This paper extends classical work on economics of doping into a multi-player game setting. Apart from being among the first papers formally formulating and analysing a multi-player doping situation, we find interesting results related to different types of Nash-equilibria (NE). Based mainly on analytic results, we claim at least two different NE structures linked to the choice of prize functions. Linear prize functions provide NEs characterised by either everyone or nobody taking drugs, while non-linear prize functions lead to qualitatively different NEs with significantly more complex predictive characteristics. Doping in competitive sport is a peculiar phenomenon. The need for performance enhancement is emerging from the desire to maximise or even expand human capacities From the system’s point of view, the current detection-based anti-doping policy does not automatically eradicate the use of prohibited substances, but rather presents a barrier with a quantifiable risk of being caught. It is easy to see that such a system leads to two primary strategies employed by the athletes: i) compliance, driven by respect for the rules, desire to compete clean or fear of being caught and ii) circumvention, i.e. outwitting the system by using not-yet-known or undetectable substances, masking or simply betting on chances of not being selected for testing. Just before the creation of the World Anti-Doping Agency, then-International Olympic Committee president Juan Antonio Samaranch voiced his opinion that doping should be acknowledged and allowed as long as it is safe In order to design effective anti-doping measures, gaining insight into the driving forces behind doping behaviour is vital. Whilst the doping decision is very complex involving moral, economical and health considerations, theoretically this complexity can be distilled into a simple decision situation where pros and cons are weighed against each other in the context of unknown but assumed choices of the opponents. Based on the assumption that eradicating doping from sport requires a significant change in this decisional balance; by formulating and formally solving multi-player doping games, we aim to make a contribution to developing a better understanding of doping decisions.In this paper, first we review the literature pertaining to the game theoretical approach to sport and in particular, doping. We set up a model for a doping game involving more than one pair of opponents. This extends the classic doping game to a multi-player situation, which resembles many actual doping situations better than a game restricted to two players. We then report analytical solutions for For athletes entering a high level sport competition, it is a requirement to abide by the rules as set by the relevant governing body. Philosophers would argue that at this point, the question whether the athlete ought to follow these rules or whether they have a reason to do so is contested and depending on the outcome, a decision is made about the action which follows. With regard to doping, athletes must decide whether they feel that they are under obligation (normative) or have a compelling reason  to obey the rules and refrain from using a prohibited performance enhancing substance.Lay explanation for doping rests on the assumption that for those who engage in doping practices “winning is everything”, hence they use prohibited methods to ensure this outcome. This approach assumes that the choice is purely rational-economical and responds to externally imposed incentives and deterrents Contrary to the detection-sanction based deterrence methods, economical models recognise the importance of the prize structure, considering both benefits and costs. Prize structures can also be manipulated so the monitoring cost is kept low The nature of doping makes policing difficult and leads to an imperfect but costly monitoring system, where externally imposed sanctions may inadvertently motivate doping use by indicating that doping is widespread, hence the need for harsh sanctions In addition to the mainstream doping research, a distinct direction (economics of doping) evolved around game theoretical approaches, focusing on different combinations of the normative determinants. Early theoretical exploration of the doping dilemma focused on typical game strategies athletes may engage in, starting from 2-player symmetric and asymmetric games An empirical cross-sectional investigation, using ranked outcomes of best, next best, next worst and worst, concluded that “ruthless”, winning-at-all-cost-type athletes clearly dominated the doping user group but drug users were also found among Naessian-type athletes who value the process more than the outcome Although several authors have discussed the obvious extension into a multi-player setting, few formal attempts on modeling or solving multi-player games exist in sports economics research. Berentsen and Lengwiler’s doping evolution game Considering the existing models to date, sports economic research appears to be lacking formal analysis of multi-player doping games. The aim of this paper is to address this gap by extending the classical doping game theoretical model to a multi-player game.For this model, we consider athletes as those who are in the Registered Testing Pool (RTP) by being identified as elite performer by their respective governing bodies (thus subject to doping testing at any time including sport events) or based on their ranking at the sport events. The RTP includes international level athletes being tested by their international sport federations, and international and national level athletes being tested by national anti doping organisations, or in some cases, national governing bodies of sport, National Olympic Committees and Regional Anti-Doping Organizations. In this respect, there is no distinction between amateur or professional sport. Consequently, we consider prize as the chance of winning vs. the chance of being caught and sanctioned. However, we recognise the fact that in real life, both affects the athlete’s ability to benefitting from sponsorships, endorsement deals and prize money.We start with the performance-enhancing drug game of We assume the following:Each athlete has a binary choice between taking performance enhancing drugs  assumed equally good. This “cloning” assumption is merely a direct extension of the original assumption of Drug testing is routinely performed on all athletes, leading to a certain probability of exposure of The cost of exposure is again extended similarly as in 4) above to Drug tests are assumed perfect in the sense that anyone who have not consumed drugs will not be exposed through a test.There is only one drug available, and the effect of the drug is assumed to be equal for all athletes The given drug is assumed effective (with certainty) in the sense that if any athlete on drugs compete against any “clean” athlete, the outcome is a certain victory in the sports competition for the drug consumer.So far, no really important assumptions have been made compared to the original model However, the utility structure of this model needs some careful remodelling. In the original model, a binary type of pay-off structure was defined. In the multi-player case, it seems obvious that a “winner takes all” pay-off structure is neither realistic nor practical. Hence, it seems evident that a more flexible pay-off structure is necessary. Let us furthermore define:9   10 Furthermore, it seems reasonable to assume:Let us now formally define decision variables for the players/athletes:Given the definition of the binary decision variables Given the above assumptions 3) and 8), there will be two groups, one group taking drugs and another group keeping clean. The first Now, given assumptions 1) - 10) as well as derived We performed a full scale computation  for the three-player case  is viewed as important related to doping regulation, general knowledge related to when (under what parametric conditions) will NEs be pure or mixed might prove valuable in a practical context. These questions (and others) are to some extent answered by the following theorem  start events. The actual reason for this changer is of course labelled on demand and TV-viewers - who presumably prefer to see a competition where the first man crossing the finish line is the winner. The fact that the time spent in mass start events is significantly shorter is of course also judged positive when competing to get TV-time. In an interval start event, even though you principally compete with the same number of competitors, your actual number of real competitors  is in practice way below the total number of participants. However, in a mass start event, the number of possible winners increase dramatically, meaning that you efficiently compete with a much larger group. Hence, based on this argument, if doping is to be controlled, this development is perhaps not the best. Still, again, this is a totality, and doping neither could nor should be fought regardless of possible adverse demand effects. This underlines the complexity of the task at hand.Finally, the role of the prize function becomes evident when a doping game is extended from a two-player to an Handling multi-player games is in general far more difficult, both model- and solution-wise compared to 2-player games. Definitely, this is the situation we have faced conducting this research. As a consequence, we have carefully chosen to make assumptions which may seem crude compared to reality. Assumptions 2), 6), 7) and 8) (see the section on Assumptions and basic modeling), as well as the inherent not explicitly mentioned assumptions of information completeness and non-existent prize re-awards Assumption 2) - homogeneity of player’s ability is a prime candidate. Most two-player research - see e.g. Unfortunately, this is not the only problem involved in relaxing the homogeneity of player’s ability assumption. Note that the share existence of a common knowledge ability 2-dimensional probability distribution actually means that all players must know (and agree) on such a distribution and not only that, they must also know and agree on all possible  drug effects. So, to sum up: relaxing this assumption leads, in our opinion, to an obvious question related to the inherent complete information assumption. In practice one would expect that an individual player would know more on his abilities as well as the effect of taking drugs than his opponents. As a consequence, a game of incomplete (and asymmetric) information becomes even more appropriate. This is, as we see it, obviously a candidate for further research.Similar types of  problems emerge if other assumptions are to be relaxed. For instance a relaxation of 7) - introducing more than one drug and/or individual drug effects - creates totally different modelling environments. This also holds for an introduction of prize re-awards. Prize re-awards We have focused on modelling and solving a multi-player doping game. We have found interesting results related to the shape of prize functions and some results related to NE behaviour as a function of Figure S1The original pay-off matrix from (TIF)Click here for additional data file.Figure S2Nash equilibria in the three-player doping game as a function of the value of (TIF)Click here for additional data file.Figure S3Nash equilibria in the four-player doping game as a function of the value of (TIF)Click here for additional data file.File S1(PDF)Click here for additional data file.


Kip via AR. Higher dosages of testosterone cause greater growth inhibition of relapsed tumors. Manipulating androgen/AR signaling may therefore be a potential therapy for AR-positive advanced prostate cancer.Prostate cancer is the most frequently diagnosed non-cutaneous tumor of men in Western countries. While surgery is often successful for organ-confined prostate cancer, androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, this therapy is associated with several undesired side-effects, including increased risk of cardiovascular diseases. Shortening the period of androgen ablation therapy may benefit prostate cancer patients. Intermittent Androgen Deprivation therapy improves quality of life, reduces toxicity and medical costs, and delays disease progression in some patients. Cell culture and xenograft studies using androgen receptor (AR)-positive castration-resistant human prostate cancers cells  suggest that androgens may suppress the growth of AR-rich prostate cancer cells. Androgens cause growth inhibition and G1 cell cycle arrest in these cells by regulating c-Myc, Skp2, and p27 In 1941, Huggins and Hodges reported that androgen ablation therapy causes regression of primary and metastatic prostate cancer . PrimaryAndrogens are male sex hormone and include several steroids, such as testosterone, dehydroepiandrosterone, androstenedione, androstenediol, androsterone, and dihydrotestosterone (DHT). 90-95% of androgens are produced by the testes, while some androgens are produced in the adrenal glands. Testosterone is the main circulating androgen in human body, while DHT is a more potent androgen that has 5-fold higher affinity for the androgen receptor (AR) than does testosterone -9. When The average serum testosterone level declines with age and elderly men usually have the condition as partial androgen deficiency. It decreases from approximately 620-670 ng/dl at age 25-44 to 470-520 ng/dl at age 65-84 . A low sAndrogen receptor (AR), an androgen-activated transcription factor, belongs to the nuclear receptor superfamily. Binding of DHT to the androgen receptor (AR) induces dissociation of AR from heat-shock proteins (HSPs) and stimulates AR phosphorylation . AR dimeBased on gene microarray studies of seven different human prostate cancer xenograft models, an increase of AR mRNA was the only change consistently associated with the development of the castration-resistant phenotype . IncreasAndrogen ablation therapy, using luteinizing hormone-releasing hormone agonists (LH-RH)  or bilateral orchiectomy, has become a primary treatment for metastatic prostate cancer . More thIn addition, androgen deprivation therapy is associated with several undesired side-effects, including sexual dysfunction, osteoporosis and bone fractures, hot flashes, fatigue, gynecomastia, anemia, depression, cognitive dysfunction, increased risk of diabetes, and cardiovascular diseases ,40-42. AClinical and basic studies have shown that in comparison with continuous androgen ablation (CAB) therapy, Intermittent Androgen Deprivation (IAD) therapy substantially prolongs the time to development of castration-resistant prostate cancer ,46-48. IThe growth of Shionogi mammary carcinoma is stimulated by androgens and was the first experimental model to test IAD therapy. Hormone-dependent Shionogi mammary carcinoma become androgen ablation-resistant following IAD therapy using cycles of transplantation into intact male mice followed by castration . HoweverIn a Canadian Prospective Trial, Bruchovsky et al. showed that IAD therapy causes repeated differentiation of prostate tumors with recovery of apoptotic potential, inhibition of tumor growth after rapid restoration of serum testosterone, and restraint of tumor growth by subnormal levels of serum testosterone . Pether in vitro [The delay of progression toward androgen-independency in IAD treatment might be related to the suppressive effect of androgen on AR-positive hormone-refractory prostate cancer cells that is observed in the LNCaP and other prostate cancer cell models. LNCaP is one of the most commonly used cell lines for prostate cancer research and was derived from a human lymph node metastatic lesion of prostate adenocarcinoma ,50. LNCain vitro ,20. Aftein vitro ,20.in vitro and in vivo [During the transition of 104-S cells to 104-R1 and 104-R2 cells, AR mRNA and protein levels increase. AR transcriptional activity also increases several fold ,18-20,51 in vivo ,18-22,51 in vivo . R2Ad ce5) as most of the cells appear to undergo senescent cell death [3 cells form colonies of androgen-insensitive cells that are not repressed by R1881 or Casodex. These sublines, designated IS cells, show greatly reduced AR expression [To further mimic the clinical situation of combined androgen deprivation and anti-androgen therapy, LNCaP 104-S cells were incubated with 5 μM Casodex in androgen-depleted medium. After four weeks, Casodex-resistant colonies (CDXR cells) appear at low frequency  that displays relaxed ligand binding specificity ,59. Howelutamide . PC-3 islutamide . Physiollutamide -64. Muchin vitro [in vitro, as well as reduces ARCaP tumor growth in athymic mice [Castration causes regression of 104-S xenografts, but tumors begin to regrow after 8 weeks as androgen ablation-resistant relapsed tumors called 104-Rrel with elevated AR mRNA and protein expression . Low serin vitro . It is win vitro ,21,51  has been examined in 104-S, 104-R1, and 104-R2 cells treated with or deprived of androgen. p21waf1/cip1 and p27Kip1 levels are induced by androgen in 104-R1 and 104-R2 cells [waf1/cip1 is induced transiently in 104-R1 cells only, while p27Kip1 is induced persistently about 3-fold in both 104-R1 and 104-R2 cells [waf1/cip1 and p27Kip1 is repressed by androgen in 104-S cells. Androgens regulate expression of the F-box protein Skp2 that binds phosphorylated p27Kip1 [Kip1. Androgen treatment down-regulates c-Myc mRNA and protein expression in hours in 104-R1 and 104-R2 cells  who received androgen ablation plus anti-androgen therapy and withdrew without metastatic disease into three groups. The three groups of patients were given treatment of three different dosages of transdermal testosterone: 2.5, 5.0, or 7.5 mg/day. Testosterone increased from castration levels to median concentrations of 305 ng/dl, 308 ng/dl, and 297 ng/dl for dosages of 2.5 mg/day (n = 4), 5.0 mg/day (n = 5), and 7.5 mg/day (n = 5), respectively. One patient was taken off due to grade 4 cardiac toxicity. One patient experienced symptomatic progression, and three (20%) patients demonstrated a decrease in PSA (largest was 43%). Median time to progression was 9 weeks (range: 2-96), with no detectable difference in the three dose cohorts . The conA phase 1 clinical trial was performed to determine the safety of high-dose exogenous testosterone in patients with castration-resistant metastatic prostate cancer. Patients with progressive castration-resistant prostate cancer who had been castrated for at least 1 yr received three times the standard replacement dose of transdermal testosterone by skin patch or topical gel. No adverse effects were reported. Cohorts of 3-6 patients received testosterone for 1 week, 1 month, or until disease progression. Average testosterone levels were within normal physiological concentration. The serum testosterone ranged from 330-870 ng/dl. One patient achieved a PSA decline of > 50% from baseline, although no other significant effect was observed. No difference was observed between different cohorts . This stAlthough our observations suggested that androgen suppress growth of AR-positive advanced prostate tumors while Vancouver group use IAD to show that cessation of anti-androgen therapy allowed tumor cells to recover their androgen-sensitivity and be sensitive to subsequent rounds of anti-ablation treatment. We believe that our LNCaP progression model may provide the molecular explanation for IAD treatment. As most prostate tumors relapsed from androgen ablation therapy express AR and expression of mRNA and protein level of AR are frequently elevated -25, restin vitro and in vivo progression model, patients developing relapsed hormone-refractory prostate tumors after androgen ablation therapy should be biopsied for expression level of AR protein in tumors. IAD and/or administration of exogenous androgen at a concentration 2500-3500 ng/dl will benefit patients with AR-rich relapsed tumors by suppressing tumor growth, improving quality of life, and reducing risks for cardiovascular diseases and diabetes. Combined treatment of androgen ablation therapy with anti-androgen cause a rapid and irreversible selection of more aggressive advanced prostate cancer cells [Based on the results from our er cells , possibler cells . Androgeer cells ,20, thuser cells ,48,72,73The authors declare that they have no competing interests.All authors contributed to the writing, read, and approved the final manuscript.th birthday. He is a member of America Academy of Art & Science (U.S.A.) and academician of Academia Sinica (Taiwan).This article is dedicated to our dear mentor Dr. Shutsung Liao, professor at Ben May Department for Cancer Research of The University of Chicago for his 80


ISO  was given to make the heart failure models. G-1(120 µg/kg·d×14 day) was used to activate GPR30 and G15 (190 µg/kg·d×14 day) was used to inhibit GPR30. Concentration of brain natriuretic peptide in serum, masson staining in isolated heart, contractile function and the expression of β1 and β2- adrenergic receptor (AR) of ventricular myocytes were also determined. Our data showed that ISO treatment led to heart failure in OVX rats. G-1 or E2 treatment decreased concentration of brain natriuretic peptide, reduced cardiac fibrosis, and enhanced contraction of the heart. Combined treatment with β1 (CGP20712A) and β2-AR (ICI118551) antagonist abolished the improvement of myocardial function induced by G-1. We also found that chronic treatment with G-1 normalized the expression of β1-AR and increased the expression of β2-AR. Our results indicate that chronic activation of the GPR30 with its agonist G-1 attenuates heart failure by normalizing the expression of β1-AR and increasing the expression of β2-AR.G protein-coupled receptor (GPR) 30 is a novel estrogen receptor. Recent studies suggest that activation of the GPR30 confers rapid cardioprotection in isolated rat heart. It is unknown whether chronic activation of GPR30 is beneficial or not for heart failure. In this study we investigated the cardiac effect of sustained activation or inhibition of GPR30. Female Sprague–Dawley rats were divided into 7 groups #2Q1: sham surgery (Sham), bilateral ovariectomy (OVX), OVX+estrogen (E Despite significant advances in understanding the mechanisms underlying this disease, current treatments for HF have not been satisfied. It is recognized that sympathetic nervous system is one of the most important mechanisms regulating cardiac function, mainly through activation of β-AR Pre-menopausal women have reduced risk for cardiovascular disease, and the incidence of cardiovascular disease increased after menopause. Studies on animal models have also suggested that estrogen played an important role in cardioprotection 1- and β2-AR In addition to the rapid effects caused by activation of GPR30, its chronic effects have also been identified. It was reported that genetic deletion of GPR30 was associated with visceral adiposity in both male and female animals 2 group, OVX treatment increased heart weight, but it was not significant. ISO plus OVX increased heart weight and heart weight/body length ratio compared with OVX group. G-1 or E2 but not E2+G15 could eliminate the increasing of the heart weight caused by OVX plus ISO. E2 and E2+G15 but not G-1 could increase uterine weight  (in µg/L) in plasma before the animals were sacrificed. Ovariectomy induced a slight increase in the release of BNP compared with the Sham or OVX+ESO group .2 increased LVDP, ±dp/dt, RPP, and decreased LVEDP, however G15 treatment can not cause such changes (Hearts were equilibrated for 30 minutes with KHB (Krebs–Henseleit buffer), and then we recorded the cardiac function data of each group. We found that there were no significant differences before ISO treatment among all groups. ISO treatment decreased LVDP, ±dp/dt and RPP, and increased LVEDP significantly. Administration of G1 or E changes .2 groups, stained sections showed increased fibrosis in OVX+ISO and OVX+ISO+E2+G15 group. OVX+ISO+G-1/E2 group has lower fibrotic areas than OVX+ISO group , ISO treatment caused significantly damage to the heart compared with E2 treatment, which indicated that estrogen played an important role in protection of the heart. To study the protective effect of GPR30, G1, E2+G15 and E2 were administrated. The amount of drugs was determined following the method mentioned in Lindsey SH.’s article 2 5.7 nM; second determine the concentration of G-1 and G15 according to the concentration of E2 which our laboratory used before 2 40 µg/kg·d.In our study, continuously and chronic effect of G-1 was observed. GPR30, as one of the estrogen receptor, can be activated by E2 or E2+G15 treatment decreased weight gain induced by ovariectomy which in line with our previous study 2+ G15 treatment didn’t play cardiac protection roles which indicated that the chronic activation of GPR30 is responsible, and not ERα and ERβ.We measured animals’ weight before they were killed, G-1 treatment didn’t change weight gain induced by ovariectomy, which was consistent with the results of Lindsey SH.’s research 2 treatment didn’t increased the phosphorylation of AKT. This indicated that the special agonist G-1 activated GPR30.PI3K-AKT pathway is the downstream pathway of GPR30, and G-1 treatment increased phosphorylation of AKT. In our experiment, we determined the phosphorylation of AKT and found that G-1 or E2 treatment increased the phosphorylation of AKT, G15+EBNP is mainly present in the left and right atria, the physiologic actions of it are similar to ANP  and include decrease in systemic vascular resistance and central venous pressure as well as an increase in natriuresis. The level of its secretion is closely related to the changes of ventricular filling pressure, when heart failure occurred, ventricular filling pressure raised and the secretion of BNP increased. The increase of the secretion was positively correlated to the degree of heart failure. So the concentration of BNP in serum could be an indicator to assess the severity of heart failure.In the experiment, the concentration of BNP in OVX+ISO group increased significantly compared with OVX group, this is in according with the hemodynamics resulst. In OVX+ISO+G-1 group, the concentration of BNP decreased compared with OVX+ISO group, this indicated that G-1 treatment conferred cardiac protective effect in ISO induced heart failure model.We have detected hemodynamic in organ levels, found that ISO treatment diminished cardiac ejection and G-1 treatment enhanced the ability of the cardiac ejection, this indicated that G-1 conferred cardiac protective effect. As G-1 could reduce vascular tone and dilate rodent arterial blood vessels 1-AR antagonist CGP20712A, β2-AR antagonit ICI118551, we found that treatment with CGP or ICI separately could not abolish the improvement of the cell contraction., but combination treatment with CGP and ICI could abolish the improvement completely. This indicated that the protective of G-1 may associate with both β1-AR and β2-AR.We isolated cardiac myocytes of OVX+ISO and OVX+ISO+G-1 group, cultured with β#3Q3Although there is a group with antagonist group, the ligand specificity in vivo is still limitation in vivo study, for example the antagonist drugs may reach to the liver, brain or other organs, which confer the systolic changes of the bodies.1-AR results in augmentation of cardiac activity (positive inotropic effect), including an increase in heart rate and atria-ventricle conduction velocity and enhancement of myocardial contraction 1 receptors caused cardiac damage 1-AR increased in ovariectomized female rats compared with the Sham group 1-AR thus conferred cardiac protection effect. In this paper, we found that the expression of β1-AR increased in OVX group, G-1 or E2 treatment decreased it, and we didn’t observed cardiac damage indications in OVX group, here we speculated ovariectomized is just a risk factor for hearts. However ISO treatment decreased the expression of β1-AR and produced injury effect which may be attributed to continuous stimulation of catecholamine led to decline in receptor number and reduce of the function 2 treatment could reduce the injury and increased the expression of β1-AR compared with OVX+ISO group. Taken together, G-1 or E2 treatment regulated protein β1-AR in the protective effects.The sympathetic nervous system is critically involved in the regulation of cardiac function through β-AR. Activation of β1AR, activation of β2-AR plays a beneficial role in hearts. Sustained β1-AR stimulation promotes apoptotic death of cardiomyocytes, sustained stimulation of β2-AR protects myocytes against a wide range of apoptotic insults 2-AR conferred cardiac protective effect in the heart Unlike β2-AR. Interestingly, other hormones or models could also regulate the expression of β2-AR in the body. For instance, Penna C has reported sub-chronic nandrolone pretreatment increased the expression of β2-AR 2-AR in heart 2-AR decreased 2-AR is direct or indirect effects such as regulating the secretion of other hormones is unknown, the mechanisms remain to be further studied.In our opinion, treatment with the estrogen hormone agonist G-1 could increase the expression of β2-AR and normalized the expression of β1-AR in ovariectomized rats. This is the first time we have reported chronic treatment with G-1 is beneficial for the heart failure.Taken all together, in this study we found that chronic treatment with G-1 attenuated heart failure by increased the expression of β1-AR, β2-AR, p-AKT, t-AKT , brain natriuretic peptide (BNP) ELISA Kits , Masson’s Trichrome Stain Kit .Female Sprague-Dawley (SD) rats weighing 180–220 g (200±20 g) were obtained from the Experimental Animal Centre of Xuzhou Medical College and all studies were approved by the Animal Ethics Committee of the Medical College of Xuzhou (permit number: xz11-12540) and conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of health . The main reagents used in the experiments are as follows: isoproterenol hydrochloride , G-1 , G15, 17β-estradlol , ICI118551 , CGP20712A , wortmannin , in situ cell death detection kit, pod , rabbit polyclonal anti β2+G15 or E2 was pumped via the minipump at the dose of 120 µg/kg·d, 190 µg/kg·d, 40 µg/kg·d respectively, treatment lasted for 2 weeks. The dose of G-1, G15, E2 based on the affinity to GPR30 2, OVX+ISO, OVX+ISO+G-1, OVX+ISO+E2+G15, OVX+ISO+E2.The animals were anesthetized with sodium pentobarbitone , corneal reflex disappeared, sensation of pain reduced and muscle tension decreased was the sign of the success of anesthesia. Incision was chosen at the side of the spine, separated the muscle layer and opened the peritoneum, then the ovaries were exteriorized, ligated and removed in OVX group. In Sham group, visualization of the ovaries through incisions into the abdominal cavity and then closure of the wounds. All surgeries were carried out under sterile conditions. ISO  treatment was continued for 17 d to get the heart failure model We collected the blood from tail vein of the rat before it was killed, added an appropriate amount of heparin and then centrifuged at the rate of 2500 rpm for 15 min. The concentration of BNP was assayed as the instructions of ELISA kits. All samples were assayed in triplicate.2+ free buffer as we have reported before Intraperitoneal injection with 5000 U/kg heparin was implemented, 15 minutes later, 150 mg/kg pentobarbital was injected for anesthesia, corneal reflex disappeared, sensation of pain reduced and muscle tension decreased was the sign of the success of anesthesia. A transverse incision was made, the abdominal cavity was exposed, hearts were quickly isolated and rinsed with ice-cold CaLinked aortic side of the isolated heart to perfusion device, left ventricular pressure was recorded using a Biopac system (BIOPAC) via a pressure sensor Millar transducer instrument . Hearts were equilibrated for 30 minutes with KHB. Heart rate (HR), left ventricular systolic pressure (LVSP), left ventricular enddiastolic pressure (LVEDP), and the rate in rise and fall of ventricular pressure  were recorded as left ventricular functional parameters. The left ventricular developed pressure (LVDP) were calculated as LVDP = LVSP-LVEDP; rate pressure product (RPP) was calculated as RPP = HR×LVDP.Rat hearts were perfused with KHB for 20 minutes to remove the residual blood, and then fixed with 10% formalin before embedding in paraffin. All hearts were embedded in a cross section orientation, and all slices were cross sections of the heart. All slices were taken from the midpoint of the left ventricles. Four-micrometer slices were deparaffinized and rehydrated. Then the instruction of masson’s trichrome stain kit was followed. Microscope  was used to get the pictures. The normal cardiac myocytes were stained red, and fibrotic areas were stained green.5 cells per well, finally took them to carbon dioxide incubator , parameters were set as below: 5% CO2, 37°C, cultured for 1 hour to stabilize the cells. The rat cardiomyocytes were incubated with vehicle (veh), CGP20712A , or ICI118551 , for 2 h to study the impact of different β-AR antagonists on shortening amplitude in rat myocytes.Ventricular myocytes were isolated from the hearts as we described before 90), the duration to reach 90% relengthening, an indicative of diastolic duration.After myocardial cells were isolated, each group was divided into three subgroups: veh, CGP, ICI. A few drops of solution containing ventricular myocytes were added to an open chamber on the stage of an inverted microscope . After 5 min, to allow cells to spontaneously attach to the floor of the chamber, they were superfused at 2 ml/min with KHB (containing 2.0 mM Ca2+ and 100 nM ISO) adjusted to pH 7.4 by equilibration with 95% O2–5% CO2. The ventricular myocytes were paced with an electrical field stimulation (0.5 Hz). Myocytes used were rod shaped with clear sarcomeres, and 10 consecutive contractions were averaged. The whole process was recorded by a video recorder  connect to the microscope. Then the video was sent to a computer, cut into pictures by the software VirtualDub, the process of contraction and relaxation was recorded and transformed into digital data by the software OpticalMeasure . Contractile function was assessed using the following indices: shortening amplitude, the amplitude myocytes shortened upon electrical stimulation, an indicative of peak ventricular; contractility time-to-peak contraction (TTP), the duration of myocyte shortening, an indicative of systolic duration; and time-to-90% relaxation . The contraction of the myocytes were analysed by two-way ANOVA, other data were analysed by one-way ANOVA followed by Bonferroni post hoc tests.


The aim of this study was to evaluate the short- term effects of percutaneous mitral valvuloplasty (PMV) on coexisting AR.Clinical, echocardiographic and catheterization data from hospital records of a total of 327 patients with rheumatic mitral stenosis who underwent PMV at a tertiary centre were retrospectively reviewed and aortic regurgitation changes 48 hours post PMV was recorded.The study population consisted of 282 females and 45 males. Mean age at the time of intervention was 47.13±11 years. Before PMV, 142 (43.3%) patients had no AR, 124 (37.9%) had mild AR and 61 (18.7%) had moderate AR. There was no change in AR severity in post- PMV follow-up. AR progression after PMV and during the follow-up was not significant and there was no increase in the need for aortic valve replacement (AVR) procedures.Our findings indicated that a considerable number of patients with rheumatic mitral stenosis had concurrent AR. At the time of PMV concomitant AR does affect procedural success and is not associated with inferior outcomes. Patients with moderate degrees of AR remain good candidates for PMV. Percutaneous mitral valvuloplasty (PMV) was first described by Inoue et al and has since been widely used to treat mitral stenosis (MS) , 2. PMV Most previous studies have evaluated patients with mild AR and very few have been done in patients with moderate or severe AR and altogThis retrospective study comprised 327 patients who underwent PMV at a tertiary centre between 1994 and 2007. Patients with available data regarding the AR degree of pre-PMV were included in the analysis. The clinical and echocardiographic data were acquired from hospital records. All the patients had undergone transthoracic echocardiography (TTE) and transesophageal echocardiography (TEE), and atrial size, pulmonary artery pressure, presence of LA thrombus, and MR were recorded. Patients with moderate to severe MS who had a valve area <1cm²/m ² of the body surface area and suitable valve morphology (MV score < 9) with no or mild mitral regurgitation (MR) and no left atrial clot were selected for PMV. All the procedures were performed via the Inoue method .Balloons were selected according to patient’s height and body surface area.Hemodynamic parameters, left and right atrial pressures, and gradient changes were recorded during PMV. Mitral valve area, MR, AR, tricuspid regurgitation severity, LVEF, and left atrial diameter were measured via TTE and TEE before the procedure. All the patients underwent TTE 48 hours after PMV.Color Doppler study and effective regurgitant orifice area, vena contracta width and pressure half time were used to define the quality and quantity of AR. Based on these findings, AR was classified into three groups of none, mild and moderate AR, 10. Nont-test was used for the interval variables and chi-square test for the categorical variables. Wilcoxon signed rank test was used to assess the relationship between AR before and after PMV.All the statistical tests were two-sided using α<0.05 level of significance. The data were processed and analyzed using SPSS 15 version.The continuous variables were expressed as mean±SD, and the categorical variables as percentages. The The mean age of patients was 47.13±11 years . The stuAR was measured before the procedure via echocardiography; and of a total of 327 patients, 142 (43.2%) had no AR, 124 (37.9%) had mild AR and 61(18.7%) had moderate AR. Echocardiographic evaluation 48 hours post-PMV showed unchanged aortic regurgitation severity in all patients. Follow-up data showed that after PMV, None of our patients had progressed to severe AR. No patient underwent AVR .P=0.02).There was also a significant relationship between the EF and rhythm (P=0.01), and the patients with sinus rhythm showed a higher mean of EF compared to those with atrial fibrillation(52.9±4.1 and 51.3±4.9 respectively). There was no significant relationship between rhythm and AR severity before PMV (P=0.2).The mean of the EF was 52.5±4.3. There was a statistically significant relationship between EF and AR  continues to be a common health problem in the developing world, causing morbidity and mortality among children and adults . ConcurrAccording to this study, we think PMV could be beneficial even in patients with severe AR who do not yet serve the true indication of AVR. Future studies might better clarify this point.Older patients were more likely to have atrial fibrillation (AF) before and after PMV. These findings are similar to those in some other studies , 13.MostAlthough there was no significant relationship between the patients’ rhythm and AR before and after PMV, it seems that the patients without AR were more likely to have sinus rhythm, which may be due to milder forms of the disease.As pointed in the results section, no post procedure AVR was performed as a result of increased AR severity. Our findings indicate that among patients with rheumatic MS and planned PMV, a considerable number have concomitant aortic regurgitation. Based on these results, we therefore suggest that PMV is an effective treatment for patients with MS and concurrent trivial to moderate AR and is not associated with inferior outcomes.Limitations: In our study, most of the patients had mild or moderate AR and it was not possible to perform a comprehensive assessment of patients with severe AR. One patient with severe AR before PMV tolerated the procedure well, but we do not have corresponding long-term follow- up data.


Rehabilitation in Finland is a good example of functions divided among several welfare sectors, such as health services and social services. The rehabilitation system in Finland is a complex one and there have been many efforts to create a coordinated entity. The purpose of this study is to open up a complex welfare system at the upper policy level and to understand the meaning of coordination at the level of service delivery. We shed light in particular on the national rehabilitation policy in Finland and how the policy has tried to overcome the negative effects of institutional complexity.In this study we used qualitative content analysis and frame analysis. As a result we identified four different welfare state frames with distinct features of policy problems, policy alternatives and institutional failure.The rehabilitation policy in Finland seems to be divided into different components which may cause problems at the level of service delivery and thus in the integration of services. Bringing these components together could at policy level enable a shared view of the rights of different population groups, effective management of integration at the level of service delivery and also an opportunity for change throughout the rehabilitation system. The Finnish institutional system of rehabilitation policies has been presented as an example of a complex welfare system with several problems , 2. In mBoth national and international studies on the Finnish rehabilitation system have acknowledged several problems. The OECD has conducted a broad study on disability service systems in several countries where they reported that the Finnish rehabilitation system is a complex one with several subsystems and lack of coordination . A recenWhat is needed in complex systems is an appropriate approach to achieve successful policy implementation . One of At the higher organisational level of care systems the concepts of governance and coordination are used more in the social science and political science literature than the concept of integration , 19. DefThe Finnish institutional system of rehabilitation has numerous functions and is linked in several different welfare systems. In this article these linked welfare systems are referred to as subsystems. The various rehabilitation functions in Finland are: the provision function, the financing function, the social rights function, the steering function, the income compensation function and the professions function. The subsystems are: 1) decentralized health and social care service system, (2) active employment services, (3) obligatory sickness insurance and first pillar (basic) pension system , (4) obligatory second pillar (earnings related) pension system, (5) obligatory traffic and work accident insurance systems (private insurance companies and private voluntary insurance systems) and (6) voluntary accident insurance system. Thus the subsystems are part of different welfare policies, such as social and health care and pension policies and the functions are usually grouped under medical, vocational and social rehabilitation  decentra, 22.One of the major goals in Finnish rehabilitation policies has been to create a more coordinated entity from the different and rather separate subsystems , 11, 12.Several barriers to integration and also to coordination could be found in complex systems. One of the barriers is a lack of communication between different parts of the system , 23. HowStudies of complex combinations of rehabilitation systems and their population-wide policies are marginal on a national and also on an international scale , 26. AltThe purpose of this study is to open up a complex welfare system at upper policy level and to understand the implications of coordination for the level of service delivery. We will in particular address the policies to overcome the negative effects of the institutional complexity of rehabilitation policy in Finland. We also address the influence on different rights to rehabilitation services for different population groups. In order to understand the context of our study, our first question is: how has the complexity in rehabilitation policies developed in Finland? Our second question is: what kinds of frames were used in the discussions about developing the rehabilitation system in Finland? Overall, this study focuses on the coordination of a complex system at policy level. As our approach we use governance theories rather than organization theories. This study is part of a larger research project (during 2005–2007) studying rehabilitation system in Finland from different perspectives , 32.kuntoutus) can be traced back to the 1960s and 1970s [The first observation regarding the history of rehabilitation services is that there is no history of rehabilitation as such, because there was never a shared concept of the practices and policies that are currently grouped under the concept of rehabilitation. The beginning of the broad use of a single Finnish concept of rehabilitation  in their service options and coverage , 40.The rehabilitation system in Finland is based on several financing schemes . In the The Finnish welfare state expanded rapidly from the 1960s onwards and new institutional options emerged . EconomiIn the 1980s the old social assistance type of invalidity welfare was incorporated into the expanding universal Beveridgean service system of social care. This system was also closely linked to the Beveridgean component of the health system creating a more or less fused “local social and health care system”. In the 1970s and 1980s Finland also experienced an expansion of tax revenue funded active employment services , 42.Since the 1980s different elements of the rehabilitation system have been perceived as parts of a whole, although they have also been developed separately. On the one hand, the elements were gathered under a single concept while on the other a government working group was established to clarify the role of the different elements from the perspective of the whole. Then the first coordinating body, the Advisory Board for Rehabilitation (KUNK), was set up with an advisory function towards the Ministry of Social Affairs and Health .Over time, new parts were thus added to the system without dismantling the old institutions. This resulted in a complex institutional entity that includes the first pillar  and the second pillar (earnings-related labour market-based) insurance systems, obligatory traffic and work accident insurance (accident insurance offered by private companies) as well as Beveridgean tax revenue funded mainly public employment, social and health care services and a Bismarckian sector? of insurance funded private health care services. The different subsystems have also been based on different governing principles and structures. The pension subsystems are governed by tripartite bodies representing labour market parties and the government. By contrast, the accident insurance subsystems tend to follow a model of government controlled private company governance. The public employment services are run by local offices directly under central government while the local social and health care services are under municipal decision-making under relatively light central government guidance.Several problems were acknowledged in the complex rehabilitation system and the problems were addressed especially in the 1970s and 1980s. One main problem mentioned in different national rehabilitation policy documents was absence of effective co-operation between the subsystems, which increased the risk of people falling through “the rehabilitation net” in the level of service delivery . At the The data in this article are derived from the last rehabilitation report of 2002 and related materials. This was the last report to focus on and describe the development of the rehabilitation system. The data consist specifically of detailed minutes of the plenary session of the parliamentary debate on the 2002 rehabilitation report. The complementary data are the main report  with itsThe main data are the detailed minutes of the parliamentary debate on the 2002 rehabilitation report. In all, the four rehabilitation reports are all an important part of rehabilitation policy in Finland. Around the rehabilitation reports a kind of a polity , 47 of tTo understand the significance of the plenary session to the rehabilitation policy in Finland the parliamentary process of the rehabilitation reports needs to be discussed. After being presented to Parliament, the reports went through a long parliamentary process. At first, Parliament had a long debate about the report in a plenary session. Every detail of the discussion was documented in the minutes. The discussion followed a specific pattern with every Parliamentary group presenting their own views on the report. The discussion is a significant and prominent part of rehabilitation polity in Finland. The documentation of the discussion is extensive, amounting to 49 pages. First the ministers concerned  described the outline of the report. After that, every parliamentary group presented their own views on it. The discussion continued with the comments of individual MPs, answered by the appropriate ministers. In 2002 the parliamentary groups were formed by the same political parties as today: the Centre Party, the Finnish Social Democratic Party, the National Coalition Party, the Left Alliance Party, the Greens of Finland, the Swedish People’s Party, the Christian Democrats and the True Finns Party.As discussed above, the rehabilitation report of 2002 and thus the plenary discussion thereon was the last in the history of rehabilitation reports in Finland. Although the report and the discussion were published years ago, there have so far been no major reforms of the whole rehabilitation system in Finland. According to the recent national policy reports the rehabilitation system still appears as complex as it was in 2002 . Thus, tThe complementary data were the Rehabilitation Report of 2002, its background documents and interviews with key informants. The data were used to complement the findings from the primary data. At a concrete level the complementary data were used after the main findings had been made from the main data. The complementary data helped to understand the main data or elaborated the missing parts in the main data. The background documents included letters and statements from the time of the preparation of the Rehabilitation Report. We used several archives to find the needed papers. The authorities interviewed were from the Ministry of Social Affairs and Health, labour market organizations and the insurance system. In all, we interviewed five key authorities of the rehabilitation system individually for the study. We chose the authorities depending on the findings from the primary data. If we needed a more profound or wider perspective on some issues we chose a relevant authority to complete the picture of the rehabilitation system. It was important to discuss with the authorities because, for example, the insurance system was hardly touched upon in the plenary discussion although it is an important part of the rehabilitation system. All interviews also gave us a more recent perspective on the system.In this study we use the concept of coordination as a goal of governance. Governance is a concept often used to comprehend complex welfare systems and their problems –50. One The analysis was twofold. The first part was data based concentrating on the detailed minutes. The second part was based on the literature.The first part of the analysis was done using data based content analysis –56. By tAfter the data based content analysis we identified different governing perspectives among the problems and solutions using frame analysis. We saw the frames as boundaries of different views on the rehabilitation system . ConcretIn the second part of the analysis we interpreted the frames identified referring to the relevant literature on the welfare state and governance e.g. , 51. We We checked and confirmed the results using the complementary data. The complementary data were analysed in the same way as the main data. The interviews were transcribed and thus in a written format. The interviews were thematic and conducted by two researchers. The interviews were planned in advance and certain themes such as “the rehabilitation system as a whole”, “development of the system” and “roles of different subsystems” were discussed with the key authorities. The interviews were audiotaped and transcribed and the texts were first analysed by one researcher. In the next phase the analysis and results were discussed with the research group and finally the results were processed together.The discussion between the representatives in the plenary session was colourful. Clashes of opinion were easily discernible in the text. As a result we identified four different frames in the debate .In the first frame the main problems identified were inequality and inequity between individuals. The shared aim in this frame was universality and also equity of rehabilitation services. However, these aims were somewhat unsuccessful. We interpreted the institutional failure as fragmentation within and between institutions, especially financiers and providers of rehabilitation services. Failure was addressed, for instance, with regard to the pension institutions and the state administration. In all, this was a traditional welfare state frame where the aim of equality and equity between population groups resembled the notion of universality.In the second frame we identified two perspectives on the problems. First, the workforce was retiring too early. Without the tax revenue, less government money would be available for the services in the future. Second, the level of well-being and health among workers was too low. Thus it seemed that the institutions had only partially met the needs of society for rehabilitation. The focus was to extend the overall career length and address the need better. In all, the perspective of this frame was a competition state, especially its corporatist component, because in this context the trade unions exert considerable influence.In the third frame the main problems were wasteful use of resources, unfair distribution between payers and beneficiaries and lack of evaluation of the cost-effectiveness of rehabilitation. In addition to not having sufficient funds to finance the services, the services financed by the welfare state had been ineffective. We interpreted the institutional failure as a lack of discipline. For instance, some pension institutions were not paying enough for rehabilitation services although they received the benefit. In all, such a critical perspective of a wasteful and ineffective welfare state has long roots in welfare state debates.In the fourth frame the main problem was unfair competition between rehabilitation service providers. This was said to undermine some rehabilitation services. Here we interpreted the institutional failure as fragmented markets. It seems that the semi-markets have created a situation giving rise to unfair competition between rehabilitation service providers. Certain features in some of the service provider organisations may have weakened their position in the competition. The fragmentation seemed to emerge especially between the financiers and providers of the rehabilitation services. In all, this was a clear competition state frame.In different frames different policy solutions were proposed for the problems described. The solutions in the first frame were usually aimed at legislation. Several obligations were assigned to the institutions and the rights were correspondingly assigned to individuals. In the second frame legislation was called for to create new obligations for some institutions while increases in financial resources were proposed for the rehabilitation services. In the third frame the main solution was the allocation of funding on the basis of effectiveness. The aim was to “get value for money” from the expensive rehabilitation services. In the fourth frame, the MPs expected a solution, but the data provided no evidence of any suggestions on what these solutions might be.After having identified the frames we also found it important to see how they were distributed among the political parties. As can be seen in Several perspectives on rehabilitation policy have been used in the same policy discussion to overcome the problems resulting from the institutional complexity of the rehabilitation system. The four frames represent four preliminary patterns of coordination and thus the complexity of governance in the system. The existence of four different frames is essential because the way a policy problem and solution are framed assigns responsibility and creates rationales that enable some policy solutions and impede others. For instance, policymakers need to commit themselves to the priorities between the frames and thus, between the subsystems. The different frames may also lead to a preference for diverse coordination models between the subsystems or institutions and thus different solutions in practice resulting to different rights for different population groups.The different frames also highlight different thoughts on the coordination of this complex system. From one perspective the problem is missing networks, the other perspective emphasises the need for command and control and the last perspective focuses on ineffective and unfair markets. Thus, it is possible to find several different coordination models in the system which refers to some kind of hybrid coordination. Such aspects are important to address if the aim is to find an appropriate approach to policy implementation in this system of mixed coordination.Overall, the results suggest that the nature of coordination is essential in complex systems. One finding is that in the case of rehabilitation the lack of common coordination at the system level may lead to a lack of successful integration at the level of rehabilitation services. In this case the successful integration of services is achieved when the continuity of a needed rehabilitation service or process is not interrupted although a person is moving between different subsystems and thus the subsystem or institution responsible is changed. Another finding is that, in order to understand the system, its coordination or integration, it is important to acknowledge the origin and background of the complexity.It seems that within the different subsystems, different perspectives on rehabilitation policy have been sustained and largely resisted a stronger governance of the whole. While there have been efforts to create coordinating bodies such as the Advisory Board for Rehabilitation and thus create some kind of centralized coordination, the system has retained its institutionally complex characteristics to this day. Evidently the Advisory Board and the rehabilitation reports to Parliament have contributed to a shared understanding of the large complex system, but have not yet resulted in any strong governance of the whole. At the core of this governance effort are the parliamentary discussions about the rehabilitation reports. Despite the Advisory Board there seems to be no shared view about the governance . From thThe institutional complexity of the rehabilitation system could partly be explained by its history. Until the 1960s coordination seemed simple. With the expansion and the ideological adoption of the Finnish welfare state since the 1960s, the idea of a working society grew stronger and new elements were added to the institutional structure of rehabilitation so that it covered as many disabled people as possible. At the same time the emphasis on paid work and the stigmatization of unemployment put pressure on disabled people. At the system level the increasing trend of policy reforms in Finland influenced the expansion of the system. It became complex with several subsystems, several coordination models and several views on how to develop this mixed welfare system, the whole of rehabilitation in Finland.The history of this complexity has an important meaning through path dependence , 60. ThiThe perspective on the history of the rehabilitation system in Finland underlines the difficulty of achieving effective cooperation between the subsystems or institutions in such a complex system. The subsystems are decidedly self-organised with meaningful histories and strong actors. Thus, the lack of a common understanding of the main goals in developing the systems is not surprising. What is needed is something that could break down the barriers between the strong self-organised subsystems.Shared governance could open the way to changing the system. A shared vision, staying on the path or breaking the path, could be the key. At the time of the parliamentary discussion a shared vision seemed to be lacking. Even now the lack of a shared view will also challenge the development of any joint coordination of the rehabilitation system. However, this vision could be based on the present discussion about extending the age of retirement in Finland.All things considered, it seems that, depending on which of the frames is emphasized, different population groups end up receiving rehabilitation services. If the system is developed using the frame of “reproduction of workforce”, the primary group to receive rehabilitation will perhaps be the working population. The justification is that effective rehabilitation will probably enable them to return to work. Similarly, if the emphasis of development is on equality, perhaps the retired population may also have a stronger claim to rehabilitation. With regard to these examples we may ask how it is possible to achieve effective integration between different services if the coordination at the macro-level is as fragmented as described.A shared view at the macro level of the complex system and its future seems important to gain a more coordinated system, also at the level of service delivery. Rehabilitation policy in Finland seems to be divided into at least two main components which should be brought together in order to gain a shared view. These parts are the perspective of the traditional welfare state with government led centralized solutions and that of the competition state with a corporatist and market oriented component. However, no indications of such a consensus were found in the plenary debate or mentioned in the interviews.Another aim to gain a shared view could be to use a strong tripartite body to act as a coordinator as is the case in the pension system in Finland. But could strong corporatism compensate weaker government? In Finland the tripartite governance system of the government and the labour market representatives has been used in the governance of pension policies and in incomes policies. However, even with regard to these policy sectors, the tripartite governance model has met with increasing resistance e.g. . Thus, cAll things considered, none of the options for a coordinating model for such a complex system as rehabilitation in Finland is an easy one. As long as common coordination is missing at the system level, the integration of different rehabilitation services at the service user level will be challenging and the rights of different population groups in this complexity will vary.In order to achieve well-coordinated integrated services and smooth service processes “the black box” needs to be opened. It is important to understand the construction of the system. If the system is as fragmented as is the rehabilitation system in Finland with its strong history and independent subsystems, cooperation will be no easy task. Although cooperation has been one of the main tasks of Finnish rehabilitation policy at least since the 1990s, it has not removed the main problems from the system. In the rehabilitation system one way to approach the complexity could be to recognise its hybrid coordination model and the special features of each subsystem. These way appropriate tools could be found and common coordination could be achieved.Jane Hendy, Phd, Senior Lecturer in Health Management and Policy, Head of PhD programmes, The Department of Health Care Management & Policy, University of Surrey, Guildford, Surrey GU2 7XH, UKSimo Kokko, Chief Medical Officer, Director of the Unit for Primary Health Care, Health Authority of Northern Savo, Finland; Adjunct professor, University of Eastern Finland, Kuopio, FinlandUlla Wihlman, RPT, MSC, PhD. Senior Research and Evaluation Consultant, Stockholm, Sweden


An exaggerated flux of glucose through the polyol pathway (as can occur in diabetes) with the subsequent accumulation of sorbitol, was originally proposed as the basic event in the aethiology of secondary diabetic complications. For decades this has meant targeting the enzyme for a specific and strong inhibition. However, the ability of AR to reduce toxic alkenals and alkanals, which are products of oxidative stress, poses the question of whether AR might be better classified as a detoxifying enzyme, thus raising doubts as to the unequivocal advantages of inhibiting the enzyme. This paper provides evidence of the possibility for an effective intervention on AR activity through an intra-site differential inhibition. Examples of a new generation of aldose reductase “differential” inhibitors (ARDIs) are presented, which can preferentially inhibit the reduction of either hydrophilic or hydrophobic substrates. Some selected inhibitors are shown to preferentially inhibit enzyme activity on glucose or glyceraldehyde and 3-glutathionyl-4-hydroxy-nonanal, but are less effective in reducing 4-hydroxy-2-nonenal. We question the efficacy of D, L-glyceraldehyde, the substrate commonly used in in vitro inhibition AR studies, as an in vitro reference AR substrate when the aim of the investigation is to impair glucose reduction.Aldose reductase (AR) is an NADPH-dependent reductase, which acts on a variety of hydrophilic as well as hydrophobic aldehydes. It is currently defined as the first enzyme in the so-called polyol pathway, in which glucose is transformed into sorbitol by AR and then to fructose by an NAD An incrrogenase , an imbarogenase , and an rogenase -9. All tRecently, AR has been shown to be involved in ischemic and inflammatory processes -12 and ta priori overall advantage in inhibiting the enzyme.All these aspects raise doubt for an The possibility of selectively intervening on the enzyme’s catalytic action on specific substrates, such as glucose, is a clear benefit as it leaves the reduction of damaging molecules such as HNE unaffected or partially affected. These aldose reductase differential inhibitors (ARDIs) have the potential to target AR in strict relation to the substrate that the enzyme is working on. This means that damaging events (i.e. sorbitol and GS-DHN generation) could be blocked  without affecting the detoxification ability of the enzyme (i.e. HNE reduction).The fact that hydrophilic molecules, such as GAL, glycol aldehyde or L-threose, and hydrophobic molecules, such as HNE, are similarly effective as AR substrates -35, suggThe peculiar ability of AR to intervene both on hydrophilic and hydrophobic substrates without being a permissive enzyme opens the possibility to identify ARDIs to be developed as possible useful instruments to modulate AR activity. The possibility of inhibiting AR while acting on aldoses but not on toxic aldehydes, previously recommended as a valuable task to be reached , was strIn this paper evidence is presented for an effective intervention on AR activity through an intra-site differential inhibition. Our data, which represent the results of what we believe is the first experimental attempt to use ARDIs in AR inhibition studies, suggest that searching for ARDIs may be a suitable approach.NADPH, D, L-glyceraldehyde (GAL), D-glucose, dithiothreitol (DTT), sodium EDTA, were purchased from Sigma-Aldrich Italy ; HNE dimethylacetal was purchased from Enzo Life Science Inc.  and the free aldehyde was obtained with the addition of 1 mM HCl; GS-HNE came from Cayman Europe . All inorganic chemicals  were of reagent grade.Molecules tested as AR inhibitors are identified by code numbers in bold. The structures of the compounds tested as AR inhibitors are reported in a) pyrimidinones 1-8 [9 [d) isothiazole10 [a) benzimidazoles 11,12 and the benzimidazole 13 [17 [a)pyrimidines 18-20 and amides 21-25 were synthesized as reported in the The pyridoazole 13 , benzisoe 13 [17  were synkcat values were obtained on the basis of a molecular weight of AR of 34 KDa. The purified enzyme was stored at 4°C in a 10 mM sodium phosphate buffer pH 7.0 supplemented by 2 mM dithiothreitol and was extensively dialyzed against a 10 mM sodium phosphate buffer pH 7.0 before use.If not otherwise specified AR activity was determined at 37°C using 4.7 mM D, L-GAL as a substrate in 0.25 M sodium phosphate buffer pH 6.8 containing 0.38 M ammonium sulfate, 0.5 mM EDTA, and 0.2 mM NADPH. AR was purified from bovine lens to electrophoretic homogeneity as described . The finThe nominal concentrations of HNE and GS-HNE solutions used as substrates of AR were determined through a colorimetric assay . These vProtein concentration was determined according to Bradford  using bokcat and KM for GAL and HNE of 47 ± 3 and 48 ±1 min-1 and 39 ± 4 and 37 ± 1 µM, respectively.GAL and HNE used as substrates for AR displayed a very similar kinetic behaviour. As shown in M for GAL is in line with previously reported data for AR from different sources, including the bovine lens enzyme [M measured for HNE is reasonably close to the one measured on human skeletal muscle (22 µM) and human placental recombinant (28 ± 9 µM) enzymes [M measured for HNE appears approximately five fold higher than the values reported for the AR from bovine tissues, namely the lens (8.8±0.9 µM) and the heart (7 ±2 µM) [+ has been reported to exert protection [kcat (41 ± 2 min-1) with approximately a 3 fold increase of the KM (105 ± 6µM) with respect to the parameters measured for HNE  ,52. In a7 ±2 µM) . In factotection , enzyme otection  to the Aotection , a modes for HNE . Howevert to HNE . Concerns enzyme .M and kcat) displayed by GAL and HNE represents an ideal condition to compare the ability of different molecules to behave as ARDIs. Thus, the susceptibility of AR to the inhibition exerted by a series of compounds (including some well-known ARIs) was tested at various inhibitor concentrations, using both GAL and HNE as substrates. The results of this screening , to -25% (i.e. GAL reduction is favored), exerted by compound 16. Despite the number of compounds tested, it is difficult at this point to find a relationship between structure and ARDI behavior, mainly because of the structural heterogeneity as well as of the wide range of inhibitory power of the compounds. In this regard, the available PDB data of AR structures co-crystallized with very strong inhibitors are, as such, less helpful in finding special structural requirements for ARDIs than one would expect. In fact, they are lacking of the most relevant information concerning the reciprocal influence between the substrate and the inhibitor, both interacting with the enzyme.The similarity of the kinetic parameters (Kcreening  are expr21-27). Indeed, a significantly lower value of ”GAL/HNE differential inhibition” was observed (approximately 7%) when L-glyceramide (compound 22) was used instead of the D-enantiomer , appears to be confirmed for inhibitors of the same class as well. In fact, starting with GAM, a variety of differential inhibitory effects was observed for different aldonamides (compounds 50 of approximately 8 mM) more efficiently than the HNE reduction  [M value.However, the kinetic behavior of both glucose and GS-HNE is very different from HNE (and consequently from GAL), which must be taken into account when directly comparing the differential inhibition data. In the case of glucose, for instance, the concentration used in the assay (7 mM), which is the blood concentration occurring in moderate hyperglycemic conditions, led to sub-saturating conditions considering the rather high K 212 mM) ,50,56-5850 of approximately 10 mM with GAL as substrate), we failed to target high differential intra-site specificity and high inhibitory power at the same time.These data clearly show the potential of a specific AR intra-site differential inhibition. However, considering the differential inhibition values and the inhibitor concentrations required for GAM to exert inhibition , whose negligible GAL/HNE differential inhibition , they appear to have the potential to be developed as effective ARDIs.The problem of underestimating the possible differential inhibition ability of molecules is illustrated in see also  an almosM values  and S5. 18 and 19 are also able to inhibit, to a different extent, the AR catalyzed reduction of GS-HNE  as a pro-inflammatory signal ,25, we bin vivo inhibition of glucose reduction, then glucose itself, possibly combined with GAL, should be used to screen the ARDI potency in vitro. Similarly, when looking for molecules that can differentially inhibit GS-HNE reduction with respect to HNE reduction, GS-HNE itself should be used as a substrate of AR.We believe that the evidence presented here opens the way for a new strategic approach to AR inhibition. The focus of this work is not only on the features of potential ARDIs, but also on the type of experimental approach to be adopted in looking for ARIs. Indeed, by extending the same arguments used to predict the potential of a differential inhibition between substrates of different classes to different substrates of the same class , our results strongly suggest that whenever the aim of the enzymological study on AR is the Finally, we firmly believe that it is time to switch from ARIs to ARDIs. In fact, ARDIs could be valuable for investigating AR-based useful/deleterious mechanisms and possibly for clarifying the relative impact of the double-sided action of this enzyme in experimental models. We also propose that a new strategic approach to controlling enzyme activity against diabetic complications and/or inflammation could be adopted.Figure S1Enzymatic titration of HNE and GS-HNE.(DOCX)Click here for additional data file.Figure S2Effect of pH on the reduction of HNE and GS-HNE catalyzed by AR.(DOCX)Click here for additional data file.Figure S3Selwyn test for AR acting on HNE and GAL as substrates.(DOCX)Click here for additional data file.Figure S4Inhibition models of compound 18 on the AR dependent reduction of different substrates.(DOCX)Click here for additional data file.Figure S5Inhibition models of compound 19 on the AR dependent reduction of different substrates.(DOCX)Click here for additional data file.Protocol S1Synthesis of AR inhibitors.(DOCX)Click here for additional data file.Table S1Compounds Tested as Differential Aldose Reductase Inhibitors.(PDF)Click here for additional data file.Table S2Compounds Tested as Differential Aldose Reductase Inhibitors.(PDF)Click here for additional data file.


The relative gain in amplitude after TS vs. SS was negatively correlated with the SSR amplitude after SS ipsilateral (p < 0.0005) and contralateral to the stimulus site (p < 0.01). The increase in SSR amplitudes after TS compared with SS is in line with temporal summation of the excitatory synaptic input in neurons generating the SSR. Driving the SSR with TS is of possible relevance for the investigation of disorders of the peripheral or central autonomic nervous system.It is well known that the size of the sympathetic skin response (SSR) depends on the stimulus strength. In the present investigation train stimuli (TS) were employed to study the behavior of the SSR when recruited above the usual level. The SSR was obtained in healthy human subjects over the palm of the hand after supramaximal single stimuli (SS) and trains of three  over the ipsilateral superficial radial nerve in 15 healthy volunteers. Ipsilateral to the stimulus site SSR amplitudes were 5.7 ± 5.3 (SS) and 7.7 ± 5.9 mV (TS;  The sympathetic skin response (SSR) is one of the most important and intensively studied neurophysiological investigations of the autonomous nervous system. The method is easy to perform  was used. The recording electrode was placed on the palm, while the reference electrode was placed on the back of the hand. Filter settings were 0.05 Hz and 40 Hz. Three SS were delivered without warning at intervals of 2–4 min. After a break of 10 min, to recover the starting conditions three TS were given at similar time intervals. SSR N1/P1-amplitudes were measured throughout. All experiments were done by the same examiner at a similar daily time window between 5 and 7 p.m. in the evening, in a quiet, semidark room, with a constant temperature of 24.0°C and humidity of 30–35%. Skin temperature was between 31°C and 32°C. Subjects were asked to remain calm and quiet throughout the stimulating procedure.Informed consent was obtained from all volunteers examined. The Ethical committee approval was granted for this investigation from the Ethics committee of the University Hospital of the Martin-Luther-University Halle-Wittenberg, Germany. The SSR was obtained with surface electrodes over the palm vs. the back of the hand after SS and TS over the right superficial radial nerve at the wrist with an interstimulus interval of 3 ms in 15 healthy volunteers at the age between 21 and 45 years. The Multiliner E-Systempost hoc. Wilcoxon’s test was furthermore used to compare the amplitudes obtained after SS and TS. p-Values below 0.05 were considered as significant.The non-parametric Friedman ANOVA was used to test for statistical significant differences when results obtained after the first, the second, and the third stimulus were compared. Wilcoxon’s test was then taken In almost all instances SSR could be obtained in the 15 subjects after each of three SS and TS from the ipsilateral and contralateral hand, respectively Figure . There wp all <0.005, Wilcoxon’s test), except for the response following the first stimulus contralateral to the stimulus site. When the gain in amplitude was plotted as the ratio of SSR after TS divided by the according responses obtained after SS, it became obvious that this gain was larger contralateral to the stimulus site than ipsilateral . Furthermore, there was a negative relation of SSR amplitudes measured after TS compared with SS  and contralateral (p < 0.002) to the stimulus site .Sympathetic skin response amplitude values were significantly larger after TS as compared with SS on either hand side  but not from the preceding to the next TS, respectively. Thus, the decremental response with subsequent stimuli was less large after TS than after SS, as can also be seen from Figure p < 0.05, Friedman test; p < 0.05, Wilcoxon’s test), and no additional significant change was noted between the responses obtained after the first and second SS or within the SSR amplitude-results after TS.Sympathetic skin response amplitudes declined significantly on the stimulus site from the preceding to the next SS (The SSR is widely used among other tests for routine purposes to assess the integrity of peripheral and central autonomous functions. The normal values measured for amplitude and latency of the SSR obtained in the present study are well within previously given figures (Shahani et al., p < 0.05; Figure The SSR amplitude-augmentation was significantly larger in the case of SSR of low amplitudes obtained after SS. This relation may be due to the fact, that eventually there are limited numbers of sweat glands available to take part in forming the SSR. When only few sweat glands are recruited by SS, then comparatively large additional numbers of sweat glands may be recruited by way of TS. When, however, a large number of sweat glands is already recruited by SS, the number of sweat glands that can be additionally activated by TS may be small. This inference is supported by the fact that the gain in SSR amplitudes from SS to TS was significantly larger when the amplitudes after SS were lower, i.e., contralateral as compared to ipsilateral to the stimulus site (Sympathetic skin response amplitudes in the present study showed a statistically significant decline when responses to subsequent stimuli were compared. This data confirms previous reports on habituation of the SSR investigated using SS alone (Cariga et al., Taken together, TS can be used to augment SSR significantly. It has to be elucidated further whether this SSR-augmentation phenomenon will prove to be clinically meaningful.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.


Using microarray analysis, we found that c-Myc and AR expression levels strongly correlated with each other in tumors from patients with castration-resistant prostate cancer (CRPC) progressing despite ADT. We confirmed that AR directly regulates c-Myc transcription in a ligand-independent manner, that AR and c-Myc suppression reduces ligand-independent prostate cancer cell growth, and that ectopic expression of c-Myc attenuates the anti-growth effects of AR suppression. Importantly, treatment with the bromodomain inhibitor JQ1 suppressed c-Myc function and suppressed ligand-independent prostate cancer cell survival. Our results define a new link between two critical proteins in prostate cancer – AR and c-Myc – and demonstrate the potential of AR and c-Myc-directed therapies to improve prostate cancer control.The androgen receptor (AR) is the principal therapeutic target in prostate cancer. For the past 70 years, androgen deprivation therapy (ADT) has been the major therapeutic focus. However, some patients do not benefit, and those tumors that do initially respond to ADT eventually progress. One recently described mechanism of such an effect is growth and survival-promoting effects of the AR that are exerted independently of the AR ligands, testosterone and dihydrotestosterone. However, specific ligand-independent AR target genes that account for this effect were not well characterized. We show here that  Prostate cancer is the most common cancer in men in the United States with 241,740 new cases anticipated this year ADT works by lowering levels of the potent AR ligands testosterone and dihydrotestosterone (DHT) or interfering with binding of androgen ligands to the androgen receptor (AR) protein, the principal therapeutic target in prostate cancer There are several possible explanations for AR-dependent mechanisms of progression despite the suppression or interference with androgen ligands. These include intratumoral androgen synthesis, the generation of constitutively active AR transcript variants, AR gene amplification, activating AR mutations, or activation of the AR by growth factors c-Myc. This is because: 1) c-Myc overexpression promotes prostate cancer development c-Myc is upregulated in androgen ligand-dependent prostate cancer and further upregulated in CRPC c-Myc gene c-Myc was a direct AR target gene and whether androgen ligands were necessary for AR regulation of c-Myc expression.To understand genes that might account for that effect, we focused on c-Myc upregulation in human CRPC tumors correlates with AR upregulation, and we confirmed that c-Myc is a direct AR target gene using chromatin immunoprecipitation (ChIP) assays. c-Myc suppression achieves the same overall effects as AR suppression, and c-Myc overexpression attenuates the anti-growth effects of AR suppression. While AR promotes c-Myc expression, treatment with androgen ligands did not increase c-Myc expression. Thus, AR promotes the expression of c-Myc in a ligand-independent manner, and c-Myc is a key AR target gene.We determined that c-Myc RNAi and reduced prostate cancer cell survival in androgen ligand-depleted conditions.Finally, we treated prostate cancer cells with the BET bromodomain inhibitor JQ1 that suppresses c-Myc expression c-Myc is a key androgen ligand-independent AR target gene that contributes to androgen ligand-independent but AR-dependent prostate cancer cell survival. Our results also demonstrate the potential of AR-directed therapies or c-Myc-directed therapies in prostate cancer as adjuncts to ADT.Our studies clarify that AR and c-Myc are commonly increased in human CRPC tumors progressing despite ADT AR and c-Myc was linked with the other in human CRPC tumors. Therefore, we determined the expression levels of AR and c-Myc using gene expression microarrays in 140 human CRPC tumors versus 15 normal prostate samples. Next, we examined the association of AR upregulation and c-Myc upregulation in the human CRPC tumors. AR mRNA levels in CRPC samples were strongly associated with c-Myc mRNA levels    . We alsoct Test) .AR with RNAi in prostate cancer cells grown in charcoal-stripped, androgen ligand-depleted serum. AR RNAi reduced cell growth of both androgen ligand-dependent LNCaP cells and their CRPC derivatives called LNCaP-abl  and in another CRPC cell line 22RV1 that expresses both full-length AR and an AR transcript variant. Although we cannot exclude a role for the AR transcript variant in also promoting c-Myc expression, our results with AR RNAi in LNCaP and Abl and our results with AR overexpression in M12 cells demonstrate that full-length AR is capable of activating c-Myc expression  and grown in 10% charcoal-stripped fetal bovine serum for all experiments. Abl cells, a CRPC derivative of LNCaP, were a kind gift from Zoran Culig, PhD and were grown in RPMI with 10% charcoal-stripped fetal bovine serum. M12 prostate cancer cells expressing empty vector or AR were a kind gift from Stephen R. Plymate, PhD, and grown as described previously AR: 5′-GACCUACCGAGGAGCUUUCUU-3′, and c-Myc: 5′-GAGCUAAAACGGAGCUUUUdTdT-3′) by using DharmaFECT 3 (Dharmacon) transfection reagent for a final concentration of 50 nM For RNAi experiments, LNCaP and abl cells were transfected with RNAi oligonucleotides (R1881 (Sigma) was resuspended in 100% ethanol. MDV3100 was purchased (Selleckchem) and resuspended in DMSO. JQ1 was purchased from Sigma and resuspended in DMSO. Cell growth was determined at the end of treatment with the trypan blue exclusion method (Life Technologies) following manufacturers’ instructions. All cell culture experiments were performed with biological triplicate samples and confirmed in repeat experiments.c-Myc  or pCMV6-AN-His (Origene) and selecting with G418.LNCaP cells with stable overexpression of c-Myc or empty vector were generated by transfecting LNCaP cells with pCDNA-DEST40-c-Myc were plated in 10 cm dishes. RPMI with 10% charcoal-stripped fetal bovine serum supplemented with 300 ug/ml G418 and 10 ug/ml bicalutamide treatment was added to the cells every other day for 14 days. Next, the cells were fixed with 4% formaldehyde and stained with syto60 (Invitrogen). Images were taken with Licor Odyssey imaging system.200,000 LNCaP cells with stable overexpression of empty vector (EV) or Immunoblotting experiments were performed as described previously RNA was extracted from cell pellets stored in RNAlater reagent (Life Technologies) using a MagMAX Total RNA Isolation kit (Life Technologies) or Trizol (Invitrogen) according to the manufacturer’s instructions. RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop). 250 ng–1 ug RNA  was reverse-transcribed using a High-Capacity cDNA Reverse Transcription kit (Life Technologies) with random primers. Realtime (QRT)-PCR was performed using a 7500 Fast thermocycler (Life Technologies) with the following cycling program: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 sec dissociation, 60°C for 1 min annealing/extension/read. 10 µL singleplex RTPCR reactions contained 1X Taqman universal standard mastermix, 1X Taqman hydrolysis probe, and 10 ng RNA-equivalent cDNA template. Human beta-actin (#4326315E) was used as an endogenous control. Primer information is included in 6 cells to immunoprecipitate DNA overnight at 4°C. 1% of chromatin was removed prior to immunoprecipitation as input. Immune complexes were collected with protein A magnetic beads . After extensive washing, immune complexes were released, cross-links were reversed, and DNA was purified with mini-elute PCR purification kit (Qiagen) and eluted with 60 µl EB. Realtime QPCR was performed as described above using 1X SYBR GreenER mastermix (Life Technologies), 750 ng of each primer, and 2 µl of the immunoprecipitated DNA or 2 µl of the 1% input in a 20 µL reaction. Immunoprecipitated DNA was calculated as “fold enrichment over IgG” with ddCt method. Primer information is included in 5 µg of anti-AR antibody , anti-acetylated histone H3 (AcH3) antibody (Millipore) or normal rabbit (IgG) antibody (Millipore) were added to sheared, formaldehyde cross-linked chromatin derived from 1 to 2×10Data are expressed as standard deviation of the mean. All PCR results and cell count results represent one single experiment performed in triplicate. p-values were calculated from one single experiment with two-tailed unpaired student’s T-test in Excel (Microsoft) or Study Results 1.0 software. Each experiment was confirmed in two to three separate experiments.http://research4.dfci.harvard.edu/brownlab/datasets/index.php?dir=Wang_AR_Data/for both abl and LNCaP cells. We filtered the data requiring that each peak have a FDR <5% and be within 50 Kb of either the 5′ or 3′ end of a RefSeq gene. Using these criteria, we found overlapping peaks with proximity to the c-Myc locus at chr8∶128844491–128845592 (∼21.6 Kb from c-Myc) in abl cells and chr8∶128844572–128845592 (∼21.7 Kb from c-Myc) in LNCaP cells.The AR ChIP-microarray data was downloaded from AR or c-Myc z-scores greater than 2.Agilent 44 K whole human genome expression oligonucleotide microarrays  were used to profile 140 human castration-resistant prostate cancer soft tissue metastases from 55 patients. Tissue samples were collected from autopsies performed at the University of Washington Medical Center under the rapid autopsy program with Institutional Review Board approval as described previously Figure S1Co-suppression of AR and c-Myc does not lead to greater anti-tumor activity than suppression of either protein by itself. A) LNCaP cells were transfected with 50 nM of NTC, AR, c-Myc, or both AR and c-Myc RNAi oligonucleotides. Cells were switched to charcoal-stripped serum on the day of transfection. Cell growth was determined 5 days later with the trypan blue exclusion method. *denotes p<0.01 compared to NTC. B) Immunoblotting was performed to determine the levels of AR, c-Myc, and actin.(TIF)Click here for additional data file.Figure S2c-Myc over expression promotes ligand-independent prostate cancer growth. A) The same number of LNCaP cells with stable overexpression of empty vector or c-Myc were grown in 10% charcoal-stripped fetal bovine serum. Cell number was determined 1, 4, and 7 days after plating with the trypan blue exclusion method. Cell growth was calculated compared to day 1. p<0.01 for both time points. B) The same number of LNCaP cells with stable overexpression of empty vector or c-Myc was plated. Cells were grown in charcoal-stripped serum supplemented with bicalutamide for 14 days. Colony formation was determined.(TIF)Click here for additional data file.Figure S3MDV3100 treatment does not reduce c-Myc expression. LNCaP, abl, and 22RV1 cells were grown in androgen-replete serum and treated with 10 µM MDV3100 or vehicle for 24 hours. QRT-PCR was performed to determine the mRNA levels of KLK3 and c-Myc relative to actin. *denotes p<0.001 compared to vehicle.(TIF)Click here for additional data file.Figure S4AR overexpression promotes c-Myc upregulation. AR or empty vector was stably overexpressed in M12 prostate cancer cells that do not express endogenous AR c-Myc relative to actin. B) Immunoblotting was performed to determine the levels of AR, c-Myc and actin. *denotes p<0.003 compared to empty vector.(TIF)Click here for additional data file.Table S1Primers used for ChIP-PCR and QRTPCR.(DOCX)Click here for additional data file.