limma .et al. (The ChIP assays were performed using a HighCell ChIP kit with IPure 2.0 kit elution (Diagenode) according to the manufacturer's protocol. The antibodies used for ChIP assays are AR_PG-21 (Millipore Cat. # 06-680); TBP (Abcam Cat. #ab63766), and Normal Rabbit IgG Control (Diagenode). LNCaP cells were grown in charcoal-stripped serum containing media for 48 h followed by 12 h of vehicle or 1 nM R1881 and dox treatment  for all ChIP-qPCR and ChIP-seq conditions. ChIP-seq sample preparation for sequencing was performed according to the manufacturer's instructions (Illumina). Further experimental details as well as information on ChIP-seq enrichment analysis and overlaps of AR binding are in Asangani et al. . Transcret al. .P-values between individual conditions, and used a one-way ANOVA with Pair-wise Multiple Comparison Procedures (Holm–Sidak method) across multiple conditions. Growth data was analyzed using multiple pair-wise Student's t-tests.Data was analyzed using GraphPad Prism software version 7.0 . EMSA quantification and qPCR experiments were performed in triplicate to determine mean standard error, and are representative of repeated experiments of multiple biological replicates. Student's t-tests were performed with normalization to control analyses to obtain We developed a screen to test whether genes related by function may be co-regulated by distinct androgen response elements (AREs), which could account in part for differences in genes regulated by AR in normal or early disease prostate cells from those in castration-recurrent PCa (CRPC) . While mDifferential AR activation was assessed by transient transfection of fluorescent reporter genes driven by multimerized cAREs or sAREs into HeLa-A6 cells that have high AR levels from a stably integrated expression vector  Figure . CitrineFresh powder samples of the five leads were tested for inhibition of cARE- or sARE-driven luciferase reporters transiently transfected into CV-1 cells. One compound, dox, suppressed cARE but not sARE activation by AR plus R1881 ligand Figure . Dox alsGiven the remarkable finding of dox as a selective AR modulator, we screened an additional 7600 compounds from ChemDiv 100K, Prestwick, LOPAC, MS2400 and Biofocus natural products libraries. There were 124 compounds that showed >70% inhibition of cARE-citrine activity and <50% cell toxicity. Of these, 109 compounds had reasonable dose response, and 15 inhibited cARE- but not sARE-citrine expression. One of these compounds was also a topoisomerase inhibitor that intercalates into DNA, strengthening the notion that a DNA response element sequence difference could differentially modulate AR signaling. However, none of these additional hits were readily amenable to further development.To confirm that dox exerted a differential effect on AR due to the response element, we examined protein–DNA interaction by electrophoretic mobility shift assay (EMSA) Figure . HEK293TKLK3), TMPRSS2, and FKBP5, were inhibited by dox in a dose-dependent manner and have well characterized cAREs. In contrast, genes that increased expression above hormone-induced levels with low dose dox were called ‘sARE-like’. SGK1 and SARG  are prototypical sARE-driven genes, identified by their altered expression in mice bearing a mutated AR that cannot activate sAREs  or 1.7 (medium) mg/kg dox delivered twice weekly, corresponding to about one-tenth and one-third human doses, respectively Figure . A high To view how global gene expression varied with dox concentration and to discover additional sARE-like genes, transcriptome analysis was performed for LNCaP cells Figure . The majThe addition of 0.4 μM dox Figure . At 0.7 Comparing dose-dependent effects of dox on androgen regulated gene expression helped to distinguish effects due primarily to dox rather than dox modulation of androgen induced genes Figure . NormaliA further indication that low dose dox treatment led to differential biology and was not simply sub-toxic was seen by displaying differentially affected genes across treatment groups as a heat map with nonbiased clustering Figure . The lowvs. cAREs, as shown by EMSA  analysis (Figure In order to link the effect of low dose dox on gene expression to underlying differences in AREs, we examined AR binding within chromatin. The dox-dependent difference in AR binding to sAREs A Figure  was alsos Figure . AR binds Figure . The ChITo obtain a genome-wide view of differential effects of dox on AR binding, we undertook ChIP-seq analysis using published methods . To confDox disruption of AR binding was first globally viewed by principal component analysis Figure . SamplesThat the effect of dox is specific for AR and not all transcription factors is evident in the read peak heat maps comparing AR and TBP Figure . The majOf greater interest were the AR binding motifs increased by low dose dox Figure , right. Our goal was to redirect AR in prostate cancer to promote differentiation over proliferation by identifying compounds that selectively inhibit AR action on genes driving cancer but not normal growth. Our screen relied on the hypothesis that these genes differ in their response elements, in accordance with genomic studies correlating transcriptional outputs at different PCa stages with differences in AR binding sites . Most ARThe anthracycline dox, also called Adriamycin, is one of the oldest and still most widely used chemotherapeutics, but the precise mechanism of action remains unclear . Dox intThe mechanism of dox action highlights the functional differences in AR response elements, and underscores that AR is prodifferentiative in development and homeostasis but pro-proliferative in oncogenesis. This is modeled by studies in cell lines, which indicate that low dose dox can induce genes with sAREs, like SGK1 and SARG, in contrast to inhibiting classic AR targets such as PSA and TMPRSS2 Figures . Both EMThe differential effects of dox are emphasized by ChIP-seq results highlighting the role of other factors in distinguishing sARE-like from cARE-like responses Figure . Under lAn unanticipated clinical opportunity derives from this study. Low dose dox is unlikely to impact late stage PCa, yet the ability to enhance the differential effect between cARE- and sARE-driven genes may favor normal over tumorigenic functions of AR, and may thus be beneficial in watchful waiting or early stage disease. The low dose dox effect cannot reverse aggressive prostate tumor growth, as seen in mouse xenografts Figure , althoughttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE137056.GEO accession to all of our data is as follows: GSE137056, gkaa178_Supplemental_FileClick here for additional data file.