The research results showed that adsorbent has the high adsorption capacity . Optimal experimental conditions including pH, initial arsenite concentration and contact time were determined. Langmuir, Freundlich and Dubinin– Radushkevich isotherm models were applied to analyze the experimental data. The best interpretation for the experimental data was given by Langmuir adsorption isotherm equation and the maximum arsenite adsorbed by synthesized nano γ–alumina (qe) was found to be 40 (mg/g).Adsorption is considered a cost-effective procedure, safer to handle with high removal efficiency. Activated alumina is the most commonly used adsorbent for the removal of arsenic from aqueous solutions. However, activated alumina has a low adsorption capacity and acts kinetically in a slow manner. An ideal adsorbent should have a high surface area, physical and/or chemical stability and be inexpensive. To meet this requirement, nanomeso porous γ-alumina with a high surface area (201.53 m Arsenic contamination has brought about severe health problems, such as lung, skin, liver and kidney cancers . The chrand hair -4.The World Health Organization (WHO) sets a maximum allowable value of 10 mg/L for the arsenic concentration in drinking water .There are several methods available for removal of arsenic from water and wastewater. The most effective treatments are reverse osmosis , ion excCoagulation has also been used for Arsenic removal. The most commonly used metal salts are ferric salts such as ferric chloride or ferric sulfate and aluminum salts such as alum ,12. But Because of the high removal efficiency, convenience and simplicity, the adsorption process is disputably one of the more popular methods for the removal of arsenic ions. Among the adsorbents, nano γ–alumina with a high surface area has a high adsorption capacity and has been used for reducing different contaminants including nitrate  and As. Therefore, a pre-oxidation step is usually required to transform the arsenite to As (V). The oxidation procedure is mainly performed by the addition of chemical reagents, such as potassium permanganate, chlorine dioxide, monochloramine, hypochlorite, ozone, hydrogen peroxide, or manganese oxide ,18.The objective of this article is offered in two sections, in the first section, a simple method for synthesizing of nano γ–alumina from impure kaolin is proposed, X-ray diffraction (XRD), scanning electron micrographs (SEM), Fourier Transform Infrared Spectroscopy (FTIR) and Brunauer Emmett Teller (BET) have been conducted for characterization of synthesizednano γ–alumina; and in the second section, the application of synthesized nano γ–alumina for arsenite removal is discussed. Adsorption studies were conducted under various experimental conditions, such as pH, contact time and initial arsenite concentrations. The data from the experiments were fitted with different models to identify the adsorption mechanism.2O3 (Merck).All chemicals used are analytical reagents. The kaolin used came from Hamedan province, west of Iran. The arsenite stock solution was prepared using reagent-grade As2O3[2O3.At the first step, the kaolin was purified. Description of the purification of the kaolin is given elsewhere . The kao2O3. Table 2α radiation [A BET nitrogen absorption for the surface areas of the samples and scanning electron microscopy (SEM) were used to determine the particle size and morphology. Sample phases were identified using X-ray diffractometry (XRD) with nickel-filtered Cu Kadiation .-1.To determine arsenite sorption mechanisms, two representative samples of nano γ–alumina before and after the arsenic sorption were subjected to FTIR, and compared . The sampzc  has important effects on adsorption capacity and was estimated by mass titration method [pHn method . Synthes2 adsorption/desorption experiments for synthesisedNano γ-Al2O3 were carried out using a Belsorp mini II (BelJapan), and pore size distributions were calculated using the Barret-Joyner-Halenda (BJH) model on the desorption branch [Nn branch .Arsenite in the solution was analyzed using UV – vis spectrophotometer (Cecil-model-7600) (detection limit: 3.4 μg/L with accuracy < ±5%) using the method described by Afkhami et al. .The arsenite in samples were analyzed, based on their inhibition effect on the redox reaction between bromate and hydrochloric acid. In the spectrophotometric method, the decolorization of methyl orange with reaction products was used to monitor the reaction spectrophotometrically at 525 nm. The absorbance value was compared with a standard calibration curve.Approximately 0.02 g of synthesized nano γ–alumina was added to a capped tube containing 20 milliliters of a solution prepared at a predetermined arsenite concentration using deionized water, followed by shaking at room temperature for 3 h. The solutions were stirred continuously at a constant temperature to achieve equilibrium. After equilibrium, the solid and liquid were separated using a centrifuge (6000 rpm for 6 min). The aqueous phases were analyzed for arsenite content by spectrophotometer within 24 hours. Reproducibility of the measurements was determined in triplicates and the average values were reported. Relative standard deviations were discovered to be within ±4.0%.e(mg/g)) was calculated as follows:The amount of arsenite adsorbed , v is the volume of solution (L) and m is mass of the adsorbent (g).In this equation, C2O3 was insoluble and stable within the this range of pH.Also,when the initial concentration was low, the arsenite concentration remaining in the solution after adsorption was below the detection limit of spectrophotometer, therefore, initial concentration of 5 mg/L was selected.To determine the optimum pH for the maximum removal of arsenite, the equilibrium adsorption of arsenite with an initial concentration of 20 mg/L was investigated over a pH range of 3.5–8.5 because synthesizednano γ-AlThe initial pH of the solution was adjusted by using 0.1 M NaOH or 0.1 M HCl. 0.02 g synthesized nano γ–alumina was added to 20 mL solution. The mixture was shaken using a temperature-controlled water bath shaker at room temperature. After adsorption, the equilibrium arsenite of all solutions was measured and the value providing the maximum arsenite removal was determined.For achieving the rate of adsorption of arsenite, experiments were done at different time intervals (5 min–6 h). The minimum contact time was selected 5 minutes, because before that time the adsorption capacity was low. At upper than 6 h, trend of the plots showed that arsenite uptake had slower removal that gradually reached a plateau. In kinetic studies, 20 mL arsenite solution (20 mg/L) was adjusted to have a pH of 7.5 ± 0.1 by adding 0.1 M HCl and/or NaOH, and was agitated with synthesized nano γ–alumina (0.02 g) using a temperature-controlled water bath shaker at room temperature. After a fixed time interval, the adsorbent was separated and the aqueous phase was analyzed for determining the equilibrium concentration of arsenite. For reaching the adsorption equilibrium, experiments were repeated for different periods.The adsorption of arsenite on synthesized nano γ–alumina was done at room temperature (25 ±1°C) by batch experiments. 20 milliliters of arsenite solution of varying initial concentrations (20–250 mg/L) with an initial solution pH of 7.5 in 20 mL capped tubes were shaken with 0.02 g of synthesized nano γ–alumina after adjusting the pH to the desired value, for a designated period of contact time in a temperature-controlled shaking assembly. After equilibrium, samples were centrifuged and the aqueous phase was then analyzed for residualarsenite concentration by spectrophotometer.2O3 phase are obviously observed as broad peaks at 2θ angles around 38.0◦, 46.0◦, and 66.0◦ which correspond to the (3 1 1), (4 0 0), and (4 4 0) planes, respectively. The peaks in the pattern are significantly indicated the formation of nano sized γ-Al2O3 crystallites. Figure 2O3 powders before and after adsorption of arsenite. The γ-Al2O3 before adsorption of arsenite , a mean pore diameter of 6.91 nm and a high pore volume (0.33 cm2/g). The location of the hysteresis loop in the N2 isotherm obtained nano γ-Al2O3 sample  against relative pressure) displays type IV hysteresis showing the presence of mesoporosity that can be used to determine whether the material possessed a regular framework pore or interparticle voids, such as a textural pore. The framework porosity at 0.4- 0.75 P/Po (relative pressure) in the N2 isotherm shows that the porosity was framework, while the textural porosity at 0.8-1 P/Po indicates porosity arising from the noncrystalline intra-aggregate voids and spaces formed by interparticle contacts.Synthesisednano γ-Al6, whereas the band around 900 1/cm corresponded to ν-AlO4[According to FTIR spectra Figure  the bandto ν-AlO4.The bands from 1400 1/cm to 1600 1/cm indicate the formation of alumina . After aAt the water-adsorbent interfaces, the pH is an important parameter controlling the sorption process. Figure 3AsO3 is the dominant species, and van der waal force between the solute and the synthesized nano γ–alumina surface is expected. A further experiment was conducted to get the optimum pH in range of 5.5–7.5 and at pH=7.5 the maximum percent removal (97.65%) was achieved.High adsorption was observed in the pH range of 5.5–7.5 and low adsorption was achieved at higher pH values (higher than 8). In this pH range the non-ionic H3AsO3 is 9.2 indicating arsenite exists as a neutral specie up to a pH of about 9.2 [pzc of synthesized nano γ–alumina is 8.2  at two different initial arsenite concentrations of 10 and 20 mg/L with an initial solution pH of 7.5.It was observed that in both different initial concentrations of arsenite, removal efficiency increased with time Figure .The trend of the plots shows that arsenite uptake was rapid in the beginning followed by a slower removal that gradually reached a plateau. Maximum removal of arsenite was achieved within the first 15 min of contact time and equilibrium was reached in 30 min.There was no important change in arsenite uptake by synthesized nano γ–alumina in the subsequent 6 h.To evaluate the adsorption capacity and for investigating the characteristics of adsorption, the adsorption isotherm is basically important. For this reason, the arsenite adsorption equilibrium data obtained at pH of 7.5 at initial solution concentrations from 5 mg/L to 250 mg/L. When the initial concentration was low, the arsenite concentration in the solution after adsorption was below the detection limit of the spectrophotometer, therefore, the minimum initial concentration of 5 mg/L was selected. Langmuir, Freundlich ,29 and Dqe and qm (mg/g) are the adsorbed and maximum adsorbed amount of arsenite by nano γ–alumina, respectively. Ce is the equilibrium concentration (mg/L) of arsenite and b is the Langmuir constant related to energy of sorption. For Freundlich isotherm model, constants n and Kf are the Freundlich constants for the system, which were indicators of intensity and adsorption capacity, respectively.For Langmuirisothermmodel, The important characteristics of the Langmuir isotherm are explained by a dimensionless separation factor, RL, which is indicative of the isotherm shape that predicts whether an adsorption system is favorable or unfavorable. RL is defined as :The linear form of (D–R) isotherm equation is shown as:qe, qm and β are the amount of arsenite adsorbed per unit mass of nano γ–alumina, the theoretical adsorption capacity and the constant of the sorption energy, respectively. Also, β is related to the average energy of sorption per mole of the adsorbate as it is transferred to the surface of the solid from infinite distance in the solution, and ϵ is Polanyi potential, which is shown as:−3 kJ/mol.°K. The value of mean sorption energy, E, can be calculated from D–R parameter β as follows [Where, T and R are the temperature (K) and the gas constant  was found. The plots indicate the applicability of Langmuir model in the present study. The values of monolayer capacity (qm) and Langmuir constant (b) are given in Table m calculated by the Langmuir isotherm were all close to experimental values at the given temperature. These facts suggest that arsenite is sorbed in the form of monolayer coverage on the surface of the adsorbent.Figure The RL value obtained lie between 0 and 1 confirming that the adsorption isotherm is favorable.e was plotted against lnCe; a straight line with slope 1/n and intercept ln KF was obtained. Figure 2) value, it was demonstrated that the removal of arsenite ions using the nano γ–alumina fairly obeyed the Freundlich isotherm.For achieving the Freundlich constant, lnqm, that is an indicator of the sorption capacity supports the previous notations that are discussed in the Langmuir isotherm for the sorption capacities of the studied ions removed using the synthesized nano γ–alumina.The values obtained for the Freundlich, Langmuir and D-R variables for arsenite ions removal are given in Table e versus ϵ2. The D–R plot yields a straight line with R2 value of 0.974, indicating that the D–R model is less fitting to the experimental data as comparable to the Langmuir isotherm model but is better than Freundlich isotherm model.To describe the sorption isotherms of single solute systems, D–R isotherm is commonly used. The D–R isotherm, apart from being analog of Langmuir isotherm, is more general than Langmuir isotherm as it rejects the homogeneous surface or constant adsorption potential . Figure m), sorption energy constants (β) and the main adsorption energies (E) are listed in Table −1 suggesting the physical nature of the adsorption process of arsenite on synthesized nano γ–alumina. So It is important to note that the suitability of all the three isotherm models to the studied sorption systems shows that monolayer sorption (Langmuir and D-R isotherms) of active sites on the surface of the synthesized nano γ–alumina in comparison to heterogeneous energetic distribution (Freundlich isotherm) is more significant [From the plotted D–R isotherm figure, the model parameters; sorption capacities  is the rate constant of pseudo-second order adsorption and often depends on the applied operating conditions, such as, pH of solution, initial metal concentration, temperature and agitation rate, etc. [qt against t, the initial adsorption rate, h, adsorption capacity (qe) and the pseudo-second order rate coefficient (K2) was determined and are shown in Table Where te, etc. ,39. FromThe initial adsorption rate, h, of a second order process as t→0 can be shown as:The values obtained by pseudo-second-order model Figure  were fouIt has been reported that the maximum arsenite adsorption capacity of Iron hydroxide coated alumina, activated alumina grains and manganese oxide-coated alumina are 9, 3.48 and 42.48 mg/g, respectively ,41, whicIn comparison, it is clear that maximum arsenite adsorption capacity of synthesized nano γ–alumina is bigger than Iron hydroxide coated alumina and activated alumina grains, and it is a little smaller than manganese oxide-coated alumina adsorbent.Although the adsorption capacity of arsenite onto synthesized nano γ–alumina adsorbent is lower than that of manganese oxide-coated alumina, but the main advantages of synthesized nano γ–alumina adsorbent are the availability of the materials, the substantially low cost, and its economic feasibility. This suggests that the adsorption property of synthesized nano γ–alumina gives the material great potential for applications in arsenite removal from aqueous solutions.2/g) and its application for arsenite removal without pre-oxidation of arsenite is described in this study.FT-IR analyses reveal that the arsenite cannot be adsorbed onto synthesized nano γ–alumina chemically. Besides, the results show that arsenite ions are physically adsorbed on the surface of synthesized nano γ–alumina. About 97.65% of arsenite removal is achieved within 30 min from the samples containing initial arsenite concentration up to 10 mg/L at pH =7.5. It is also revealed that the experimental results of adsorption isotherms are well fitted with the Langmuir and D-R models and the maximum adsorption capacity was 40 mg/g. The adsorption rate of arsenite is fast and equilibrium time is around 15 min. The pseudo-second-order model is suitable for all initial arsenite concentrations (10 and 20 mg/L), suggesting that the adsorption of arsenite onto synthesized nano γ–alumina follows pseudo-second-order kinetics.A simple method for producing nano γ–alumina from impure kaolin with spherical shape, with a particle size distribution ranging from 22 to 36 nm with a relatively high surface area (201.53 mThe authors declare that they have no competing interests.All authors read and approved the final manuscript.


Ritualized fighting between conspecifics is an inherently dangerous behavioral strategy, optimized to secure limited resources at minimal cost and risk. To be adaptive, potential rewards, and costs of aggression must be assessed to decide when it would be more opportune to fight or flee. We summarize insights into the proximate mechanisms underlying this decision-making process in field crickets. As in other animals, cricket aggression is enhanced dramatically by motor activity, winning, and the possession of resources. Pharmacological manipulations provide evidence that these cases of experience dependent enhancement of aggression are each mediated by octopamine, the invertebrate counterpart to adrenaline/noradrenaline. The data suggest that both physical exertion and rewarding aspects of experiences can activate the octopaminergic system, which increases the propensity to fight. Octopamine thus represents the motivational component of aggression in insects. For the decision to flee, animals are thought to assess information from agonistic signals exchanged during fighting. Cricket fights conform to the cumulative assessment model, in that they persist in fighting until the sum of their opponent’s actions accumulates to some threshold at which they withdraw. We discuss evidence that serotonin, nitric oxide, and some neuropeptides may promote an insect’s tendency to flee. We propose that the decision to fight or flee in crickets is controlled simply by relative behavioral thresholds. Rewarding experiences increase the propensity to fight to a level determined by the modulatory action of octopamine. The animal will then flee only when the accumulated sum of the opponent’s actions surpasses this level; serotonin and nitric oxide may be involved in this process. This concept is in line with the roles proposed for noradrenaline, serotonin, and nitric oxide in mammals and suggests that basic mechanisms of aggressive modulation may be conserved in phylogeny. There are many forms of aggression but no uniform definition . Crickets possess a conveniently sized and comparatively simple, segmentally organized nervous system, and above all have a rich and robust behavioral repertoire , winning, and the possession of key resources such as food, mates, and shelter  are mediated in each case by octopamine. First, the effect of flying can be mimicked by activating the octopaminergic system with CDM, but it is no longer evident in octopamine/dopamine depleted crickets, and it is also selectively blocked by octopamine receptor antagonists , the total sum of own actions (Energetic War of Attrition Model), or the total sum of opponent actions , at least partly by influencing serotonergic signaling (Nelson and Trainor, We propose that the decision to fight or flee could be accounted for in crickets by simply modulating the initiation thresholds for these two opposing behaviors relative to each other Figure . As arguThe brains of insects may be comparatively simple in terms of neuron number, they nonetheless have the integrative power to sculpture social interactions of a complexity approaching that of our own (Simpson and Stevenson, It has been shown that the tendency to fight in insects is promoted by the amine octopamine, the analog of noradrenaline. Rather than acting as a releaser of aggression, or simple “arousal” agent, octopamine appears rather to function as a selective neuromodulator that mediates the aggression promoting effect of experiences, including physical exertion, winning, and the possession of resources. In this respect octopamine represents the motivational component of aggression that drives the tendency of a cricket to fight. In correspondence with the envisaged role of dopamine on aggression in mammals (reviews: Barron et al., per se. Future studies must now be directed toward discovering how information from an opponent’s agonistic signals are summated in the nervous system and how this could promote the drive to withdraw. The first step must be to evaluate the extent to which serotonin, nitric oxide, and possibly peptides are involved in this process. At present we also do not know how the decision to flee is influenced by the energetic costs of physical fighting, which is high in crickets (Hack, The decision to flee, on the other hand, appears to be controlled in line with predictions of the cumulative assessment hypothesis Payne,  in that ts Hack, . In hermts Hack, . In cricIn conclusion, we propose that the decision to fight or flee in crickets is controlled by the action of separate neuromodulator system that set the relative behavioral thresholds for these opposing behaviors. A simple threshold mechanism also has the power to control sophisticated collective decision-making in eusocial insects (Robinson et al., While some progress has been made in elucidating the nervous centers and neuroanatomical pathways underlying aggression in rodents and non-human primates (review: Nelson and Trainor, 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.


Monte Carlo simulations reveal that this efficient AR results from charge carrier congregation in ‘Auger hot spots’: lower-energy sites that are present because of energy disorder. Disorder-enhanced AR is a general effect that is expected to be active in all disordered materials. The observed efficient AR is an issue of concern for devices that work at charge carrier densities in excess of ~10−3 charge carriers per quantum dot. At the same time, efficient carrier congregation could be exploited for fast optical switching or to achieve optical gain in the near infrared.Auger recombination (AR) can be an important loss mechanism for optoelectronic devices, but it is typically not very efficient at low excitation densities. Here we show that in conductive quantum-dot solids, AR is the dominant charge carrier decay path even at excitation densities as low as 10 Understanding Auger recombination is important for building better optoelectronics, as it is a crucial loss mechanism. Gao et al. study it in conductive quantum dot solids, and find that it is the dominant charge carrier decay path, even for very low excitation densities. Auger recombination (AR) is a process in which an electron recombines with a hole while transferring the electron–hole pair energy to a third charge carrier. It is well known to occur in (inorganic) semiconductor materials12458910−3 per QD. This decay is attributed to diffusion-mediated AR. We compare QD films with ligands of different lengths, and concomitantly differing charge carrier mobilities, and demonstrate that AR becomes faster as the mobility increases. From Monte Carlo simulations, we find that this efficient AR stems from charge carrier congregation in low-energy sites that are present because of energy disorder, which we term ‘Auger hot spots’. Disorder-enhanced AR is a general effect that is expected to be active in all disordered materials. The observed efficient AR is an issue of concern for devices that work at charge carrier densities in excess of ~10−3 charge carriers per QD, such as QD light-emitting diodes (LEDs) or solar cells under concentrated sunlight. Fast carrier congregation in low-energy sites could possibly be exploited for fast optical switching or to achieve optical gain in the near infrared (NIR).Here we present photoconductivity and transient absorption (TA) studies on QD solids over a wide range of excitation densities and observe higher-order decay at excitation densities as low as 10PbSe QD solids were prepared using a layer-by-layer procedure with 1,2-ethanediamine (EDA) or 1,4-benzenediamine (BDA) as the capping ligands to substitute for the much longer original oleylamine ligands. This results in optically smooth films with a mirror-like appearance. Their absorption spectra are shown in ΦmaxΣμ of the yield of charge carrier generation Φmax  and the sum of the electron and hole mobilities Σμ as a function of the average number of photons absorbed per QD, <Nabs>. <Nabs> is obtained as I0σ, where I0 is the photon fluence in the excitation pulse and σ is the absorption cross-section, which is taken from ref. 815Nabs> is also approximately a factor 2.The photoconductance of the QD solids was studied with the time-resolved microwave conductance (TRMC) technique12ΦmaxΣμ decreases with increasing laser intensity, whereas at low excitation density ΦmaxΣμ is independent of <Nabs>. For higher <Nabs>, the magnitude of Φmax decreases because of a higher-order charge decay process that takes place within the 3-ns excitation pulse and reduces the maximum photoconductance. The charge carrier mobilities are obtained at the lower limit of excitation density, where Φmax is not influenced by higher-order recombination. For the EDA QD solid, we have previously shown that all excitons rapidly dissociate into mobile charge carriers and that (below <Nabs>=10−3) Φmax is ~1  or two holes and an electron (ehh). Therefore, it is surprising at first sight that AR in QD solids is already active at <Nabs>=0.001. One possible explanation could be that charge trapping results in long-lived charges that survive longer than the time between two pump pulses, which is 2 ms for these experiments. This could result in a built-up of charge density giving rise to AR.We attribute the higher-order decay observed here to AR. In QD dispersions, AR only occurs if the number of excitons per QD exceeds one, as the AR process requires at least a trion: two electrons and a hole −1 for the BDA QD solid . Therefore, diffusion of charge carriers could result in AR on a picosecond timescale. Charge carrier diffusion-mediated AR can also explain why the decay is faster for EDA ligands than for BDA ligands, as the charge carrier mobility is significantly higher for the EDA QD solid.Although at low excitation density Auger reactive species are not photogenerated directly, they could form via charge carrier diffusion, as illustrated in Nabs>=0.15 is only 25 ps. Hence, it is unlikely that trions are responsible for the observed decay.The first Auger reactive species that can be formed via diffusion is a trion. As a starting point, we could assume that AR is due to trions only. The expected trion lifetime for this size of PbSe QDs is 70 psThe scaling of the AR rate with the number of charge carriers has been investigated for QDs in dispersion28ne and nh are the number of electrons and holes in a QD, respectively, ne,nh)→ and ktr is the trion AR rate. For an experimentally determined biexciton decay rate of (17.5 ps)−1  decay rate of (1.5 ps)−1. Such highly charged QDs could in principle explain the observed fast decay, if they would form.where )−1 ref. , equationtot, containing both electrons and holes, is equal to 2<Nabs>. The probability that three charges occupy the same QD is then given by P3=ntot3exp(−ntot)/3!, which equals 0.3% at <Nabs>=0.15. Of these QDs with three charges, only half carry trions, the other half carry three electrons or three holes.) Therefore, no significant AR is expected.At an excitation density of 0.15 excitations per QD, the probability of directly photoexciting QDs multiple times is negligible. Ultrafast dissociation of excitons and diffusion of charge carriers may generate charged QDs with various combinations of electrons and holes. If these charge carriers reach equilibrium and are distributed with equal probability over all QDs in the film, this distribution would again be Poissonian. In this case, the fraction of QDs that contain a trion is only 0.15%.  in site j, g is the 1S level degeneracy  is included to take into account that charge carriers can only hop into empty levels. Charge carriers residing within the same QD decay via AR with decay rates following equation where d is a uniform distribution between 0 and typically 1.0 nm.Electron–hole pairs are initially distributed on the lattice according to a Poisson distribution equation  to simul2 V−1 s−1. The trion lifetime matches the expected lifetime of 70 psUsing our Monte Carlo simulations, we can reproduce the experimental data, as shown in n1,n2) represents the charge carrier combination of n1 electrons and n2 holes or vice versa. We distinguish groups of charge carriers consisting of only electrons or holes , excitons  and other combinations of electrons and holes , with red, grey and blue colours, respectively. The fractions of AR-reactive charge carriers are also shown in histograms termed multiple carrier distributions (MCDs) for several delay times, In Nabs>=0.0012, is shown in i) increases up to ~25%. On a longer timescale of ~30 ps the fraction of free charges increases further to ~80%.At zero time delay, (multiple) excitons are formed following a Poisson distribution. As a result of the high hopping rate, these excitons rapidly dissociate and the charges redistribute over the various carrier combinations. First, we focus on the situation where the excitation density is very low and little AR occurs. This situation, with <2 V−1 s−1 carrier mobility used in these simulations corresponds to a (470-fs)−1 hopping rate. However, this is the hopping rate after thermalization of the charge carriers. From our simulations, we determine that the average hopping rate is 30 times higher before thermalization. Therefore, it can be understood that the simulated initial exciton dissociation occurs on a <50-fs timescale. Further exciton dissociation takes longer as the remaining excitons have a lower energy. This implies that exciton dissociation will be slowed down by both disorder and electron–hole attraction. Even so, exciton dissociation is rapid and nearly complete in 30 ps. We remark that, in our simulations, electrons and holes do not form strongly bound excitons but are in dynamic equilibrium with single charges. The probability of forming an exciton from free charges depends on the square of the charge density, and therefore the fraction of free charges is a function of charge density.The initial, rapid exciton dissociation can be understood by considering that as a result of energy disorder, excitons on high-energy sites may dissociate into charges at low-energy sites without significant increase in energy. A similar explanation was recently given for the dissociation of charge transfer complexes in disordered organic heterointerfacesNabs>=0.01, for the EDA-treated PbSe QD solid investigated here. These carrier densities are high compared with the charge carrier densities in QD solar cells and LEDs under operational conditions. The charge density in a QD solar cell with a charge carrier mobility of 1 cm2 V−1 s−1 can be estimated to be ~10−6 charges per QD, whereas the residence time of charges in the solar cell is ~5 ns , with a high modulation and a fast return to the ground stateh1Se absorption bleach of the smallest QDs exhibits a fast modulation. As charges diffuse from the small to the big QDs, the signal at 1.5 μm exhibits an exponential decay with a time constant of 1.3 ps 2]2 dissolved in 2 ml of trioctylphosphine and 6 ml of 1 M Se in trioctylphosphine were mixed in a syringe and immediately injected into the PbCl2 oleylamine solution. The reaction mixture was kept at the temperature after the injection for 2 min and cooled with a water bath. The resulting solution was filtered with a 200-nm pore size filter to remove residual PbCl2. The QDs were precipitated with butanol and finally dried under vacuum. The QDs were dispersed in tetrachloroethylene for absorption measurements and in hexane for dip coating.PbSe QDs were synthesized following ref. Quartz substrates were silanized with (3-aminopropyl) triethoxysilaneOptical absorption spectra were recorded with a Perkin–Elmer Lambda 900 spectrometer equipped with an integrating sphere. The spectra were corrected for scattering and reflection.I0 was varied between 1 × 1011 and 1 × 1014 photons per cm2 per pulse. Upon photoexcitation, the change in microwave power reflected was measured. For small photo-induced changes in the real conductance of the sample, ΔG(t), and negligible change in imaginary conductance, the relative change in microwave power can be written as ΔP(t)/P=−KΔG(t) , where K is a sensitivity factor which has been determined previously. The photoconductance ΔG(t) can be expressed as ΔG(t)=eβI0FaΦ(t)Σμ, where e is the elementary charge, β is the ratio between the broad and narrow inner dimensions of the waveguide, I0 is the photon fluence in the laser pulse, Fa is the fraction of light absorbed by the sample, Φ(t) is the number of mobile charge carriers at time t per absorbed photon and Σμ is the sum of the electron and hole mobilities. The time resolution of the TRMC set-up is limited by the 3-ns laser pulse.The photoconductance of QD solids was investigated using the TRMC technique1213The samples were excited and monitored by pump and probe pulses from a chirped-pulse amplified laser system , running at 1 kHz and delivering pulses of 60 fs, 2.2 mJ and at a wavelength of 795 nm. Tunable infrared and visible pulses (<100 fs) were generated by optical parametric amplification . Pump and probe beams were imaged onto InGaAs pin photodiodes . The two beams were spatially separated downstream of the sample. Orthogonal polarization of the beams allowed further separation by means of a polarizer.Y.G. and C.S.S.S. synthesized the quantum dots, prepared the QD films and performed TA experiments. Y.G. performed the TRMC experiments and the Monte Carlo simulations; A.J.H. and Y.G. designed the experiment and wrote the paper; Y.G., A.J.H., J.M.S. and L.D.A.S. analysed the data.How to cite this article: Gao, Y. et al. Disorder strongly enhances Auger recombination in conductive quantum dot solids. Nat. Commun. 4:2329 doi: 10.1038/ncomms3329 (2013).Supplementary Figures S1-S10, Supplementary Tables S1-S2, Supplementary Notes 1-3, Supplementary Methods and Supplementary References


Allergic rhinitis (AR) is a common problem and we sought to examine the burden of disease and its management in Canada from the perspectives of patients and physicians.Two parallel, Canadawide structured telephone interviews surveyed 1,001 AR patients and 160 physicians in July 2006.44% of patients had experienced nasal symptoms unrelated to a cold and 20% had a physician diagnosis of AR. At screening 27% reported asthma, 15% chronic or recurrent sinusitis and 5% nasal polyps. With attacks nasal congestion and runny nose were the most bothersome symptoms. Other problems experienced were fatigue (46%), poor concentration (32%), and reduced productivity (23%). Most (77%) had not seen a physician in the past year. Physicians estimated they prescribed intranasal cortico steroids (INCS) to most AR patients (77%) consistent with guidelines but only 19% of patients had used one in the last month. Only 48% of patients were very satisfied with their current INCS. 41% of AR patients reported discontinuing their INCS with the most common reason being a perceived lack of long-lasting symptom relief (44%). 52% of patients felt that their current INCS lost effectiveness over 24 h. The most common INCS side effects included dripping down the throat, bad taste, and dryness. Most AR patients reported lifestyle limitations despite treatment (66%). 61% of patients felt that their symptoms were only somewhat controlled or poorly/not controlled during their worst month in the past year.AR symptoms are common and many patients experience inadequate control. Physicians report they commonly prescribe intranasal corticosteroids, but patient’s perceived loss of efficacy and side effects lead to their discontinuation. Persistent relief of allergic rhinitis symptoms remains a major unmet need. Better treatments and education are required. Allergic rhinitis (AR) is an inflammatory disease of the nasal mucous membranes ,2. AllerAllergen avoidance and pharmacotherapy are the cornerstones of AR management ,7. PharmAllergies in Canada, a study consisting of two parallel, nationwide surveys of 3,671 adults with further questioning of 1,001 adult nasal allergy sufferers as well as 160 physicians in Canada. The objectives of the Allergies in Canada survey were to examine the breadth of symptoms, disease burden, and current management of AR in Canada from the perspective of physicians and patients.The perspectives of patients and physicians on the burden of disease for AR and its management have been described for the United States -13. HoweA cross-sectional sample of adults patients in Canada was obtained through random-digit dialing, telephone screening of households nationwide. Cooperative individuals were eligible to participate in the patient survey if they were ≥18 years old. In screening, a random subset of cooperative individuals ≥18 years old were asked if they had other common upper and lower respiratory problems including asthma, sinusitis and nasal polyps since the Canadian prevalence of these conditions is unknown.Those cooperative individuals ≥18 years old and suffering from AR as defined by:· Receipt of a physician diagnosis of AR, nasal allergies, or “hay fever”OR· Use of prescription or over-the-counter medications to treat self-diagnosed nasal congestion or symptoms (unrelated to cold or flu).A representative sample of Canadian physicians who treat AR was obtained by random screening of a Canadian physician database comprised of active clinicians who have experience treating with INCS.The goal was to obtain a sample of 1,000 patients. The maximum expected sampling error for a simple random sample of 1,001 (the patient sample) is ±1.9 percentage points at the 95% confidence level. The maximum expected sampling error for a simple random sample of 100  is ±4.8 percentage points at the 95% confidence level.Allergies in America study [Previous patient and physician questionnaires used in the ca study ,14 were Patient-reported data corresponds to the actual percentages of patients providing a particular response. Physician-reported data on the characteristics of their own AR patient population corresponds to the averages of all physician responses.An analysis was performed of symptom control in the worst month in the subgroups of patients using over-the-counter (OTC) vs prescription medication.LiveContact, the research provider for this study, guaranteed privacy and confidentiality for all study respondents and conducted the survey and data collection according to Canadian privacy legislation (PIPEDA) and marketing research guidelines established by the Marketing Research and Intelligence Association (MRIA) in Canada. Based on the anonymous nature of the survey, the lack of intervention and the implied consent by completing the survey by the subjects our research ethics board at the time felt this was a quality assurance project and deemed it did not require formal submission.30,987 telephone calls to households were made. Among 5,348 respondents to the initial telephone contact, there were 3,671 respondents who were ≥18 years old and cooperative . Of these, 44%  had experienced nasal symptoms and 20%  had been formally diagnosed with AR, nasal allergies, or hay fever by a physician. Among the subset of cooperative respondents asked further about specific comorbid conditions, 27% reported asthma, 17% reported having been diagnosed with chronic or recurrent sinusitis  and 5% had nasal polyps .1,001 respondents reported that they had used prescription or OTC medication to treat nasal symptoms OR had a diagnosis of AR and were willing to proceed with the remainder of the survey. These 1,001 individuals affected with AR completed the survey questionnaire. Unless otherwise specified, this cohort of 1,001 composes the “patients” whose perspectives are described in this report . The next most common symptoms reported to be extremely or moderately bothersome were runny nose (52%) and sneezing (47%)  while otolaryngologists (25%) and general practice/family medicine physicians (29%) felt that the percentage was less.AR symptom control was felt to be suboptimal by patients. 61% of patients felt that their symptoms were only somewhat controlled or poorly/not controlled during their worst month in the past year. Among those patients, 60% reported that their symptoms were only somewhat controlled or poorly/not controlled in the past month  and headache (37%). Poor concentration and reduced productivity are also common troublesome problems  and 8% of general practice/family practice physicians . Most physicians (73%) rarely or never use blood tests , 22% had been treated with immunotherapy at some point in time. Overall, physicians estimated 15% of their patients  were currently or ever had received immunotherapy.Among patients who had seen a physician for their nasal allergies, 58% reported that their physician had demonstrated how to use a nasal spray device. In comparison, when physicians were asked 90% of allergists, 87% of otolaryngologists, and 75% of general practice/family medicine physicians estimated that they demonstrate INCS spray technique once a year or when they prescribe a product.When symptom control in the worst month was compared between the subgroups of patients using OTC vs prescription medication, there was no apparent difference between subgroups. The use of prescription medication such as INCS was not associated with higher rates of completely or well controlled symptoms in the worst month.Among patients using INCS to control AR, only 48% reported being very satisfied with their current INCS, whereas 12% reported being dissatisfied , bad taste (32%), and dryness (31%) as shown in Figure Patients’ and physicians’ expectations for onset of action, duration of effect, and overall effectiveness of INCS were markedly different, with patients generally expecting a faster onset of action and greater degree of symptom relief  , the CanAllergies in Canada study included the largest patient and physician surveys of AR and common comorbid conditions conducted to date in Canada. These data confirm and extend findings reported for AR patient populations in other countries with regard to burden of disease [The  disease .As with all telephone surveys, certain limitations in interpretation exist. Surveys in general may be associated with bias in that the survey sample may not always be representative of the population. Certain subsets of individuals may be more likely to be unavailable to participate in a telephone survey. The criteria for AR patients in this study allowed for inclusion of AR sufferers who had not received a physician diagnosis of AR, provided they had symptoms consistent with AR that they self-treated with medication. Patients were not subjected to physical examination or skin tests. This may have permitted inclusion of some patients who did not have AR, for example, it is possible that some patients with nonallergic rhinitis could have been included. If the sample size of this study had been larger, more inferences might have been drawn regarding the impact of demographic factors on AR experiences and attitudes. The surveys were conducted in July 2006 and some currently available AR medications  were not available at that time.In this study, the prevalence of physician-diagnosed AR in Canadian adults in the general population and in the patient population seen in general practice/family practice paralleled the 20–25% prevalence of AR previously reported . The preNasal congestion and runny nose were identified as the most bothersome symptoms of AR and were reported to occur frequently. This parallels findings of a recent survey of adult AR patients in the United States . SimilarMost patients reported an adverse impact of nasal allergy symptoms on their daily life, a problem not always appreciated by physicians and patients. Major problems due to allergy symptoms cited by patients included fatigue, headache, poor concentration, and reduced productivity. These findings are consistent with the high rates of sleep disorders, daytime fatigue, memory impairment, and reduced work productivity in AR patients described in previous studies -20. OtheThis study provided a comprehensive description of current management of AR in Canada. Results show that more than one-third (37%) of nasal allergy symptom sufferers who had sought medical care for their symptoms had not received a physician diagnosis of AR. It was not determined whether these patients received alternative diagnoses  or went undiagnosed. However, the prevalence of nonallergic, noninfectious rhinitis in the general population is estimated to be low (2-4%) compared with AR (approximately 20%);therefore, this would not be expected to contribute substantially to the observed lack of AR diagnosis ,15. The Approximately half of patients surveyed were treating AR with OTC medication only and 36% were using prescription medication. The prescription medication usage rate parallels that seen in a recent study of seasonal AR in Canada in which 30% of subjects reported currently using prescription medication, specifically INCS, to control AR . Among pOnly a small subset (15%) of patients were estimated by physicians to be receiving allergy shots, consistent with guidelines that recommend immunotherapy be reserved for more severe disease and where symptoms are inadequately managed by maximum pharmacologic therapy ,8. AlthoGuidelines recommend that patients be instructed in the use of intranasal corticosteroid sprays to minimize the occurrence of side effects ,15. DemoPRN (as needed), regardless of the prescribed dosing regimen.Patients are not completely satisfied with INCS. Lack of long-lasting symptom relief is a concern and the most common reason for treatment discontinuation. Poor tolerability is the second most common reason for treatment discontinuation. Consistent with the findings of other AR studies, dripping down the throat, bad taste, and dryness are among the most commonly reported side effects . DrowsinMost patients and physicians perceived that AR causes lifestyle limitations even when properly treated. More than twice as many patients (38%) as physicians (17%) do not believe that truly effective AR treatment exists. Patients’ lack of confidence in the existence of effective treatment is perhaps attributable to their own experiences. Most patients (61%) reported that their symptoms were only somewhat controlled, poorly controlled, or not controlled during the worst month in the past year and few patients (23%) reported seeing a physician for AR management in the past year.The gaps identified between patients and physicians with respect to expectations for treatment and treatment outcomes suggest areas that might benefit from better communication. When initiating treatment, patients should be instructed on what to expect regarding a response. Patients should be encouraged to report any suboptimal treatment response so that appropriate intervention can be made.Comorbid conditions are often overlooked yet contribute significantly to the burden of AR. Asthma, sinusitis, nasal polyposis, and sleep apnea were reported in a significant number of patients in this study.) The contribution of rhinitis whether allergic or nonallergic to the development of asthma, sinusitis, and sleep apnea has been documented -25. In aUnprompted awareness of AR management guidelines was relatively low among physicians, particularly among those in general practice/family medicine, suggesting a need for more effective guidelines dissemination. Treatment consistent with expert consensus guidelines has been shown to result in significantly better patient outcomes than nonstandardized treatment . Better Allergies in America survey questionnaires [Allergies in Canada survey questionnaires. Content validity of the survey questionnaires was determined by a panel of Canadian AR experts, however, the questionnaires were not fully validated. The survey questionnaires were generally consistent with those used to study particular aspects of AR  in accepted health and rhinitis surveys [It is hoped that the questionnaires used in this study provide the basis for development of validated, standardized, comprehensive AR questionnaires. Validated, standardized, comprehensive AR questionnaires did not exist before the development of the onnaires , which f surveys -30.Many AR patients in Canada experience symptoms that could be better controlled. AR has a significant negative impact on patients’ activities of daily living. Evidence suggests that a substantial number of AR sufferers did not receive medical care for their condition in the past year and/or have not been diagnosed with their condition. Although INCS are the most commonly prescribed AR treatment, less than half of patients are fully satisfied with their INCS. The majority of patients perceive that INCS lose effectiveness over a 24-hour period. The most common reasons for patients to discontinue treatment relate to lack of long-lasting symptom relief rather than side effects. Asthma, sinusitis, nasal polyposis, and sleep apnea are common comorbid conditions and represent an often unrecognized portion of the total burden of disease. Major unmet needs of physicians and patients in AR management include the needs for better therapies and better education.AR: Allergic rhinitis; INCS: Intranasal corticosteroids; OTC: Over-the-counter; MRIA: Marketing Research and Intelligence Association; CSACI: Canadian Society of Allergy and Clinical Immunology; ARIA: Allergic Rhinitis and its Impact on Asthma Workshop Group; AAAAI: American Academy of Allergy Asthma and Immunology.Keith: Consultant/Advisory Board for: GlaxoSmithKline, Merck Frosst, Nycomed, Talecris, CSL Behring. Grant/Research support from: GlaxoSmithKline, Nycomed, Allergy Therapeutics, Merck Frosst. Honoraria for lectures from: GlaxoSmithKline, Astra, Merck Frosst, Nycomed, CSL Behring. Waserman: GlaxoSmithKline, Astra Zeneca, Merck Frosst, Nycomed, King Pharma, Paladin Labs, Novartis. Desrosiers: Speakers bureau, advisory boards, consultant: Aventis, GlaxoSmithKline, Schering-Plough, MedtronicXomed, Bayer. Research funding: Fondation Antoine Turmel: Philanthropic organisation. Laister: Currently employed at Nycomed: A Takeda Company in the position of Manager, Medical Liaison-Ontario. Schellenberg: Speakers bureau, advisory boards, consultant: GlaxoSmithKline, Merck Frosst, Novartis, Talecris, Bayer Biologics, and CSL Behring.All authors  made substantial contributions to the study design and development of the final surveys as well as the analysis and interpretation of the data and the drafting and revision of the manuscript. All authors read and approved the final manuscript.


Drosophila organs differ in their size response to developmental nutrition and this reflects differences in organ-specific insulin-sensitivity. We show that this variation in insulin-sensitivity is regulated at the level of the forkhead transcription factor FOXO, a negative growth regulator that is activated when nutrition and insulin signaling are low. Individual organs appear to attenuate growth suppression in response to low nutrition through an organ-specific reduction in FOXO expression, thereby reducing their nutritional plasticity. We show that FOXO expression is necessary to maintain organ-specific differences in nutritional-plasticity and insulin-sensitivity, while organ-autonomous changes in FOXO expression are sufficient to autonomously alter an organ's nutritional-plasticity and insulin-sensitivity. These data identify a gene (FOXO) that modulates a plastic response through variation in its expression. FOXO is recognized as a key player in the response of size, immunity, and longevity to changes in developmental nutrition, stress, and oxygen levels. FOXO may therefore act as a more general regulator of plasticity. These data indicate that the extent of phenotypic plasticity may be modified by changes in the expression of genes involved in signaling environmental information to developmental processes.Phenotypic plasticity, the ability for a single genotype to generate different phenotypes in response to environmental conditions, is biologically ubiquitous, and yet almost nothing is known of the developmental mechanisms that regulate the extent of a plastic response. In particular, it is unclear why some traits or individuals are highly sensitive to an environmental variable while other traits or individuals are less so. Here we elucidate the developmental mechanisms that regulate the expression of a particularly important form of phenotypic plasticity: the effect of developmental nutrition on organ size. In all animals, developmental nutrition is signaled to growing organs via the insulin-signaling pathway.  The ability of an organism to respond to its environment is a defining quality of life. However, why are some characteristics or individuals sensitive to environmental change while others are not? We identified the mechanism that controls the response of growing organs to a particularly important environmental factor—developmental nutrition. In all animals, a decrease in developmental nutrition reduces final body and organ size. However, the size of some organs is less responsive to changes in nutrition than others. In a male fruit fly, it is the size of the genitals that is resistant to dietary restriction. This is achieved by the male fruit fly reducing expression of a key gene in their genitalia. This gene, FOXO, forms part of the insulin signaling system, which signals food levels to tissues in all animals. By lowering the production of FOXO, the genitalia are able to “ignore” hormonal signals that tell the rest of the body to grow slowly due to limited food. The ability of tissues to become insensitive to nutritional information is a characteristic of many tumors and also underlies type 2 diabetes. Our data may therefore provide insight into the origin and treatment of both conditions. Why are some traits or individuals highly sensitive to an environmental variable while other traits or individuals are less sensitive?The ability of organisms to adjust their development., physiology or behavior in response to environmental conditions, called phenotypic plasticity, is a defining property of life. Phenotypic plasticity underlies such diverse phenomena as the relationship between childhood nutrition and adult size in humans nutritional plasticity. In animals as diverse as humans and flies, malnutrition during development reduces adult body size One of the most familiar and important examples of phenotypic plasticity is the response of body and organ size to changes in developmental nutrition, here referred to as Work over the last twenty years has identified the insulin/IGF-signaling (IIS) pathway as the major signaling pathway coordinating growth with nutritional conditions in all animals Drosophila melanogaster, to identify the mechanisms that regulate the degree of an organ's phenotypic plasticity with respect to developmental nutrition. In Drosophila, most morphological traits share the same nutritional plasticity as the body as whole. However, the male genitalia are remarkably resistant to changes in developmental nutrition – like the mammalian brain they are more or less the same size in large and small individuals FOXO. FOXO expression is necessary to maintain organ-specific differences in nutritional-plasticity and insulin-sensitivity, while organ-autonomous changes in FOXO expression are sufficient to autonomously alter an organ's nutritional-plasticity and insulin-sensitivity.Here we use the fruit fly, allometric coefficient to compare the nutritional plasticity of different organs within the Drosophila body. The allometric coefficient (b) is the slope of the linear scaling relationship between two traits plotted on a log-log scale; that is where log (trait 1 size) = b log (trait 2 size)+c. The coefficient gives the extent to which variation in the size of trait 1 is accompanied by variation in the size of trait 2. When size variation is due to variation in developmental nutrition, the allometric coefficient captures the nutritional plasticity of trait 1 relative to the nutritional plasticity of trait 2We used the Drosophila may therefore be a consequence of their relative insensitivity to change in insulin signaling. This could be because the developing genitalia are exposed to elevated levels of circulating insulin-like peptides (dILPs) even when nutrition is low. dILPS are released into the hemolymph from insulin-producing cells (IPCs) in the brain, although it is possible that their distribution is modified by localized production of dILPs Imp-L2The IIS pathway is the major regulator of size with respect to nutrition in all animals. The low nutritional plasticity of the genitalia in Inr (E19Inr) and its substrate chico (1chico) genocopy starvation and result in a more substantial reduction in the size of the wing and maxillary palp than the genitalia  and wild-type control clones generated in the imaginal discs of late first-instar larvae. While mutation of Inr decreased the rate of cell proliferation for clones in all the discs, the suppressive effect was significantly greater in the wing and eye-antennal discs than in the genital disc   . CollectInr in the IIS pathway. The logic for this deduction is as follows. Because nutritional-insensitivity of the genitalia appears to reflect a reduction in their organ-autonomous response to changes in Inr activity, the mechanisms that regulate this insulin-insensitivity should lie within the insulin-signaling pathway itself. These mechanisms modify systemic inputs into the insulin-signaling pathway into organ-specific outputs. One method to identify where in the IIS pathway this mechanism acts is to perturb the IIS pathway at different points and assay the size effect on the genitalia compared to other organs, in well fed larvae. If the perturbation acts upstream of the mechanisms that regulate insulin-sensitivity, the perturbation will have less of an effect on the size of the genitalia compared to other organs and genocopy starvation. Conversely, if the perturbation does not act upstream of the mechanisms that regulate insulin-sensitivity the perturbation will have the same effect on the size of the genitalia as other organs. Since mutation of Inr and chico had less of an effect on the size of the genitalia than other organs, it follows that the mechanisms that account for this reduced sensitivity lie downstream of Inr and chico on the IIS pathway.These data suggest that the mechanism that reduces the genitals' response to changes in IIS and account for their reduced nutritional plasticity act downstream of P{GawB}NP6333 (here referred to as NP6333). For negative regulators of IIS we perturbed signaling by driving UAS-mediated expression of the gene, again using NP6333.To determine where in the IIS pathway the mechanisms that regulate insulin-sensitivity act, we used a variety of genetic method to systematically perturb signaling at genes increasingly downstream in the IIS pathway . For eacPerturbation at Chico, phosphoinositide 3-kinase (PI3K) 92E, PTEN, TOR, raptor (a co-factor of TOR), S6 Kinase (S6K) and Akt all genocopied dietary restriction and had less of an effect on the size of the genitalia than on the wings . In contInr, chico and Akt mutants, loss of FOXO attenuates any decrease in size The next gene downstream of Akt in the canonical IIS pathway is the Forkhead Box O transcription factor (FOXO) . FOXO isFOXO in the imaginal discs using NP6333 genocopied dietary restriction, reducing the size of the adult wings and maxillary palps by ∼30% but only reducing the size of the genitalia by ∼15%  in the imaginal discs of well-fed larvae had the same effect on the genitalia, wing and maxillary palps, causing a ∼30% reduction in size  reporter construct luciferase gene under the transcriptional control of the herpes simplex minimal promoter and 8 direct repeats of the FOXO Response Element (FRE) To confirm this, we measured FOXO activity in the wing and genital imaginal discs of fed and starved third instar larvae using the 21/FOXO25FOXO) 21/FOXO25FOXO mutants produce no detectable protein FOXO-mutant rather than FOXO-null If variation among organs in their nutritional- and insulin-sensitivity is mediated by FOXO, then loss of FOXO should result in all organs showing the same level of nutritional- and insulin-sensitivity. To test this we examined the nutritional plasticity of the wings, palps and genital in flies mutant for FOXO   . Howevert clones . The resDrosophila have a limited response to changes in nutrition and IIS is because they are able to limit the transcriptional activity of FOXO when nutrition and IIS is low, effectively restricting the genitals' size-response to one that is independent of FOXO. Other organs only show this reduced sensitivity to changes in nutrition and IIS when mutant for FOXO.Collectively these data suggest that the reason the male genitalia of Akt null cells (1Akt) in the developing imaginal discs Akt had less of an effect on the rate of cell proliferation in the genital discs compared to the wing and the eye-antennal discs  to measure the expression of r organs .FOXO expression were indeed the mechanism by which the genitalia reduce their insulin-sensitivity and nutritional plasticity, then increasing expression of FOXO in the genitalia should increase their size response to changes in nutrition. Conversely, decreasing FOXO expression in the wings should reduce their size response to changes in nutrition.If reduced FOXO in the developing wing and genital imaginal discs and assayed the extent to which adult wing and genital size responded to changes in developmental nutrition, that is their nutritional plasticity. We used NP6333 to drive FOXO.wt and FOXO.RNAi expression, increasing and decreasing FOXO expression respectively . The effects of FOXO expression on plasticity were organ autonomous. NP6333 does not drive expression in the leg imaginal discs and the nutritional plasticity of the legs were unaffected by changes in FOXO expression in other imaginal discs , which resulted in increasingly elevated levels of FOXO expression in their wing discs , substantial increases in FOXO expression (>FOXO.wt at 25°C) reduced plasticity to a level below that observed when FOXO expression is down-regulated (>FOXO.RNAi at 20°C)  . These eC) . Such factors exist in mammals  plays a key role in limiting the response of the CNS to changes in developmental nutrition in Drosophila show reduced plasticity not only in response to developmental nutrition but also developmental temperature and density FOXO lies at the nexus of a number of other signaling pathways involved in size regulation FOXO expression is a common mechanism by which organs regulate their response to environmental factors that reduce size. Further, if genetic variation in size is a consequence of allelic variation in these different signaling pathways, then low levels of FOXO may also limit an organ's response to genetic factors that reduce size.Work over the last decade has established FOXO as a major regulator of longevity, diabetes, and organ and body size. Our study expands this role to include regulation of nutritional plasticity and insulin-sensitivity. However, FOXO may be a more general plasticity gene FOXO as a key regulator of morphological scaling in Drosophila. However, the importance of nutrition as a regulator of size in animals and the evolutionary conservation of the IIS suggests that FOXO may be a proximate target of selection on morphological scaling in animals in general.By altering an organ's nutritional plasticity we affected how that organ's size scaled with body size, as both varied with nutritional condition. The scaling relationship between organ and body size controls body proportion and defines the shape of an animal FOXO expression and nutritional plasticity means that ostensibly similar scaling relationships may be achieved either through increases or decreases in FOXO expression. In Drosophila, nutritional-insensitivity of the genitalia is achieved through a reduction in FOXO expression, with flies maintaining a near maximum genital size even in poorly-fed individuals. In the horned beetle, Onthophagus nigriventris, horn size in small males and females is also nutritionally-insensitive and is more-or-less constant across a range of body sizes FOXO expression were relatively high in the developing horns of small males and females. Indeed, this is supported by the finding that expression levels of Inr, a transcriptional target of FOXO, are elevated in these horns FOXO expression may prove to be a proximate target of selection on morphological scaling, its response to selection will depend on the nature of the selective pressure.The non-linear relationship between Plasticity is a fundamental biological process that ensures that individuals' morphology, behavior and physiology match their environment. An essential aspect of this process is how these pathways are modified to either amplify or attenuate the environmental signal to which an individual is responding, thereby modulating the extent of the plastic response. Understanding the mechanisms that regulate the extent of trait plasticity is important for two reasons:Foxo1 expression appears to be a positive regulator of insulin-sensitivity in mammalian kidney cells FOXO expression and nutritional- and insulin-sensitivity.First, an understanding of how phenotypic plasticity is regulated has important consequences for the study of diseases that result from changes in plasticity. One particularly relevant example is type 2 diabetes, characterized by a reduction in insulin-sensitivity. Interestingly, Second, phenotypic plasticity – and its inverse environmental canalization – are increasingly recognized as playing a central role in evolution. Numerous studies have demonstrated that trait plasticity varies within and between species  processes. The generality of this mechanism in regulating the extent of phenotypic plasticity, however, requires further investigation.P{GawB}NP6333 (113920) is expressed in the wing, eye-antennal, and genital imaginal discs and was acquired from the DGRC, Kyoto, Japan. UAS-Akt.RNAi (2902), UAS-PI3K.RNAi (38986 & 38986), UAS-Inr.RNAi (992 & 993), UAS-FOXO.RNAi (30556), and UAS-raptor.RNAi (13112) were from the VDRC . GC25Inr (9554), E19Inr (9646), UAS-GFP (5430) UAS-Inr.DN (8253), UAS-TOR.TED (7013), UAS-TOR.WT (7012) and FRT82B arm-lacZ (7369) were from the Bloomington stock center . l-1S6K was the kind gift of George Thomas. 1chico, 21FOXO and 25FOXO was the kind gift of Ernst Hafen. 1Akt was the kind gift of Hugo Stocker. UAS-PTEN was a kind gift of Bruce Edgar. UAS-FOXO.wt was the kind gift of Jamie Kramer. UAS-FOXO.wt (m3-1), UAS-FOXO.TM (f3-9) and UAS-FOXO.TM (m6-15) were the kind gift of Marc Tater. y,w,UAS-GFP; tub-GAL4, FRT82B, tub-GAL80 was the kind gift of Melissa Gilbert. P{GAL4}NP6333, UAS-FOXO.wt (Kramer), and UAS-GFP, were used to assay the affect of FOXO expression on morphological scaling, and were made coisogenic through backcrossing into a wild-type SAM background for 5 generations. FRE-Luc was the kind gift of Brian Staveley.The following flies were used in this study (stock numbers are in parentheses): The GAL4-driver smatrNP6333>FOXO.RNAi larvae were reared at 20°C, since larvae reared at higher temperatures did not eclose as adults, while NP6333>FOXO.wt larvae were reared at 17, 20, 23, 24 and 25°C, as a means to control the expression of FOXO.All scaling relationships were for isogenic flies where variation in size was due to variation in developmental nutrition NP6333>FOXO.RNAi (20°C), >FOXO.wt (23°C) and >FOXO.wt (25°C) flies were normalized for temperature using the wing-body scaling relationships of the control flies (NP6333>GFP) at 20, 23 and 25°C. We first transformed the data for experimental and control flies reared at 20 and 25°C so that the bivariate mean of wing and body size for the un-starved control flies was equal to that of the un-starved control flies reared at 23°C. We then used this bivariate mean as an anchor point around which we rotated the data for the 20 and 25°C experimental and control flies such that the slope of the scaling relationship for the controls flies was equal to that of the control flies reared at 23°C. In sum, these transformations resulted in a common control scaling relationship at all three temperatures, against which the experimental scaling relationships were plotted.For InrE19), RNAi (NP6333>UAS-Inr.RNAi) and by expressing a dominant negative of Inr (NP6333>UAS-Inr.DN); at Chico using mutation (1chico) and RNAi (NP6333>UAS-chico.RNAi); at PI3K using RNAi (NP6333>UAS-Pi3K.RNAi); at PTEN by over-expressing pten (NP6333>UAS-PTEN); at AKT using RNAi (NP6333>UAS-Akt.RNAi); at raptor using RNAi (NP6333>UAS-raptor.RNAi); at TOR by over-expressing Tor (NP6333>UAS-TOR.WT and NP6333>UAS-TOR.TED); at S6 Kinase by mutation (l-1S6K) and RNAi (NP6333>UAS-S6K.RNAi), and at FOXO by over-expressing wild-type and constitutively active FOXO (NP6333>UAS-FOXO.wt and NP6333>UAS-FOXO.TM respectively). All larvae were reared at low density on standard cornmeal/molasses medium at 25°C. Body parts were measured as described in The IIS pathway was perturbed at Inr using mutation , E19Inr, 1Akt, E19+FOXO21Inr, or 1+FOXO21Akt. Females were left for 2 h to clear retained eggs and then allowed to lay a 6 h cohort of larvae. Larvae were heat-shocked at 37°C for 1.5 h, 42 h after egg laying to generate mitotic clones. Clones were left to develop for ∼48 h before the wing, eye-antennal and genital imaginal discs from each larva were dissected and fixed. The timing of each dissection was recorded to calculate the precise age of clones within each larvae. Discs were dissected from eight to 10 larva for each genotype. The discs were imaged using standard methods and the number of cells within each clone was recorded. The number of clones per disc ranged from five to 30. The rate of cell proliferation for each clone was calculated as log(N)/t where N is the number of cells in each clones and t is the age of the clone. A mixed model analysis of variance (ANOVA) with disc type and genotype as fixed effect and larvae as random effect was used to estimate the mean rate of cell division for each genotype/disc type combination whilst controlling for variation in the rate of cell division among larvae. A subsequent Tukey HSD test was used to compare specific rates of cell division between specific genotype/disc-type combinations. We also estimated the size of cells within each clone by measuring their cross-sectional area at the surface of the disc. The data were again analyzed using a mixed model ANOVA to calculate mean cell size for each genotype/disc type combination. All analyses were conducted with JMP (SAS Institute).Clones were induced using the MARCM system and marked using GFP 28S rRNA. Primers for assaying FOXO expression levels were AGGCGCAGCCGATAGACGAATTTA (forward) and TGCTGTTGACCAGGTTCGTGTTGA (reverse). Primers for assaying 28S expression levels were TAACGAACGAGACTCAAATATAT (forward) and GAATGAAGGCTACATCCGC (reverse). Standard curves were generated using seven serial dilutions of total RNA extracted from 2× 1st instar larvae, 2× 2nd instar larvae, 2× 3rd instar larvae , 2× pupae  and 2× adult flies . The same methods was used to assay gene expression in the imaginal discs of NP6333>FOXO.wt, NP6333>FOXO.RNAi, and NP6333>GFP larvae. However, because these larvae were reared at different temperatures, wing imaginal discs were dissected at a specific developmental stage (wandering) rather than a specific larval age.qPCR was conducted on imaginal discs from male SAM wild-type third instar larvae reared at low density on standard cornmeal/molasses medium at 25°C and dissected 39 hours after ecdysis from the second to the third larval instar. Gene expression was assayed on four to five biological replicates, using a standard curve and normalized against expression of FRE-luc larvae were reared on standard cornmeal medium at 25°C and staged into 4 hour cohorts at ecdysis to the third larval instar. Larvae were then reared at 25°C for an additional 15 hours before being either starved for 24 hours or left to continue feeding. Larvae were then dissected in PBS and their wing and genital imaginal discs were stored in minimal PBS at −80°C. One hundred wing and 100 genital imaginal discs from both fed and starved larvae were homogenized in 50 µl of PBS with protease inhibitor (Roche) and then centrifuged at 13,0000 rpm for 5 minutes. We then tested 10 µl of the supernatant for lucifersase activity using the Promega Luciferase Assay System. We measured the protein concentration for each sample using a standard BCA assay and normalized the luciferase activity as activity per mg.Figure S1NP6333 drives expression equally in the wing and genital imaginal discs. Expression of GFP (assayed by qPCR) 39 hours after ecdysis from the second to the third larval instar is not significantly different in wing and genital discs of NP6333>GFP larvae .(TIF)Click here for additional data file.Figure S2FOXO attenuates the effects of loss of Akt on the rate cell proliferation in the eye-antennal and wing discs but not in the genital discs. Columns with the same letter are not significantly different . Error bars are 1 standard error.Mutation of (TIF)Click here for additional data file.Figure S3FOXO expression in the developing wing imaginal disc. Driving expression of FOXO.RNAi and FOXO.wt in the wing imaginal disc using NP6333 (gray bars), results in a significant decrease or increase in FOXO expression respectively, compared to wild-type controls (open bars) . Error bars are 1 standard error.Manipulating (TIF)Click here for additional data file.Figure S4FOXO expression in the wing imaginal discs have organ autonomous effects. (A) The scaling relationship between leg length and body size for wild-type (open diamonds) and NP6333>FOXO.wt (closed diamonds) flies reared at 23°C are not significantly different . (B) Final body size was the same for NP6333>FOXO.wt and N6333>GFP control flies reared at 23°C. Two-sample t-test, p = 0.175, N = 26 (>FOXO.wt), 28 (>GFP).Changes in (TIF)Click here for additional data file.Figure S5FOXO expression using the temperature dependence of GAL4 activity. Rearing NP6333>FOXO.wt larvae at higher temperatures results in a significant increase in FOXO expression in the wing imaginal discs .Altering (TIF)Click here for additional data file.Figure S6FOXO expression in wild-type Drosophila (A) In wild-type larvae, an increase in rearing temperature leads to a decrease in FOXO expression in the wing imaginal discs  (B) As temperature increases there is a significant trend towards decreased nutritional plasticity of the wing in wild-type flies (permutation test p = 0.033). The permutation procedure was to first transform the raw data so that the bivariate mean for the wing and body size at each temperature was zero. The transformed data for all rearing temperatures were pooled and re-sampled without replacement, to create four new permuted data sets, one for each temperature. The slope of the SMA for each of these datasets was then regressed against temperature, to generate a regression coefficient (Ab). This was repeated 1000 times to produce a distribution of regression coefficients under the null hypothesis that there is no relationship between plasticity and temperature. The position of the observed regression coefficient (Ob = −0.0105) was determined among the ordered coefficients (Ab) from the permuted datasets. The proportion of Ab less than or equal to Ob was used as the p-value under the null hypothesis.The relationship between temperature, wing nutritional plasticity and (TIF)Click here for additional data file.


AKR1C3 rs12529 and AR-CAG repeat length remained significantly associated with prostate cancer-specific mortality (PCSM) after ADT (P≤0.041). Furthermore, individuals carrying two unfavorable genotypes at these loci presented a 13.7-fold increased risk of PCSM compared with individuals carrying zero (P<0.001). Our results identify two candidate molecular markers in key genes of androgen and estrogen pathways associated with PCSM after ADT, establishing the role of pharmacogenomics in this therapy.Although most advanced prostate cancer patients respond to androgen-deprivation therapy (ADT), the efficacy is widely variable. We investigated whether the host genetic variations in sex hormone pathway genes are associated with the efficacy of ADT. A cohort of 645 patients with advanced prostate cancer treated with ADT was genotyped for 18 polymorphisms across 12 key genes involved in androgen and estrogen metabolism. We found that after adjusting for known risk factors in multivariate Cox regression models,  AR gene is amplified in about one third of cases AR mutations in CRPC allow the receptor to be activated by weak androgens, other steroid hormones, or drugs Since the sex hormone signaling pathways play an important role in prostate cancer development, androgen-deprivation therapy (ADT) is still the standard systemic treatment for advanced prostate cancer. The majority of patients treated with ADT, which suppress androgen production or androgen receptor (AR) activity, show clinical improvement. Unfortunately, many patients relapse with a more aggressive form of prostate cancer termed castration-resistant prostate cancer (CRPC). Several mechanisms have been proposed for explaining the development of CRPC. The CYP19A1, HSD3B1, HSD17B4, SLCO2B1, and SLCO1B3 on time to progression during ADT Based on the above findings, genetic variants in genes of sex hormone metabolic pathways have been investigated as candidates for prostate cancer risk in many association studies Six hundred and forty-five advanced prostate cancer patients were recruited between 1995 and 2009 from three medical centers in Taiwan: Kaohsiung Medical University Hospital, Kaohsiung Veterans General Hospital, and National Taiwan University Hospital, as previously described P>0.01). Ten percent of samples were blind duplicated for quality control and the genotype concordance was 100%.We selected 18 polymorphisms in 12 androgen and estrogen pathway genes with functional association with cancers according to the literature review. Genomic DNA was extracted from peripheral blood of patients and stored at −80°C until the time of study. Genotyping was performed by Sequenom iPLEX matrix-assisted laser desorption/ionization-time of flight mass spectrometry technology at the National Center for Genome Medicine, Academia Sinica, Taiwan. The average genotype call rate for these polymorphisms was 93.0% and each of the polymorphisms was in Hardy-Weinberg equilibrium , which uses recursive partitioning to identify subgroups of individuals with similar risk q values) were calculated to determine the degree to which the tests for association were prone to false-positives q values were estimated using the R q value package. Statistical Package for the Social Sciences software version 16.0.1  was used for other statistical analyses. A two-sided P value of ≤0.05 was considered statistically significant.Patient clinicopathologic characteristics were summarized as number and percentage of patients or median and interquartile range of values. The continuous factors were dichotomized at the median value within the cohort, with the exception of PSA nadir, which was dichotomized at 0.2 ng/mL because of its correlation with disease progression and PCSM P≤0.007). Age was only associated with ACM, and the PSA at ADT initiation was associated with PCSM and ACM.The study cohort consisted of 645 prostate cancer patients treated with ADT and the characteristics of patients were summarized in P value of ≤0.05 for association with time to progression, PCSM, and ACM respectively, according to the univariate log-rank test. Median AR-CAG repeat length was 22 , and there was an association with time to progression  when analyzed as quartile groups   . To asseAKR1C3 rs12529 and AR-CAG repeat length were associated with PCSM (P≤0.029), and had a q value of 0.232 (AKR1C3 rs12529 and AR-CAG repeat length remained significant predictors for PCSM in patients receiving ADT (P≤0.041). A significant combined genotype effect on PCSM was also observed, and the hazard ratios (HRs) for PCSM increased as the number of unfavorable genotypes increased  1.20–4.18, P for trend = 0.011, P<0.001) compared with individuals carrying zero. Since metastatic disease typically has a poor prognosis, a substratification of high-risk patients based on the metastasis status at diagnosis was performed. The combined genotypes particularly had significant effects on PCSM in patients with distant metastasis , and had a q value of 0.436 .of 0.436 . HoweverAKR1C3 rs12529, following by AR-CAG repeat length, and resulted in 3 terminal nodes with low-, medium-, and high-risk for PCSM (AKR1C3 rs12529 and AR-CAG repeat length <21), the HR was 1.77  for medium risk node 3, and 9.11  for high risk node 1. The time to PCSM decreased as the increase in risk classification .We further used survival tree analysis to explored higher order SNP-SNP interactions among the SNPs that were associated with PCSM. The tree structure was first split by for PCSM . When usAKR1C3 and CAG repeat in AR, retained their associations with PCSM after ADT while controlling for known prognostic factors, age at diagnosis, clinical stage, Gleason score, PSA level at ADT initiation, PSA nadir, and time to PSA nadir, suggesting that these host genetic factors add information above and beyond currently used predictors. Intriguingly, patients possessing a greater number of unfavorable alleles had a shorter survival following ADT.We have identified two genetic polymorphisms, rs12529 in AKR1C3 rs12529 has also been associated with lung and bladder cancer risk A critical step in the synthesis of AR ligands involves the conversion of androstenedione to testosterone, which is catalyzed by 17β-hydroxysteroid dehydrogenases type 3 (HSD17B3) and type 5, also called aldo-keto reductase (AKR) 1C3. HSD17B3 is the predominant enzyme in catalyzing testosterone formation in testis, but synthesis of active androgens proceeds via AKR1C3 in prostate AR-CAG repeat length on the outcomes of ADT, the results remain uncertain. Some studies showed that shorter CAG repeat length was correlated with better responses to hormonal therapy AR-CAG repeat lengths in peripheral blood samples, but the actual repeat lengths within the prostate tumors might play a more critical role in response to ADT. Finally, AR has recently been suggested to function as a tumor suppressor in epithelium to suppress prostate tumor invasion and metastasis AR amplifications, alteration of its coregulators rendering AR signaling sensitive to low concentrations of androgen, and AR mutations allowing the receptor to be reactivated by other steroids as well as by antiandrogens. Therefore, other factors that might influence the activity of AR, such as AR coregulators and AR mutations, should also be studied in conjunction of AR-CAG repeats to allow a more comprehensive analysis.AR plays a pivotal role in prostate cancer development and progression. The factors that modify the function of AR might influence the progression of tumor to a castration-resistant state during ADT. The N-terminal transcriptional activation domain of the AR protein contains a CAG repeat, highly polymorphic in length, that affects the transactivation function of AR. Prior studies have shown an inverse relationship between CAG repeat length and AR transcriptional activation ability AKR1C3 and AR that were associated with PCSM. Our cohort consisted of only Chinese Han population, and the results reported here are limited by multiple comparisons. Further work is necessary to characterize these polymorphisms and determine how to ultimately translate these findings into clinical practice.In conclusion, most prostate cancer patients will have an indolent form of disease, but aggressive prostate cancer is still the second leading cause of cancer deaths in men of the United States. New biomarkers to help distinguish between lethal and indolent prostate cancer are urgently needed. Of the 18 polymorphisms in the 12 sex hormone pathway genes, we identified two polymorphisms in Table S1P values of their association with time to progression, PCSM, ACM during ADT.Genotyped polymorphisms and the (DOC)Click here for additional data file.


PSA and TMPRSS2) was examined. Two novel patient-derived PCa xenograft models from high grade PCa specimens were established by implanting the specimens into nude mice and passing tumor pieces through subcutaneous injection in nude mice, and then treated with kava extract and flavokawain B to examine their effects on tumor growth, AR expression and serum PSA levels. The kava extract and flavokawain B effectively down-regulated the expression of both the full-length AR and AR splice variants. The kava extract and kavalactones accelerated AR protein degradation, while flavokawain B inhibited AR mRNA transcription via decreasing Sp1 expression and the binding of Sp1 to the AR promoter. The kava root extract and flavokawain B reduce tumor growth, AR expression in tumor tissues and levels of serum PSA in the patient-derived PCa xenograft models. These results suggest a potential usefulness of a safe kava product or its active components for prevention and treatment of advanced PCa by targeting AR.Men living in Fiji and drinking kava have low incidence of prostate cancer (PCa). However, the PCa incidence among Fijian men who had migrated to Australia, increased by 5.1-fold. We therefore examined the potential effects of kava root extracts and its active components  on PCa growth and androgen receptor (AR) expression. PCa cell lines  with different AR expression, and a transformed prostate myofibroblast cell line (WPMY-1), were treated with a commercial kava extract, kavalactones  and flavokawain B. Expression of AR and its target genes ( Asian/Pacific men who consume a low fat and plant-based diet have the lowest rates of clinical PCa in the world Piper methysticum Forst) is a perennial plant indigenous to the Pacific Islands. Kava root and rhizome are used to prepare a non-fermented and ceremonial beverage with relaxant effects in the Pacific Islands for thousands of years Kava  down-regulate AR expression. Depletion of AR protein occurred through two different mechanisms: 1) enhanced AR protein degradation, and 2) reduced Specificity protein 1 (Sp1)-mediated AR transcription. Kava extract and flavokawain B treatment of mice with patient-derived xenografts reduced tumor growth and expression of AR and its target genes in tumor tissues, and lowered serum PSA levels.Use of mice and their care for this study was specifically approved by the University of California, Irvine Institutional Animal Care and Use Committee .The LNCaP, LAPC4, 22Rv1, PC3, DU145, and WPMY-1 cell lines were obtained from American Type Culture Collection (ATCC) , and C4-2B cell line was from Urocor Inc. . These cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS). All cell lines used in this study were within 20 passages after receipt. The cell lines were tested and authenticated by ATCC or Urocor Inc. All cells lines were also tested for known species of mycoplasma contamination using a kit from LONZA Inc. . Pure kawain, 5′, 6′-dehydrokawain, yangonin, methysticin, and flavokawain B (99%) were isolated from kava extracts by LKT Laboratories, Inc. . Kava root extract at a concentration of 150 mg/ml kavalactones in 50% ethanol was obtained from Gaia Herbs . Antibodies against AR and tubulin were from Santa Cruz Biotechnology, Inc. . PSA and Sp1 antibodies were purchased from Thermo Scientific . 3--2, 5-diphenyltetrazolium bromide (MTT) was from Sigma. RNAzol B was purchased from Tel-Test . The Reverse Transcription System kit and was from Promega . A quantitative RT-PCR kit was from Bio-Rad .The kava extract was diluted 400 times by acetronitrile, and then filtered with 0.45 µm solvent resistant filters and stored at −80°C until further analysis. Chromatography was performed on an Acquity Ultra Performance Liquid Chromatography (UPLC) system  with an auto-sampler at 8°C. Separation of compounds was carried out at 50°C using a Waters Acquity UPLC ®BEH C18 1.7 µm (2.1×50 mm) column with a gradient elution: (A) Water: Acetonitrile: acetic acid (97.8∶2∶0.2 v/v/v), and (B) Acetonitrile: acetic acid 99.8∶0.2 (v/v) as the mobile phase.e−3 mbar with a selected reaction monitoring transition for flavokawain A of m/z 315>m/z 181, flavokawain B of 284>181, kawain of 231>115, 5′, 6′-dehydrokawain of 229>131, mythysticin of 275>159, Yangonin of 259>161.The elution program was as follows: 10% B , 90% B (1.0 min), 90% B (2.0 min), 10%B (2.05 min), and 10% (3 min). The flow rate was 0.3 mL/min and the injection volume was 10 µL. The UPLC was coupled to Micromass Quattro Micro Liquid chromatography–mass spectrometry (LC/MS/MS) triple quadrupole mass spectrometer (Mass Range: 2∼2000 m/z) with electrospray ionization (ESI) interface in positive mode. The instrument was operated in a positive ion mode with an ESI voltage of 3.8 kV, and a desolvation gas flow of 700 L/h. Argon was used as the collision induced dissociation gas at a pressure of 7.14 per well in 24-well culture plates for 24 hours, and then treated as indicated in the figures. After treatment for 72 hours, 1 mg/mL MTT was added to each well for 2 hours, and the absorbance was determined at 570 nm. Dose–response curves for growth inhibition were generated as a percentage of vehicle-treated controls.Cells were plated at a density of 2×10Clarified protein lysates (20–100 µg) were denatured and resolved by 8–16% SDS-PAGE. Proteins were transferred to nitrocellulose membranes, and probed with indicated antibodies and visualized by an enhanced chemiluminescence detection system.AR, prostate specific antigen (PSA) and Transmembrane protease, serine 2 (TMPRSS2) mRNA levels were carried out using MyiQ system (Bio-Rad) as described previously AR, PSA and TMPRSS2 are available upon request. Data were analyzed by using the comparative Ct method, where Ct is the cycle number at which fluorescence first exceeds the threshold. The Ct values from each sample were obtained by subtracting the values for beta-actin Ct from the target gene Ct value. The variation of beta actin Ct values is <0.5 among different samples. A one cycle difference of Ct value represents a 2-fold difference in the level of mRNA. Specificity of resulting PCR products was confirmed by melting curves and agarose gel.Real-time quantitative PCR amplification reactions for The PSA-Luc and plARS-Luc plasmids are a kindly gift from Dr. Wang Longgui (New York University Cancer Institute). Human Sp1-HA tagged plasmid was kindly provided by Macus Tien Kuo (The University of Texas M. D. Anderson Cancer Center). C4-2B and LNCaP cells were co-transfected with PSA-Luc or plARS-Luc and Renilla luciferase plasmid pGL 4.71 (Promega) or with Sp1-HA plasmid by Lipofectamine 2000 (Invitrogen). After 48 hours, flavokawain B was added as indicated with triple replications. Then cells were harvested and luciferase activity was measured with the Dual-Glo Luiferase assay system (Promega). Renilla luminescence was used as an inner control for cell numbers and transfection efficiency. The relative ratio of luminescence from interested gene promoter to Renilla luminescence was shown in the figures as promoter activity. For Sp1 transfection, cells were harvest for an immunoblotting assay.LNCaP and C4-2B cells were cross-linked by formaldehyde. The protein/DNA complex was sheared to 500–1500 bp fragments by sonication. Equal amounts of protein (4 mg) were incubated with Sp1 antibody, or with a mouse anti-GFP antibody. Protein G sepharose beads (Sigma) was employed to pull down the complex, followed by washing with High Salt Wash Buffer, LiCl, and TE buffers). The DNA-protein immunoprecipitates were eluted from the beads and the DNA was extracted and purified. Expand High-Fidelity PCR System (Roche) was used to amplify the sequence from +248 to +487 nucleotides of the AR 5′-UTR region, containing two Sp1 binding sites. Primer sequences are available upon request.3, mice were randomized into treatment groups. For the kava extract treatment, mice were fed a standard diet supplemented with either vehicle control or 6 g/kg kava extract in the diet. For the flavokawain B treatment, mice were injected intraperitoneally each day with vehicle control  or 200 mg flavokawain B/kilogram mouse body weight. Treatment was continued from day 0 to day 17 for the kava extract and to day 27 for flavokawain B. Tumor growth was monitored by both serum PSA and caliper measurement of tumor size weekly. Body weight and food intakes were recorded during the experiments. At the end of the experiments, tumor tissues were weighed and cut in half for mRNA isolation and immunohistochemistry staining, respectively. The tumor volume was calculated by the formula: 0.5236×L1×(L2)2, where L1 is the long axis and L2 is the short axis of the tumor. Serum PSA concentration was determined by PSA Enzyme-linked immunosorbent assay kit  following the kit's instruction.Prostatectomy tissues were obtained through an IRB-approved protocol at UCI Medical Center. Small fragments of histologically-proven tumor were implanted into 8-week-old male Severe combined immunodeficiency (SCID) mice using a subrenal xeongraft technique Antigen was retrieved using 0.05 M Glycine-HCL buffer, pH 3.5, containing 0.01% (w/v) EDTA, at 95°C for 20 min and stained with anti-human AR (1∶100). Staining was visualized with diaminobenzadine using the Cell and Tissue Staining kit (R&D Systems).t test. For tumor growth experiments, repeated-measures ANOVA was used to examine the differences in tumor sizes among treatments, time points, and treatment-time interactions. Additional post-tests were done to examine the differences in tumor sizes between control, the kava extract, and flavokawain B treatment at each time point by using the conservative Bonferroni method. All statistical tests were two sided.Comparisons of quantitative RT-PCR and promoter assays between treatment and control experiments were conducted using Student's Kavalactones and chalcones (i.e. flavokawains) are two important classes of bioactive compounds identified from kava extracts. Using UPLC, we measured the contents of kavalactones and flavokawains. The commercial kava extract dissolved in ethanol and used here contained 2.7% kawain, 1.75% 5, 6-dehydrokawain, 3.08% Yangonin, 1.4% methysticin, 0.33% flavokawain B, and 0.21% flavokawain A . To examPSA and TMRSS2) but not mRNA expression of AR in C4-2B cells. We next examined the stability of AR protein by treating the cells with cycloheximide to inhibit new protein synthesis. AR and its target genes (PSA and TMPRSS2) (Flavokawain B is the most potent agent for inhibiting cell growth among the examined bioactive components of the kava extract. We therefore investigated the effect of flavokawain B on AR expression. TMPRSS2) . These rWe next investigated potential mechanism of flavokawain B mediated AR transcription. Sp1 is a putative transcriptional factor for AR regulation. Sp1 directly binds to the GC-box in the AR promoter through a zinc-finger protein motif in vivo anti-PCa effect of the kava extract and flavokawain B, we developed two more clinically relevant, patient-derived PCa xenograft models, GM0308 and RC0309. The GM0308 patient-derived PCa xengrafts line was derived from a high-grade (gleason score sum = 5+5) PCa prostatectomy specimen obtained from a patient who was previously treated with androgen deprivation therapy (ADT), docetaxel plus carboplatin, and etoposide plus cisplatin. The xenograft tumor growth was initially established by using a subrenal xenograft technique, and then passed through subcutaneous injection of tumor pieces. The RC0309 line was derived from a high-grade (gleason score sum = 5+4) PCa prostatectomy specimen obtained from a patient who had no previous treatment. These human cancer xenografts faithfully preserve the histopathological characteristics of the original clinical sample. Both lines secrete PSA into the host mouse serum  by about 84%, 86% and 87%, respectively, compared to vehicle control treatments  alpha binds to a site near Sp1 in the AR promoter and suppress AR expression, and that PUR alpha expression was associated AR overexpression in androgen-independent cell lines The mechanisms of kavalactones and flavokawain B mediated AR down-regulation are different. Kavalactones act through a mechanism of AR protein degradation, while flavokawain B down-regulate Sp1 expression leading to a decrease of In summary, the kava root extract and flavokawain B inhibited tumor growth in clinically relevant xenograft models, decreased AR expression in tumor tissues, and lowered serum PSA levels in tumor-bearing mice. The down-regulation of AR expression by kavalactones is associated with protein degradation. In contrast flavokawain B decreases expression of both the full-length AR and AR splice variants via inhibition of Sp1 mediated AR transcription. Flavokawain B is more potent to castration-resistant PCa. Since traditional kava preparation has been safely consumed for thousands of years, it is unlikely that active components of kava extracts, including kavalactones and flavokawains, are all toxic to human. Therefore, the results obtained from this study clearly suggest a need for further developing a safe and inexpensive kava product or its active components for treatment and prevention of PCa and CRPC.