Beta-blockade is contraindicated in severe aortic regurgitation (AR) due to the fear of prolonging diastole and thus aggravate regurgitation. However, this has never been scientifically proven and positive effects of targeting the sympathetic system in AR has been demonstrated in several studies.Thirty-nine Sprague–Dawley rats with AR were randomized to ten weeks of medical treatment with carvedilol or no treatment. Treatment was initiated either early or late after AR induction. The effect of carvedilol was assessed by serial echocardiography and invasive hemodynamic measurements.AR resulted in eccentric hypertrophy and left ventricular (LV) dysfunction. LV remodeling and function as measured by echocardiography was unaffected by treatment. LV dimensions were similar between treated and untreated groups and measures of LV performance (including strain and strain rate) were also unaltered. This result was confirmed by invasive measurements showing maximal and minimal pressure–time development, LV volumes, and LV pressures, to be unaltered by treatment. On the contrary, despite relative bradycardia carvedilol did not reflect any negative impact on the heart.Carvedilol did not improve left ventricular remodeling or function in rats with surgically induced AR. Despite relative bradycardia, we did not find carvedilol to negatively impact the heart, either when treatment was initiated early or late in the course of disease. Chronic aortic regurgitation (AR) is a common heart valve disease with high morbidity and mortality rates once symptoms occur  group, two from the AR + CAR (early) group, one from the AR (late) group, and one from the AR + CAR (late) group. All rats dying were found dead in the morning with no preceding signs of heart failure. At sacrifice heart weight in sham (early) operated rats was significantly lower than in rats with AR (early), while no significant difference was found between AR and AR + CAR in either the early or the late treated groups, Table At baseline no echocardiographic differences were observed between groups. Rats with AR developed eccentric hypertrophy with increased LV dimensions (LVEDD and LVESD) and wall thickness, Table Thus, we did not find carvedilol to alter the development of eccentric remodeling or LV dysfunction as measured by echocardiography, regardless of treatment–start.Invasive hemodynamic measurements were performed at baseline and repeated immediately before sacrifice. Although only borderline significant in the late treated group we observed a lower heart rate in carvedilol–treated compared to untreated rats.max and dp/dtmin) or LV volumes (relative end diastolic or end systolic volume) compared to untreated rats. Furthermore, end–diastolic and end–systolic LV pressures were unaltered by treatment in both study-legs, Table Carvedilol did not result in any significant improvement in LV maximum or minimum pressure–time development  and carvedilol slightly reversed this mild decrease (71 ± 1.4% in treated vs. 66 ± 0.5% in untreated rats). In another study invasive hemodynamic measurements were not performed but echocardiography showed improved systolic and diastolic function in rats treated early with metoprolol for twenty–four weeks. However, in this study LV systolic function was also only marginally affected by treatment. We did not find carvedilol to improve LV systolic function. In the studies showing positive effects systolic function, LVEF was within the limits of reference and did not decrease significantly during the course of disease. This may reflect a more benign type of AR where six months of severe volume overload does not impair systolic function. Our model somehow resulted in a reduction in LV function measured by FS and speckle tracking echocardiography and may represent a more progressive type of disease less susceptible to the positive effects of beta–blockade.Previous studies have shown inconsistent effects on LV performance. In one study, long–term treatment with metoprolol did not change pressure development or LVEF after 6 and 12 months  animal studies cannot and should never be extrapolated to humans.Using a higher dose of carvedilol or a different beta-blocker (e.g. a selective) may show more pronounced results. However, we sought to only bluntly inhibit the sympathetic system in fear of increasing regurgitation. The dose of carvedilol chosen resulted in a heart rate drop of 5-10% compared to non-treated AR groups. This is similar to what has previously shown significant effects on the heart on other experimental studies. Although selective and non-selective beta-blockers have pharmacological actions in common they differ greatly, and cannot be directly compared. Thus, we interpret our results in the scope hereof.Due to a small sample size it is hard to conclude definitively on our results and thus we suggest a larger scale study to investigated this interesting field. However, we believe that we have interpreted our findings accordingly.Carvedilol did not improve left ventricular remodeling or performance in rats with surgically induced severe AR. Despite relative bradycardia, we did not find carvedilol to negatively impact the heart, either when treatment was initiated early or late in the course of disease.−1 and Xylazine 10 mg kg−1) injection. AR perforation and severity was confirmed by echocardiography during the procedure. Treatment was performed by mixing carvedilol (10 mg/kg/d) in transgenic dough–diet , which was fed to rats daily for the duration of medical treatment. All animal experiments were approved by the Johns Hopkins University Institutional Animal Care and Use Committee.AR was created in rats by echocardiography–guided closed–chest operation, as described elsewhere  included in this part of the study were divided into three groups: Sham–operated rats receiving no treatment (Sham (early), n = 8), rats with AR receiving no medical treatment (AR (early), n = 8), and rats treated with carvedilol beginning two weeks after AR induction (AR + CAR (early), n = 9). In sham–operated rats all procedures were performed, except perforation of aortic valves. All rats were sacrificed twelve weeks after induction of AR.Fourteen male Sprague Dawley rats (age 9–10 weeks) were subjected to severe AR and divided into two groups: Controls with severe AR receiving no medical treatment (AR (late), n = 6) and rats with severe AR treated with carvedilol beginning at twelve weeks after induction of AR (AR + CAR (late), n = 8). All rats were sacrificed twenty–two weeks after induction of AR.Early study: 0 (baseline), 2, 6, 9 and 12 weeks after AR induction. Late study: 0 (baseline), 2, 6, 9, 10, 12, 16, 19, 22 weeks after AR induction. Regurgitation was confirmed by a color-Doppler ratio of regurgitant jet >50% of the LV outflow tract diameter. LV measurements were assessed by conventional M-mode echocardiography; end diastolic diameter (LVEDD), end systolic diameter (LVESD), shortening fraction (FS) and wall thickness (WT). Measurements were averaged from four beats, two parasternal long–axis (PLAX) and two short–axis (SAX) planes. FS was calculated as [LVEDD–LVESD]/ [LVEDD]. As a measure of WT, septum and LV posterior wall were averaged. Speckle tracking was performed by acquiring 2D cine loops from at least six cardiac cycles, in mid–ventricular SAX plane at frame rates >70 s−1. Tracking was performed by using at least three consecutive cardiac cycles in EchoPAC’s strain modality analysis software . Averaged circumferential deformation represented by the global trace was used. For strain–rate measurements the global traces were used.Echocardiography was performed on a Vivid 7 machine  with a 14 MHZ linear vascular probe in unconscious rats anesthetized with gas (Isoflurane 1.5–2.0%). Examinations were performed at following time points: A micro–tip conductance pressure–volume catheter  was inserted into the heart by right–sided carotid arteriotomy using a classic closed–chest approach  and PASW statistics 18.0 .


Liquidambar formosana, considered as an AR-sensitive tree species, was largely injured by AR, compared with Schima superba, an AR-tolerant tree species. To clarify the different responses of these two species to AR, a comparative proteomic analysis was conducted in this study. More than 1000 protein spots were reproducibly detected on two-dimensional electrophoresis gels. Among them, 74 protein spots from L. formosana gels and 34 protein spots from S. superba gels showed significant changes in their abundances under AR stress. In both L. formosana and S. superba, the majority proteins with more than 2 fold changes were involved in photosynthesis and energy production, followed by material metabolism, stress and defense, transcription, post-translational and modification, and signal transduction. In contrast with L. formosana, no hormone response-related protein was found in S. superba. Moreover, the changes of proteins involved in photosynthesis, starch synthesis, and translation were distinctly different between L. formosana and S. superba. Protein expression analysis of three proteins  by Western blot was well correlated with the results of proteomics. In conclusion, our study provides new insights into AR stress responses in woody plants and clarifies the differences in strategies to cope with AR between L. formosana and S. superba.Acid rain (AR) is a serious environmental issue inducing harmful impacts on plant growth and development. It has been reported that  Acid rain (AR) emerged as a serious environmental issue as a consequence of the increasing industrial activities throughout the world Liquidambar formosana and Schima superba are both dominant broad-leaf tree species and are distributed over large surface areas in the forest of southern China S. superba, L. formosana was largely injured by AR during the past decade, which had negative impacts on forest ecosystem L. formosana seedlings than S. superba’s seedlings, e.g., seed germination, seedling growth, photosynthesis, antioxidant system, etc. L. formosana is considered as an AR-sensitive species, while S. superba is an AR-tolerant species. Although the differential responses of L. formosana and S. superba to AR have been analyzed at the morphological and physiological level, a comprehensive elucidation of the molecular mechanisms underlying the different strategies to cope with AR between two tree species is still needed.Arabidopsis thalianaProteomics is a powerful tool for providing new insights into complete proteomes at the organ, tissue and cell levels L. formosana and S. superba, that are different sensitive to AR tolerance, when submitted to simulated AR (pH 3.0) treatment for one month. Based on two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) analysis, a comprehensive inventory of proteins regulated by AR was established in the two tree species. The overall objectives of this study are (1) to provide valuable insights into AR stress responses in woody plants; (2) to clarify the differences in strategies to cope with AR between L. formosana and S. superba.In the present study, we initiated a comparative proteomic study to systematically investigate the changes in protein profile in two broad-leaf tree species, L. formosana and S. superba were purchased from Tree Seed Centre of Shuyang County in Jiangsu Province, China. The seeds have been mixed together when they were collected from independent individuals and families. Seeds were surface-sterilized with 0.5% hypochlorite for 30 min, then washed thoroughly with distilled water. Then the seeds were germinated in a soil/vermiculite (1∶1) mixture in an environmentally controlled growth chamber. For each species, three weeks old healthy seedlings with similar size were randomly transplanted into individual pots, each with a dimension of 24 cm (open top) × 13 cm (height) × 15 cm (flat bottom), and filled with soil/vermiculite (1∶1) mixture. Fifteen seedlings were planted in one pot. All seedlings were cultivated in the same controlled growth chamber with a daily temperature regime of 28/25°C (day/night), relative humidity of 60–70% and a 12-h photoperiod at 210 µmol m−2 s−1 photosynthetically active radiation (PAR). Three months later, the seedlings were divided into control group (CK) and simulated AR treatment group (AR). Each group had at least three replicates. The control group and simulated AR treatment group were sprayed once per day with the control (pH 5.6) solution and AR (pH 3.0) solution, respectively. The ion compositions of the control solution was adapted from Fan and Wang 2SO4 and HNO3 in the ratio of 5∶1. The final concentration of H2SO4 and HNO3 were 0.45 and 0.09 mM, respectively, which represents the average ion composition of rainfall in southern China 2O2 content and so on. The remaining leaves were stored at −80°C for proteomic and Western blot analysis.Seeds of −1 fresh weight (FW)) was determined as described previously At least 30 fully expanded leaves of each species were randomly selected from control and AR treatment groups and photographed using a digital camera. The necrosis percentage was calculated as described previously Three seedlings per species were randomly chosen from different pots and at least two fully emerged leaves per plant were selected for net photosynthetic rate (Pn) and chlorophyll fluorescence measurements. Pn was performed with a portable photosynthesis system , as described previously 2•-) and H2O2 content was measured following the method of Chen et al Proline content was measured according to the method of Jiang et al g, 4°C) for 15 min. The phenol phase was collected and proteins were precipitated with ammonium acetate in methanol for 10 h at −20°C. After centrifugation, the supernatant was discarded and the pellet was washed for three times using cold acetone containing 10 mM DTT. The washed protein pellets were dissolved in a lysis buffer  3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), 1% (w/v) DTT and 0.5% (w/v) IPG buffer pH 4–7) at room temperature. The total protein concentration of the lysates was determined using a Bio-Rad protein assay reagent .Protein extraction was performed using phenol-based protocol described by Liu et al Two-dimensional electrophoresis was conducted according to the methods of Bai et al http://www.matrixscience.com) and National Center for Biotechnology Information (NCBI) protein database (http://www.ncbi.nlm.nih.gov) . PMF search parameters were set up as described previously http://www.uniprot.org) and/or NCBI protein database (http://www.ncbi.nlm.nih.gov) for updated annotation and homologous proteins identification. Afterwards, the successfully identified proteins were further classified using Functional Catalogue software (http://mips.gsf.de/projects/funcat).The protein spots, which were differentially displayed under AR treatment, were excised from the preparative 2-D gels and digested by trypsin. After digestion, the peptide solution was collected and peptide mass fingerprint (PMF) was acquired using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis  as described previously L. formosana and S. superba leaves were separated by 12% w/v standard sodium dodecyl sulfate polyacrylamide gel electrophoresis and then electrophoretically blotted to polyvinylidene difluoride membrane for 50 min. The membranes were blocked over-night with Western Blocking Buffer . Protein blots were probed with primary antibodies of Rubisco large subunit (RuBISCO LSU) , ascorbate peroxidase (APX)  and glutathione-S-transferase (GST) , at dilution of 1∶5000, 1∶2000 and 1∶1000, respectively, for 4 h at room temperature with agitation. Next, the membranes were washed in phosphate buffered saline with Tween-20 solution (PBST) solution  three times and incubated with anti-rabbit IgG horseradish peroxidase (HRP) conjugated to alkaline phosphatase  for 1 h at room temperature to detect primary antibodies. β-actin  was used as an internal control. After several washes with PBST solution, membranes were incubated in an enhanced chemiluminescence (ECL) substrate detection solution  according to the manufacturer’s instructions. Images of protein blots were obtained using a CCD imager . The optical density values of the protein signals were quantified using the Quantity One software .Western blot analysis was performed as described previously p<0.05) with the SPSS 20.0 package . For proteomic experiment, protein samples for 2-DE gel image analysis were extracted from three independent seedlings grown in three different pots in the same growth chamber. Thus, for each species, three independent biological replicates were performed in 2-DE gel image analysis. Statistic analysis for protein spot on 2-DE gels was performed using Student’s t-test (p<0.05) provided by PDQuest software as mentioned earlier.For physiological measurements, at lease four independent repetitions were used. Values in figures and tables were expressed as means ± se. The statistical significance of the data was analyzed using a univariate analysis of variance  in L. formosana and 6 proteins (spots S29–S34) in S. superba were annotated as predicted or unknown proteins  large subunit  in S. superba. The multiple spots might represent isoforms or different post-translation modification of individual proteins Of the 74 protein spots identified in se to AR . In S. sR stress . Among t superba  and 2. R superba  and 2. Fhttp://www.uniprot.org) and/or NCBI protein database (http://www.ncbi.nlm.nih.gov). AR-responsive proteins were found to be involved in a wide range of biological processes. After AR treatment, with the exception of photosynthesis and energy production related proteins, the abundance of most proteins were decreased in both L. formosana and S. superba , while APX (spot L40) and GST (spot L41) were increased in reatment . As showormosana . No sign superba .Eugenia uniflora. Moreover, chlorophyll content and photosynthesis in L. formosana were also remarkably suppressed by AR treatment in this study  production are commonly used as biochemical markers to monitor the damage level in plants under environmental stress L. formosana and S. superba  and S. superba (spot S11) in our study demonstrate the prime role of ATP synthase in the adaptation of two tree species to AR stress.Besides photosynthesis-related proteins, AR also affected the abundances of energy production-related proteins in L. formosana, AR affected a series of protein abundances involved in several metabolism processes, including nitrogen metabolism , starch and sugar metabolism , lipid metabolism , secondary metabolism , vitamin metabolism (spot L3) and lignin biosynthesis (spot L12) (S. superba (L. formosana than in S. superba.In pot L12) . However superba . It is cL. formosana  in S. superba were identified  and stearoyl-acyl-carrier protein desaturase-like protein , as well as secondary metabolism-related proteins including flavanone 3-hydroxylase  and chalcone synthase  in L. formosana. The abundance of SACPDLP was increased, which may contribute to lipid synthesis and membrane integrity in AR-treated L. formosanaL. formosana.Abiotic stress can also affect starch synthesis entified  and 2. ITillandsia species under 2 µM Cd2+ treatment over 30 days. In this study, H2O2 and O2•- content, and thus oxidative stress, induced by AR was remarkably higher in L. formosana than in S. superba , GST (spot L41) and class III peroxidase ATP32 (spot L44), were identified by proteomic analysis. APX plays an important role in scavenging H2O2 from cells Piuns massonianaL. formosana in this study, implying that antioxidant defense system was provoked by AR in L. formosana. However, the expression levels of APX and GST were not changed in S. superba seedlings under AR treatment , which appears to be a key enzyme in H2O2 detoxification S. superba. The increased TPx may function in resisting AR-induced oxidative damage by reducing ROS production in S. superba. In addition, endochitinase, a glycosyl hydrolase that catalyzes chitin degradation, plays an essential role in forming the fine cell-wall matrix that enhances the physical barrier against abiotic stresses S. superba , CDPK (spot S28) and 14-3-3 protein (spot S15) were increased after AR treatment, while these proteins were not identified in L. formosana. In agreement with the proteomic analysis results, the enhanced gene expression of CaM and CDPK by real-time quantitative PCR was more obvious in S. superba than in L. formosana under AR treatment  also plays a crucial role in regulating plant defense responses to various environmental stimuli reatment . These rL. formosana and S. superba, respectively  production in L. formosana than that in S. superba  and mitochondrial import receptor subunit TOM6 homolog isoform 1 (spot S5) were found in AR-treated leaves  plays important roles in response to abiotic stresses including high and low temperatures, salinity, and water deficit d leaves . ProteasL. formosana Click here for additional data file.Table S1L. formosana.Details of identified proteins and peptides list of each protein in AR-treated (DOC)Click here for additional data file.Table S2S. superba.Details of identified proteins and peptides list of each protein in AR-treated (DOC)Click here for additional data file.


Comprehensive rehabilitation, involving health professionals from various disciplines, is widely used as an adjunct to pharmacological and surgical treatment in people with rheumatic diseases. However, the evidence for the clinical- and cost-effectiveness of such interventions is limited, and the majority of those who receive rehabilitation are back to their initial health status six to 12 months after discharge.To evaluate the goal attainment, health effects and cost-effectiveness of a new rehabilitation programme compared to current traditional rehabilitation programmes for people with rheumatic diseases, a stepped-wedge cluster randomized trial will be performed. Patients admitted for rehabilitation at six centres in the south-eastern part of Norway will be invited to participate. In the trial, six participating centres will switch from a control (current rehabilitation programme) to an intervention phase (the new rehabilitation programme) in a randomized order. Supported by recent research, the new programme will be a supplement to the existing programme at each centre, and will comprise four elements designed to enhance and support lifestyle changes introduced in the rehabilitation period: structured goal-planning, motivational interviewing, a self-help booklet and four follow-up telephone calls during the first five months following discharge. The primary outcome will be health-related quality of life and goal attainment, as measured by the Patient Generated Index directly before and after the rehabilitation stay, as well as after six and 12 months. Secondary outcomes will include self-reported pain, fatigue, a global assessment of disease activity and motivation for change , health-related quality of life as measured by the Short Form 36 Health Survey (SF-36) and utility assessed by the SF6D utility index.The main analysis will be on an intention to treat basis and will assess the clinical- and cost-effectiveness of the structured goal planning and tailored follow-up rehabilitation programme for patients with rheumatic diseases.The findings will constitute an important contribution to more cost-effective- and evidence-based rehabilitation services for people with rheumatic diseases.ISRCTN91433175. Inflammatory rheumatic diseases are characterized by inflammatory processes that may lead to joint damage and various degrees of disability, thus representing a major burden to the individual patient and society. Current treatment focuses on early diagnosis and early use of disease modifying agents. Nevertheless, a major percentage of patients has a residual disability and will need some form of rehabilitation throughout their life.Rehabilitation has been defined by the World Health Organization as “a process aimed at enabling people with disabilities to reach and maintain their optimal physical, sensory, intellectual, psychological and social functional levels. Rehabilitation provides disabled people with the tools they need to attain independence and self-determination”.As such, the rehabilitation process involves several professions and services, and is characterized by being tailored to the patients’ individual needs and challenges, with the aim of reducing the consequences of illness while improving activity, participation and quality of life.Several studies have shown that patients with rheumatic diseases benefit from rehabilitation. However, the effect seems to fade over time, and most patients are back to their initial health status six to 12 months after discharge-7. A majGoal planning is considered as an essential part of rehabilitation practice and is a process directed at setting goals at various levels of function, within different life areas and in various time frames. The keyThe objective of goal setting involves a change in patient behaviour. Hence, theories of behaviour and behavioural change may guide goal-setting interventions-18, and Rehabilitation often addresses health lifestyle changes involving a process that takes a certain amount of time before being settled in as a new habit. The process involves both cognitive and behavioural elements that may by the end of a rehabilitation stay still be unclear and fragile. A folloThe treatment and rehabilitation of complex chronic conditions are often resource-intensive and expensive, both for the individual patient and for society, with muThe main objective will be to evaluate goal attainment, health effects and the cost-effectiveness of a new rehabilitation programme  compared to current traditional rehabilitation programmes  for patients with rheumatic diseases.More specifically, our study will consider the following research questions:• What is the content of patients’ rehabilitation goals throughout the rehabilitation and follow-up period, and are the goals stable over time?• Is the new rehabilitation programme more effective with regard to goal attainment and an improved health-related quality of life compared to traditional rehabilitation programmes for patients with rheumatic diseases?• Which factors  are associated with goal attainment and an improved health-related quality of life during the first year following rehabilitation?• Which of the two programmes  provides the most cost-effective use of health-care resources?The trial will be developed as part of the research activity in a Norwegian regional rheumatology research network in Health Region South-East during the period from 2010–2012, and is funded by a grant from the Regional Health Authority. The rehabilitation branch in the network consists of three clinicians from the National Advisory Unit on Rehabilitation in Rheumatology, six local study coordinators from five departments of rheumatology and one rehabilitation centre, respectively (hereafter termed centres), in addition to three representatives from the Norwegian Rheumatism Association. The group is led by two researchers from the National Advisory Unit on Rehabilitation in Rheumatology (KBH and IK). From March 2011, a research coordinator also works full-time in the project (GB). The group will meet five times during 2010 and the spring of 2011 to develop the trial, including the designing and piloting of the new rehabilitation programme. Additionally, e-mail correspondence, telephone meetings and separate meetings with two of the patient representatives will be used to exchange information and give feedback on protocol and intervention material.This will be a pragmatic, multicentre, stepped-wedge cluster randomized controlled trial,38 in whThe new programme will represent a supplement to the existing rehabilitation programmes, and all patients will, regardless of being included in the trial or not, receive the programme currently delivered at the different participant institutions when they are admitted. However, patients admitted in the intervention phase who do not participate in the trial will not receive a telephone follow-up after being discharged.st 2011 to June 30th 2012 through an invitation from health professionals at each participating centre on admission to the rehabilitation stay. Those who are interested will be screened for eligibility, and receive oral and written information about the study, and to enrol, participants will have to give their written informed consent.A minimum of 312 participants will be included in the period from August 21People will be eligible if they are 18 years or older, are admitted for rehabilitation at one of the six participating centres, have a good understanding of the Norwegian language and have one of the following inflammatory rheumatic diagnoses: rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, systemic lupus erythematosus and juvenile idiopathic arthritis. In addition, patients with generalized osteoarthritis with an affection of the hip and/or knee will be included. Each participant’s diagnosis will be verified by a clinical examination by a physician at admission, according to the diagnostic criteria for each of the specific diagnoses. The exclusion criteria will be cognitive impairment, severe psychiatric disorder or being admitted for rehabilitation after elective orthopaedic surgery.The sample size and power calculations have been estimated for the primary end point in this study by use of the sum score of the Patient Generated Index (PGI) at the six- and 12-month follow-ups. Based on results from a previous study, it is eAccording to the stepped-wedge design, the intervention will be sequentially rolled out at the participating centres over a number of different time periods ;• Exercises in how to practice MI in setting rehabilitation goals;• Using MI in telephone follow-up calls;• Content and use of the self-management booklet;• Logistics, routines for data collection and management of data in the study;• Exercises in how to invite and inform study participants.The second workshop will be held at each participating centre a few weeks before the centre switches from the control to the intervention period. Three persons from the National Advisory Unit on Rehabilitation in Rheumatology  will visit the centre and give lectures and training sessions in project organization, the methods of data collection, privacy matters and MI.Half-way through the data collection period, a third workshop with all participating centres and intervention providers, comprising more lectures and exercises in MI, setting rehabilitation goals, telephone follow-up calls and study logistics will be held. Additionally, providers from the centres that have switched to the new rehabilitation programme will share their experiences, and there will also be time for questions and group discussions related to relevant topics.The last workshop will be arranged when all telephone follow-up calls have been completed. The main aim of this workshop will be to allow for feedback from intervention providers to researchers and vice versa, and to discuss ideas and methods for implementing the new programme into clinical practice.All local study coordinators, intervention providers and representatives from the Norwegian Rheumatism Association (NRA) will be invited to participate in the first, third and fourth workshop, and the local study coordinator and a local representative from the NRA will be invited to participate at the workshops held at each centre.Both groups will receive medical treatment as usual.The control group will receive the traditional rehabilitation programme provided at each participating centre at the start of the study ,43, whicA summarized index score of PGI will be generated as follows: Each area ratings from stage 2 will be multiplied by the points given to the area in stage 3. Thereafter, the product of all areas will be summed up and divided by the number of areas . Lastly, this sum will be multiplied by 10 to yield a PGI index score between 0 and 100, with higher scores reflecting a better health-related quality of life.At follow-up after six and twelve months, participants will be asked to score the same five areas that they listed in stage 1 at baseline.In this study, the baseline PGI areas will also serve as a basis for developing individual rehabilitation goals for patients in the intervention group and for evaluating goal attainment.The psychometric properties of the Norwegian version of the PGI has recently been tested with good results in patients with rheumatic diseases undergoing rehabilitation.A number of secondary measures will be included , whereas comorbidity will be reported by asking the participant to check off the presence of 16 diseases/health problems (yes/no).The level of exercising and social activity will be self-reported using two scales with the following response categories: “3 times per week”, “1-2 times per week”, “1-2 times per month”, “not on a regular basis” and “I cannot exercise or be socially active due to functional limitations”.The level of physical activity will be self-reported using the International Physical Activity Questionnaire-Short Form, (IPAQ-SF), which is expressed as weekly energy expenditures determined by the expressed metabolic equivalent task minutes per week (METs min/wk) of different categories  and physical activity levels .Illness perception will be captured by the Rheumatic Disease Illness Perception Questionnaire (RD-IPQ), which iThe content and stability of rehabilitation goals will be explored by text analyses, and the raw text materials for coding will be the rehabilitation goals agreed upon by the participants and health professionals during the semi-structured interviews shortly after admission, at discharge and in the follow-up telephone calls  blinded for group allocation will oversee the analyses of the data, with demographic and clinical characteristics, as well as other baseline data, presented to assess the baseline characteristics of the two groups, e.g. participants who have received the traditional rehabilitation programme (control group) versus participants who received the new programme (intervention group). These variables will also be compared for both those participants who withdraw from the study and those who remain. Parametric and non-parametric statistical analysis models will be used depending on the distribution of the variables.The main comparative analyses between the two groups will be performed using an intention-to-treat (ITT) analysis, and descriptive statistics will be presented for each group as the mean change  in the outcomes from baseline to each time point. Because of the cluster nature of the randomisation, the differences in mean change will be compared between groups using Generalized Linear Repeated Measures Mixed models with random effects, adjusting for baseline values, a possible time trend and the clustering effects of centres,55. The The main analysis for exploring factors associated with goal attainment and improved health-related quality of life during the first year following rehabilitation will be a multivariate manual backward stepwise linear regression, with the PGI sum-score at one year as the dependent variable. The number of variables included in the analysis will be kept below 10% of the included sample size, and will include age, gender, diagnosis, level of education, body mass index, comorbidity, pain, fatigue, disease activity, motivation for rehabilitation, level of exercises and physical activity, and illness perception.All variables will be entered into the model and the best subsets of prognostic factors will be selected by excluding those independent variables with the smallest contribution to the model . The model will be adjusted at every step for the baseline value of the outcome and the other independent variables.The primary economic evaluation will take the form of a cost-effectiveness study of the cost per extra quality adjusted life years (QALYs), which will be calculated using the SF6D scores at six and 12 months, respectively. Differences in mean change from the baseline for the SF6D to each time point will be weighted by the time from the baseline using generalized linear regression modeling, adjusting for baseline levels of the SF6D to construct QALYs and then compared between groups.The study has been approved by the Norwegian Regional Committee for Medical Research Ethics  and by the Norwegian Social Science Data Services (2011/6602).The research will be carried out in compliance with the Helsinki Declaration, personal confidentiality will be guaranteed and declarations of voluntary participation with detailed information on the study purposes and processes will be signed by each participant, thereby emphasizing the right to withdraw from the study at any time without any explanation.The randomization procedure is deemed as being ethically acceptable because all participants will receive a rehabilitation stay comprised of either the rehabilitation programme currently provided at the participating centres or the new and potentially more effective programme. Hence, no participants will receive an intervention that is below the standard currently delivered at each centre.Comprehensive rehabilitation, involving health professionals from various disciplines, is widely used as an adjunct to pharmacological and surgical treatment in people with rheumatic diseases. However, the evidence for the clinical- and cost-effectiveness of such interventions is limited.This paper outlines the protocol for a study where the main aim will be to assess the effects of a structured goal planning and tailored follow-up programme in rehabilitation for patients with rheumatic diseases, using a multicentre, stepped-wedge randomized controlled design.According to Brown et al., two keyFurthermore, all participants in the trial will receive rehabilitation, regardless of group allocation. This may reduce the chance that participants refuse participation for fear of ending up in a control group with an inferior treatment. The design may therefore increase the inclusion rate, and also to a large degree keep participants blinded for group allocation.Based on current knowledge and evidence, we will develop a new rehabilitation programme that includes structured goal planning and tailored follow-up after discharge. The main objective of the study will be to evaluate goal attainment, health effects and the cost-effectiveness of this new programme compared to current traditional rehabilitation programmes. We will seek to include a minimum of 312 participants from six rehabilitation centres, which will make this one of the largest rehabilitation studies conducted within this patient population. The findings will therefore constitute an important contribution to more cost-effective- and evidence-based rehabilitation services for people with rheumatic diseases.In a review of the efficacy of multidisciplinary team care programmes in rheumatoid arthritis, Vliet Vlieland et al. advocate the use of patient-oriented outcome measures and procedures to help enhance the role of the patient in the team care process and communication among health professionals. The priBecause the PGI captures the aspects of life that are of direct concern to the individual, the “noise” that is present in standardized instruments will be reduced, which in theory will have the potential to make it more responsive to capture the effects of rehabilitation. In addiA possible limitation in the PGI is that participants will select and score those areas of life that are experienced as problematic at baseline. The scores may therefore be prone to regression to the mean. As this probably will occur in both groups, it will, however, not affect the effect estimate, calculated as the differences between groups.In this trial, we will also use the SF-36 as a standardised generic measure of health related quality of life. The combination of a patient-specific measure and a questionnaire with standardised items may ensure that different of aspects of health related quality of life are captured, and will also allow for comparison of populations and results across studies.Regarding scientific benefits, the multicentre design will ensure that the new programme will be implemented in various settings, which enhances the generalizability of the results. Lastly, the study may increase the knowledge of how the structure, content and follow-up of rehabilitation stays can contribute in optimizing the effect of such stays. If proven effective, the programme may also be applied within other fields of rehabilitation, as well as in less complex interventions directed at lifestyle changes and self-management.The authors declare that they have no competing interests.IK and KBH conceived the project idea and designed the study together with GB, IB, TD, AD, JH, BH, SGE, EF, MN, RWR, ALSS, BS and SHW. PM has performed the power calculations and determined the random order in which the different centres will switch from control to intervention period, and will perform the statistical analyses. IK, GB, IB, AD, BH, SGE, BS, and SHW administer the trial and coordinate assessments. TD, GB and IK have trained the health professionals involved in assessments and provision of the intervention. IB, AD, BH, SGE, BS and SHW recruit and screen the participants. IK has drafted this manuscript. All authors have provided feedback on drafts and have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/15/153/prepub


AR gene, but is insufficient to maintain cell growth as evidenced by marked anti-proliferative effects in FOXA1 knockdown cells. In all, our data suggests that AR-Vs are dependent on FOXA1 for sustaining a pro-proliferative gene signature and agents targeting FOXA1 may represent novel therapeutic options for CRPC patients.Retention of androgen receptor (AR) signalling in castrate-resistant prostate cancer (CRPC) highlights the requirement for the development of more effective AR targeting therapies. A key mechanism of resistance to anti-androgens is through expression of constitutively active AR variants (AR-Vs) that are refractory to next-generation therapies, including Enzalutamide and Abiraterone. By maintaining an androgenic gene signature, AR-Vs drive tumour survival and progression in castrate conditions. Critically, however, our understanding of the mechanics of AR-V-driven transcription is limited, particularly with respect to dependency on pioneer factor function. Here we show that depletion of FOXA1 in the CWR22Rv1 CRPC cell line abrogates the oncogenic potential of AR-Vs. Gene expression profiling reveals that approximately 41% of the AR-V transcriptome requires FOXA1 and that depletion of FOXA1 attenuates AR-V binding at a sub-set of analysed co-regulated genes. Interestingly, AR-V levels are elevated in cells depleted of FOXA1 as a consequence of attenuated negative feedback on the  Prostate cancer (PC) is the most prevalent male cancer in the Western world and represents the fourth most common cancer worldwide. PC growth is initially androgen-dependent hence the mainstay for treatment is hormone-ablation therapy using anti-androgens and/or androgen-deprivation therapies (ADT) , 2. UnfoThe androgen receptor (AR) is a member of the nuclear hormone receptor family of transcription factors that transmits androgenic signals to drive prostate growth and cellular transformation. Like other nuclear receptors, the process of AR-mediated transcription is complex and is tightly orchestrated within the nucleus by a plethora of co-regulatory proteins, a number of which directly acetylate and methylate the receptor to control the strength and duration of androgenic signalling 13. Our Several aberrations in the AR signalling cascade have been identified in CRPC, including AR mutation and non-androgenic activation of the receptor, that facilitate AR activity in castrate conditions and contribute to conventional and next-generation anti-androgen treatment failure , 21. MorPSA and KLK2, that is a direct consequence of elevated AR-V levels. Irrespective of this, however, FOXA1 knockdown markedly reduces cell growth and is shown to be an important regulator of the pro-proliferative function of AR-Vs in CRPC. In all, our data supports the notion that AR-V activity is regulated by pioneer factors akin to the full-length AR (FL-AR), and FOXA1 may represent a suitable target for therapy in advanced disease.Our understanding of how AR-Vs are regulated remains limited, particularly with respect to their dependency on pioneer factors for transcriptional activity. To this end, we assessed the role of FOXA1 in regulation of AR-Vs in CWR22Rv1 cells and show that 41% of the AR-V transcriptome overlaps with a FOXA1-dependent gene signature. Consistent with previous findings in LNCaP cells , depletiPSA enhancer in both VCaP and CWR22Rv1 cells  in intron 2 of the aP cells . By recrpromoter  and was promoter . To asse Figures  and 4D, promoter , but notpromoter . Our finIn order to establish a role for FOXA1 in regulating global AR-V transcriptional activity, we individually depleted AR and FOXA1 in CWR22Rv1 cells grown in the presence of Enzalutamide and assessed differential gene expression signatures in both experimental arms. To confirm that the effect of AR knockdown was a specific read-out for AR-V function, we firstly conducted analysis on CWR22Rv1 cells grown in steroid-depleted conditions with and without Enzalutamide to assess the activation status of FL-AR in these conditions. Importantly, we found that maintaining cells in the absence of DHT for 72 hours was sufficient to completely abrogate FL-AR activity as treatment with Enzalutamide failed to impact on global gene expression (data not shown). We therefore reasoned that all genes found to be de-regulated upon AR knockdown can be attributed to AR-V activity. In support of this, Figure Depletion of AR and FOXA1 resulted in differential expression of respective 3085 and 2224 probes  and S2, UBE2C, that was defined as being specifically up-regulated by ectopically-expressed AR-V7 in LNCaP cells . IntegrA recent and more physiological study identified a total of 285 non-duplicated probes to be up-regulated by AR-Vs in CWR22Rv1 cells . CompariUBE2C, NKx3.1 and ATAD2 that were identified from the micro-array as being down-regulated in response to FOXA1 and AR knockdown. As shown in Figure UBE2C and ATAD2 expression was modestly elevated in response to Enzalutamide, suggesting that FL-AR may act to repress the function of AR-Vs at these genes , Cyclin B1 (CCNB1), Cyclin E2 (CCNE2) and Cyclin-dependent Kinase 1 (CDK1) were all found to be significantly down-regulated in response to individual depletion of AR and FOXA1, indicating a role for FOXA1 in controlling the activity of AR-Vs at pro-proliferative genes in CRPC. To confirm this, CCNA2 expression was assessed in CWR22Rv1 cells depleted of either FOXA1, AR or AR-Vs in the presence and absence of Enzalutamide. As shown in Figure CCNA2 mRNA levels were unaffected by Enzalutamide treatment, indicating a dependency on AR-V-mediated transcription for CCNA2 expression, that was confirmed by AR-V knockdown  analysis ) to our AR-V and FOXA1-co-regulated genes identified cell cycle control as the highest-ranked pathways dually controlled by AR-V and FOXA1 Figure . Cyclin nockdown . Importar Figure  indicati2/M- and sub-G1- phases of the cell cycle  [The mechanisms that regulate AR-V generation in cells are well characterised with splicing aberrations and genomic deletion of exons 4–8, that encode the LBD, being largely responsible for the production of receptor variants in CRPC . Howevermic loci . Moreoveelements . Our anapression . It will, (2014) .AR gene regulates the generation of AR-V7 in both VCaP cells, and the androgen-independent LNCaP cell derivative LN95, in ADT conditions [A key observation from our CWR22Rv1 experiments was of selective up-regulation of AR-V7 and AR-1/2/3/2b, but not FL-AR, in response to FOXA1 knockdown  [UBE2C that is an established AR-V target gene [et al., (2015) [Expression analysis demonstrated that 41% of AR-V target genes were also co-regulated by FOXA1 indicating a considerable overlap and potential dependency of the receptor variants on pioneer factor activity for both AR-activated and—repressed transcriptomes. This was confirmed at several target genes, including PC cells , 19. We , (2012)  to be reget gene . Moreoveget gene . Consist, (2015)  were als, (2015) , we used, (2015) ) is an iin vitro and in vivo [CCNA2, CCNB1, CCNE2 and CDK1 suggesting that the pioneer factor represents an additional tractable therapeutic target in AR-V-expressing CRPC. In support of this, several studies have demonstrated up-regulated FOXA1 protein in advanced disease and knockdown attenuates growth of additional PC cell lines [bona fide therapeutic target in this disease setting [There remains a paucity of agents/drugs available to attenuate receptor variant activity in the clinical setting hence components of the AR-V signalling network could represent novel therapeutic targets. The recent identification of the anti-helminthic drug Niclosamide as a selective destabiliser of AR-Vs in CWR22Rv1 cells and an AR-V7 expressing C4–2B cell line derivative suggests that selective targeting of these aberrantly functioning receptors is achievable at least in pre-clinical studies . Consist in vivo . Althoug in vivo , there rll lines , 41. Out setting , 42. Tar setting . In all,CWR22Rv1, LNCaP and VCaP cells were maintained in RPMI1640 media (Sigma) supplemented with 10% foetal calf serum (FCS) and 5% L-glutamine at 37°C. Transient transfection of FOXA1 and AR siRNAs  using the Benjamini–Hochberg method. Gene ontology analysis was performed as described in [RNA was extracted from CWR22Rv1 cells grown in steroid-depleted media subjected to control, AR or FOXA1 knockdown for 24 hours prior to vehicle or 1 μM Enzalutamide treatment for 24 hours. Samples were hybridised onto an Illumina HT12 v4 BeadChip Array  using variance stabilizing transformation. The robust spline normalisation (RSN) was used as the array normalisation method. Outlier samples, poor quality probes (detection threshold < 0.01), and probes that are not detected at all in the remaining arrays were removed prior downstream analysis. The remaining probe  normalised intensity, VSD was used in the differential expression analysis. Differential expression analysis was performed using the R package ‘Limma’, and ribed in .


Ultimately, there exists a complex interplay between FoxO proteins and their signal transduction pathways that can significantly impact programmed cell death pathways of apoptosis and autophagy as well as the development of clinical strategies for the treatment of neurodegenerative disorders.Acute as well as chronic disorders of the nervous system lead to significant morbidity and mortality for millions of individuals globally. Given the ability to govern stem cell proliferation and differentiated cell survival, mammalian forkhead transcription factors of the forkhead box class O (FoxO) are increasingly being identified as potential targets for disorders of the nervous system, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and auditory neuronal disease. FoxO proteins are present throughout the body, but they are selectively expressed in the nervous system and have diverse biological functions. The forkhead O class transcription factors interface with an array of signal transduction pathways that include protein kinase B (Akt), serum- and glucocorticoid-inducible protein kinase (SgK), I Nervous system disorders lead to disability and death in a significant proportion of the world's population. For example, almost ten percent of the global population suffers from the sporadic form of Alzheimer's disease (AD) while familial cases of AD account for less than 2% of all presentation , 2. In tYet, the availability of treatments that can prevent the initiation of acute or chronic neurodegenerative disorders or block the progression of these diseases is scarce. Therapeutic strategies that can aggressively treat AD and stroke continue to remain limited for most individuals. Furthermore, multiple other neurodegenerative disorders also greatly impact the global population with treatments that are not always optimal. By the year 2030, epilepsy is predicted to affect over 50 million people, neuropathies are estimated to afflict almost 300 million individuals, and neurological injuries may alter the lives of 243 million individuals . Drosophila melanogaster gene forkhead, greater than 100 forkhead genes, and 19 human subgroups that range from FOXA to FOXS is now known to exist [ Drosophila gene fork head (fkh), Forkhead Related Activator- (FREAC-) 1 and FREAC-2, and the acute leukemia fusion gene located in chromosome X (AFX) (FOXO4) [Given the need for novel directions that can potentially diminish or resolve the onset and progression of neurological disorders, mammalian forkhead transcription factors are surfacing as potential effective targets that can offer new developments for drug discovery. Since the documentation of theto exist . Prior t (FOXO4) , 14. For (FOXO4) . All let (FOXO4) –19.α-helices, three β-sheets, and two loops that compose the “wings” of the domain [α-helices and β-sheets have high sequence homology with variations in either absent β-sheets and loops or additional α-helices. As transcription factors, FoxO proteins bind DNA through the FoxO-recognized element in the C-terminal basic region of the forkhead DNA binding domain [α-helix H3 [Mammalian FOXO proteins are assigned to the O class of the forkhead box class transcription factors and consist of FOXO1, FOXO3, FOXO4, and FOXO6 . With a e domain  which ise domain . The α-hg domain , 26. Tarhelix H3 . Phosphohelix H3 . Howeverhelix H3 –31. Caenorhabditis elegans, Drosophila melanogaster, and mammals. FoxO proteins are homologous to the transcription factor DAuer Formation-16 (DAF-16) in the worm Caenorhabditis elegans that can determine metabolic insulin signaling and lead to lifespan extension [FoxO proteins are expressed in all tissues of the body . In relaxtension , 33. Furxtension , 34. Foxxtension , since ixtension , 37. Foxxtension , modulatxtension , regulatxtension , vasculaxtension , and motxtension . FoxO3 mxtension , cerebraxtension , 43, oxixtension , neonataxtension , erythroxtension , and hipxtension , 48.β) toxicity [Activity of FoxO proteins is controlled by epigenetic , 49 and toxicity –70, oxidtoxicity , inflammtoxicity , cerebratoxicity , experimtoxicity , flavonotoxicity , lipoic toxicity , 77, epitoxicity , neurointoxicity , tau homtoxicity , senile toxicity , and grotoxicity , 82–89. toxicity , 90, 91.toxicity , 92–101.toxicity , 102–104toxicity . Howevertoxicity , 106.In addition to Akt, other pathways can lead to the phosphorylation and inactivation of FoxO proteins. The serum- and glucocorticoid-inducible protein kinase (SgK), a member of a family of kinases termed AGC (protein kinase A/protein kinase G/protein kinase C) kinases that includes Akt and phosphorylates FoxO3a and maintains this protein in the cytoplasm . ImportaS. cerevisiae) (SIRT1) [κB kinase (IKK) also can directly phosphorylate and block the activity of FoxO proteins that results in the proteolysis of FoxO3a via the Ub-dependent proteasome pathway [Pathways associated with ubiquitylation and acetylation also control posttranslational modification of FoxO proteins , 111. Fo (SIRT1) , 113. In (SIRT1) , 114–117 (SIRT1) . FoxO pr (SIRT1) . In some (SIRT1) . IκB kin pathway . Acetyla pathway . Further pathway . FoxO pr pathway , 124. Hi pathway . FOXO3A gene, FOXO3A levels and catalase enzyme activity in vitiligo patients were decreased compared with control groups, suggesting in this case that FoxO proteins may confer protection. In other systems such as the maternal decidua, FoxO proteins may function independently in regards to oxidative stress with FOXO1 preventing oxidative stress damage and FOXO3a promoting oxidative cell death [FoxO proteins are important components in the control of cell survival and neurodegenerative disorders determined by apoptosis and autophagy in the presence of oxidative stress , 125–128ll death . In addill death .Autophagy is a process that recycles cytoplasmic components while removing dysfunctional organelles for tissue remodeling , 153–156β exposure [+) precursors [In regards to the programmed cell death pathway of apoptosis, a later phase that leads to genomic DNA degradation is preceded by an early phase with the loss of plasma membrane lipid phosphatidylserine (PS) asymmetry , 168. Thexposure , promoteexposure , increasecursors , and proecursors , 46, 52 ecursors –185. Howecursors . Other secursors , suggestβ-catenin [β-catenin during oxidative stress can protect hepatocytes from apoptotic cell death [β-catenin may increase FoxO transcriptional activity and competitively limit β-catenin interaction with members of the lymphoid enhancer factor/T cell factor family [FoxO proteins such as FoxO3a can lead to the induction of “proapoptotic genes” and disrupt proliferative pathways of Wnt signaling . A conve-catenin . Wnt sig-catenin –192. Wnt-catenin , metabol-catenin , nonneur-catenin , 193–195-catenin , stroke -catenin , and oxi-catenin , 197. Th-catenin , impaire-catenin , and apo-catenin . Wnt1 si-catenin , 201, is-catenin , dopamin-catenin , toxic e-catenin , 193, an-catenin . Wnt sigll death . Osteoblll death . In micrll death . Neuroprll death , 207, alll death , 208. Dull death . Wnt1 inll death , 209–213ll death . Overexpll death , 214. Thll death . It shour family . Foxo3a −/− and Foxo4 −/− mice can develop without incidence and have similar weight gain [ Foxo1 die by embryonic day eleven and lack development of the vascular system [ Foxo3a −/− mice are known to become infertile with ovarian follicles that are depleted of oocytes [ FOXO3a and FOXO1a have been reported in a small percentage of women who suffer from premature ovarian failure [ Foxo1, Foxo3a, and Foxo4 or a single deletion of Foxo3a also blocks the repopulation of hematopoietic stem cells in murine models [β (TGFβ) is required for oligodendrocyte progenitor development and myelination in the central nervous system [FoxO proteins have a prominent role not only in new cell development and differentiation, but also in determining the survival of mature cells in the nervous system . Each ofght gain . Yet, mir system . Overexpr system . On furt oocytes . FoxO3a  oocytes , indicat oocytes . Mutatio failure . Deletioe models , 222, ile models . Nuclears system .β-catenin and inhibiting Wnt signaling [In contrast, other studies suggest that inhibition of FoxO protein activity or prevention of Wnt pathway disruption may be necessary for stem cell survival. FoxO1 may negatively affect pancreatic beta cell survival . Work thignaling .β-catenin for the differentiation of marrow derived mesenchymal stem cells [β-catenin and prevent stem cell development similar to the previously described pathways with Wnt signaling [In the nervous system, FoxO proteins similarly determine the fate of neuronal precursors and the maintenance of neurons , 229. Stem cells . During ignaling , 235. Ceignaling –241. EPOignaling , 243. Otignaling .β toxicity [In relation to neurodegenerative disorders and neuronal cell survival, activation of FoxO proteins under most conditions leads to cell death , 245. Matoxicity  employs toxicity . Indepentoxicity , 52. Durtoxicity  and FoxOtoxicity . Toxin etoxicity . In micrtoxicity , 104. Dutoxicity  and vasctoxicity , 53, 58 However, it is important to recognize the antiproliferative and anticancer effects of FoxO proteins that make these transcription factors attractive targets for the inhibition of tumor growth , 50. Incβ toxicity, loss of FoxO results in decreased survival and locomotive activity [α-synuclein, inhibition of FoxO3 is protective. However, a small degree of FoxO3 activity prevents nigral neuron cell death in the presence of human α-synuclein accumulation by reducing the amount of α-synuclein and fostering the accumulation of autophagic vacuoles containing lipofuscin [In some experimental scenarios, FoxO protein activation may be required for neuronal protection. Blockade of neurodegenerative disease and adverse behavioral deficits during selenium exposure that may be linked to the development of amyotrophic lateral sclerosis occurs during increased FoxO protein expression . FoxO3a activity . FoxO practivity . In dopapofuscin . Interespofuscin . Levels pofuscin . Activatpofuscin . This expofuscin .β-catenin to result in apoptotic cell death. Conversely, Wnt signaling that includes WISP1 can phosphorylate, limit deacetylation, and sequester FoxO proteins in the cytoplasm to block apoptotic pathways that include caspase activation. FoxO proteins can promote autophagy to preserve cell survival during oxidative stress and clear toxic proteins from the cell. Yet, under some conditions, FoxO proteins may be tied to enhanced cell survival that is independent of autophagy. These observations do not always provide crisp conclusions and suggest the presence of a complex interplay between FoxO proteins and multiple signal transduction pathways in the cell. Furthermore, the degree of FoxO activity as well as companion pathways that involve SIRT1 can significantly impact cell development and survival. Elevated FoxO or SIRT1 activity can be detrimental to cells, but a minimal level of activity that can shepherd autophagic accumulation of toxic proteins may be beneficial. Importantly, these considerations provide further insight for the targeting of FoxO in the nervous system that may involve Wnt signaling, SIRT1, and trophic factors such as EPO to block cellular injury during oxidative stress. In addition, one should be cognizant of the nonproliferative role FoxO proteins play in tumorigenesis. Inactivation of FoxO proteins could yield unexpected cell growth not only in the nervous system but also in other regions of the body. Focusing upon FoxO proteins for the consideration of new therapeutic strategies against neurodegenerative disorders that oversee early cell development as well as differentiated cellular function can offer potentially high returns for new drug development.Neurodegenerative disorders result in significant death and disability for millions of individuals throughout the world but remain for the most part with limited treatment options and palliative therapies. Forkhead transcription factors and especially those of the FoxO subgroup are increasingly being identified as potential targets for disorders of the nervous system. FoxO proteins are expressed throughout the body, but their varied expression in the nervous system suggests that specific FoxO proteins may be vital for selective cellular and biological function and may be applicable for Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and auditory neuronal disease. For example, FoxO3 may be important for auditory synaptic transmission, cerebral endothelial vascular cell survival, and erythroid cell growth. In contrast, FoxO6 may be critical for memory consolidation and emotion. FoxOs are regulated by epigenetic and posttranslational modifications that involve phosphorylation, ubiquitylation, and acetylation by cellular pathways that involve Akt, SgK, MST1, IKK, SIRT1, and Wnt signaling to control the activity and integrity of these proteins. The ability of FoxO proteins to ultimately determine cell development and survival in the nervous system during oxidative stress resides with FoxO control of the programmed cell death pathways of apoptosis and autophagy. During oxidative stress cell injury, activation of FoxO proteins often leads to apoptotic cell death that initially fosters membrane PS residue externalization and subsequent DNA degradation. FoxO activity also can disrupt proliferative pathways of Wnt signaling involving


Our recent retrospective study revealed a significantly reduced risk of bladder cancer (BC) recurrence in men who received androgen deprivation therapy (ADT) for their prostate cancer. However, whether androgen receptor (AR) signals contributed to the preventive effect of ADT remained unclear because ADT could reduce serum estrogens as well. The purpose of this study is to investigate the associations between the expression of AR/estrogen receptors (ERs) and BC recurrence in patients treated with ADT. We immunohistochemically stained 72 BCs and 42 corresponding normal urothelial tissues. AR/ERα/ERβ were positive in 44(61%)/22(31%)/39(54%) tumors and 35(83%)/24(57%)/34(81%) corresponding normal urothelial tissues, respectively. There were no statistically significant correlations between AR/ERα/ERβ expression and clinicopathological features of BC. With a median follow-up of 31.3 months, 12 (43%) of 28 patients with AR-negative tumor versus 11 (23%) of 44 patients with AR-positive tumor experienced BC recurrence. Thus, patients with AR-positive tumor had a significantly lower risk of BC recurrence (P=0.031), compared with those with AR-negative tumor. Meanwhile, the expression of ERα/ERβ in tumors and that of AR/ERα/ERβ in normal urothelial tissues were not significantly correlated with BC recurrence. A multivariate analysis revealed AR positivity in tumors as an independent prognosticator  for BC recurrence. These results indicate that ADT prevents BC recurrence via the AR pathway, but not via the ERα/ERβ pathways. Men have 3-4 times higher incidence of bladder cancer (BC) than women . AlthougUsing preclinical models, we and others have revealed the involvement of androgen receptor (AR) signaling in BC development –6. HowevRecently, we demonstrated first clinical evidence indicating that androgen deprivation therapy (ADT) prevented BC recurrence . In brieThus, whether AR signals were responsible for the preventive effect of ADT on BC recurrence was still open question. The purpose of this study is to investigate the associations between AR/ERα/ERβ expression in surgical specimens and BC recurrence in patients treated with ADT.P = 0.020)/ERα (P = 0.006)/ERβ (P = 0.005) expression was significantly lower in tumors than in non-neoplastic urothelial tissues.We immunohistochemically evaluated 72 BCs and 42 corresponding normal tissues for the expression of AR, ERα, and ERβ. Positive signals for all three receptors were detected predominantly in the nuclei of neoplastic Figure  or non-nAR/ERα/ERβ expression in BCs and corresponding normal tissues, in relation to patient/tumor characteristics, is shown in Tables versus 11 (23%) of 44 patients with AR-positive tumor experienced BC recurrence. Median time to recurrence was 8.2 (interquartile range [IQR]: 4.7-20.4) months. Thus, patients with AR-positive tumor had a significantly lower risk of BC recurrence  of 28 patients with AR-negative tumor To see whether AR expression was an independent predictor of recurrence, multivariate analysis was performed with Cox model, including AR positivity and other well-known clinicopathological risk factors for BC recurrence Table . Of thesSince de novo tumorigenesis is considered to be one of the mechanisms for BC recurrence, we then investigated associations between AR/ERα/ERβ expression in corresponding normal urothelial tissues and tumor recurrence. However, as shown in Figure In the current study, we investigated very specific patients with double primary cancers of the bladder and prostate who received ADT for their prostate cancer and showed that AR positivity in BCs correlated with a reduced risk of tumor recurrence. These findings strongly support that AR signals contributed to the preventive effect of ADT on BC recurrence. On the other hand, similar to the results in patients without ADT , ERβ posAlthough a recent review article introduced some clinical trials of new targeted therapies for BC, none of these were approved for BC treatment to date . The autFor the treatment of prostate cancer, there are two major ways to target AR signals; surgical/chemical castration and anti-androgens. Castration is often associated with reduced health-related quality of life  and bone mineral density, compared with anti-androgen monotherapy . TherefoRecently, some large-scale genome-wide profiling studies proposed new classifications of BC –25. InteAR expression as a predictor of better prognosis seen in the present study appears to be contrary to the tumor promoting effect of AR signals in BC –6. A potA recent retrospective study involving patients who received ADT for prostate cancer showed a significantly lower incidence of subsequent BC development . In addiThe main limitations of the current study include its retrospective design with limited study population. However, since the incidence of primary BC in prostate cancer patients in our cohort was only 1.2%  and onlyvia the AR pathway. In addition, AR expression in BC can be a useful marker in selecting patients for ADT.We here report the expression of AR, but not ERα or ERβ, in BC is associated with tumor recurrence in patients treated with ADT. Together with our previous observations, ADT with anti-androgen monotherapy and/or LHRH analogue is suggested to prevent BC recurrence We retrospectively retrieved 72 BCs and 42 corresponding normal urothelial tissues obtained by transurethral resection from patients who received ADT for their prostate cancer from 2001 to 2012 in four hospitals . Fifteen patients received LHRH monotherapy whereas others received combined androgen blockade consisting of LHRH analogue and anti-androgen. Median duration of ADT was 25.7 (IQR: 9.8-55.1) months. None of these patients received systemic chemotherapy or underwent radical cystectomy. Appropriate approval from the institutional review board at each institution was obtained before use of the tissue samples.Immunohistochemical staining was performed, as described previously . SectionP < 0.05 was considered to be statistically significant. All statistical analyses were performed with EZR , which is a graphical user interface for R . More precisely, it is a modified version of R commander designed to add statistical functions frequently used in biostatistics.The Fisher exact test was used to evaluate the association between categorized variables. Nonparametric two group comparison was carried out using Mann-Whitney U test to assess differences in continuous variables. Survival rates in patients were calculated by the Kaplan-Meier method, and comparison was made by log-rank test. The Cox proportional hazards model was used to determine statistical significance of predictors in a multivariate setting.


Mott effect, featured by a sharp increase of ionization, is one of the unique properties of partially ionized plasmas, and thus of great interest to astrophysics and inertial confinement fusion. Recent experiments of single bubble sonoluminescence (SBSL) revealed that strong ionization took place at a density two orders lower than usual theoretical expectation. We show from the perspective of electronic structures that the strong ionization is unlikely the result of Mott effect in a pure argon plasma. Instead, first-principles calculations suggest that other ion species from aqueous environments can energetically fit in the gap between the continuum and the top of occupied states of argon, making the Mott effect possible. These results would help to clarify the relationship between SBSL and Mott effect, and further to gain an better understanding of partially ionized plasmas. A sharp rising in ionization is a remarkable feature of Mott effect68ρ ~ 1.0 g/cm3, in line with the theoretical expectation.The physical origin of Mott effect, however, has been under debate since it was first noticed in 1940’s2412131415ρN ~ 1021/cm3, i.e., ~0.07 g/cm3, and at a temperature T ~ 1 eV, i.e., 11000 K. The ρN corresponds to an average atomic distance of 10 Å, which is much larger than the length scale (~2 Å) of the outer-most 3p electronic orbital of Ar atoms. A theory developed recently by Kappus et al.However, in single bubble sonoluminescence (SBSL) experiments1718et al.et al.et al.12232627However, as was pointed out by Redmer and HolstIn this work, we concentrate on electronic structures of Ar at equilibrium, calculated using the first-principles molecular dynamics (FPMD) method. In the calculation, ions are described via classical Newtonian molecular dynamics (MD), whereas electrons are treated quantum-mechanically through density functional theory (DFT). Two levels of quantum descriptions are used in the calculation. Mean-field effects of excited electrons are accounted for through finite-temperature DFT (FTDFT) formula proposed by Mermin32The inaccuracy brought about by the methodology is also considered. It has been well known that LDA underestimates the energy gap between occupied and unoccupied states at low temperature, usually 40–60% for semiconductors and insulators27ni is approximately 4 × 1021/cm3, i.e., 0.25 g/cm3, about half of the number density at the liquid-gas critical point. However, surprisingly, the number density of electrons ne is roughly the same as or even higher than ni, making the degree of ionization α ≥ 1. The coupling constantIn a typical SBSL experiment containing Ar, a sharp increase of ionization takes place with strong visible light emitted at a pressure over 4000 bar and at a temperature ~1.5 eV (17000 K)20α is therefore overestimated by about 5 times, as estimated using α ~ exp(−I0/2T)I0 the magnitude of IP. Even with this overestimation, the calculated α is less than 3%, which is about twice the value of 1.4 % given by the self-consistent fluid variational theory(SCFVT)Testing calculation on experimental conditions shows that electronic structures and IP are essentially the same as those of an isolated Ar atom. The interaction between Ar atoms is thus negligible according to the quantum perturbation theory. Compared with experimental value of 15.7 eV, the calculation (10.3 eV) underestimates the IP by 33%. et al.4113 and 8.34 g/cm3. The upper limit of 8.34 g/cm3 corresponds to an average atomic distance ~2 Å, slightly larger than the diameter d = 1.94 Å of the 3p orbital for an isolated Ar atom. The diameter of the 3p orbital is calculated as 3 K and 2 × 104 K. Higher temperature is not considered because both theories and experiments showed that Mott effect prefers lower temperature. It can only take place below some critical temperature411121321However, the absence of Mott effect is possibly caused by the drift of temperature and density predicted by the calculation, which is inevitable for the FTDFT+MD method due to various approximations involved in the calculation, as has been noticed in the calculation of hydrogen123 K, 104 K, and 2 × 104 K, where isotherms of T = 103 K and T = 2 × 104 K define the boundary of our calculations, and the isotherm of T = 104 K indicates the median of the temperature measured in SBSL experiments. To give an estimation to the reliability of the FTDFT+MD method, also displayed in the inset is the calculated isotherm at room temperature, compared with experimental results measured by Brillouin scattering3. This deviation is expected because the atomic distance at that density is only slightly larger than the diameter (1.94 Å) of the 3p orbital of Ar atom, where tunneling effect cannot be well accounted for by the empirical pair potential fitted from experimental data at low densities. In g(r) along the isotherms are presented to reveal structural changes. When a first-order phase transition takes place, g(r) usually undergoes a significant change. This, however, is only observed at ρ = 8.34 g/cm3 and T = 103 K in 20g(r) and isotherms in T and ρ. It shows that structural transition is not a prominent feature near the experimental conditions, which is consistent with the usual understanding of Mott effect24et al.et al.In addition to thermodynamical properties, the electronic structure of Ar plasma is also calculated using the FTDFT+MD method. A strong reduction (over 75 %) in IP is expected according to the estimation of Kappus T = 104 K isotherm is presented in s states, the segment in the middle is from the 3p states, and the continuum above the chemical potential is formed by the bottom of conduction bands. The 3s and 3p states in the figure are broadened significantly due to thermal effects and tunneling effects. The calculated IP is ~5 eV in a large range of density until ρ ~ 4.3 g/cm3, from which the IP starts to shrink to zero. This tendency is similar to that observed in KCl under shock compressionρ = 8.34 g/cm3, corresponding to an atomic distance comparable with the average diameter of the outer-most 3p orbital of Ar. The small IP (~0.8 eV) suggests that the Ar plasma is close to a nonmetal-metal transition point. The merging of the 3s and 3p states also implies a remarkable change in the electronic structure. ρ = 12.45 g/cm3. The disappearing of energy gap leads to an α over 12%, which is twice of that for ρ = 8.34 g/cm3 in Total density of electronic states (TDOS) of Ar plasma along the ρ = 1 g/cm3 are displayed. When temperature increases from T = 103 K to T = 2 × 104 K, the 3s and 3p TDOS are broadened by about twice, and the IP decreases smoothly from ~7 eV to ~4 eV. No abrupt change of IP is observed in the temperature range. As has been pointed out by the PPT modelα, which is unlikely to reach an α ~ 1 as SBSL experiments suggested.The influence of temperature is presented in To understand the role of dynamical screening effect in determining electronic structures, which is not sufficiently accounted for in the FTDFT+MD method, we further calculate the ionization ratio of Ar under typical conditions with the influence of dynamical screening included using the TDDFT method. It gives an explicit description to the real-time evolution of excited electrons by solving the time-dependent Kohn-Sham equation using adiabatic local density approximation (ALDA), and has been applied to a few occasions, e.g., atomic collisionsα at two representative densities ρ = 3.6 g/cm3 and ρ = 10.5 g/cm3. α of 17.2% at T = 2 × 104 K for ρ = 3.6 g/cm3, which is similar to the value calculated using the SCFVT methodα ≥ 1) observed in the experiments. An indication of typical Mott effect is illustrated in ρ = 10.5 g/cm3. When the energy gap closes and the system becomes metallic, stepwise multi-ionization processes are observed, and α reaches 12% at 0.48 ps (20000 a.u.) for T = 5 × 103 K without artificial boost to the ionization. Further increase in α is observed when temperature rises, and the ionization process does not stop in our longest calculation at T = 2 × 104 K after 0.72 ps (30000 a.u.) In general, ρ = 1.7 g/cm3, T = 104 K. This state has a density close to the lowest density bound in our calculation at typical SBSL temperatures, and is expected to have the least possibility to present the Mott effect according to the trend illustrated in the FTDFT+MD and TDDFT+MD calculations. To distinguish the contribution of different ion species, the density of states is decomposed into projected density of states (PDOS) according to the composing elements  and their electronic orbitals. For pure Ar plasma, as displayed in 2O at the same number density (NAr = 9 : 9) is introduced, as displayed in p orbital of O, originated from OH− radicals or H2O molecules. At this temperature (T = 104 K), OH− does not have enough energy to decompose into O2− and H+. So, the contribution of O2− is negligible. As a result, the energy gap in Ar plasma is filled by energy levels associated with ionized water, and the effective IP of the mixed Ar and H2O plasma is substantially reduced, making the strong ionization possible.From the perspective of electronic structures, we have shown that the strong ionization observed in the Ar SBSL experiments is quite unlikely the result of Mott effect of pure Ar plasma. However, it is still possible to obtain an effective IP small enough to meet the requirement of Mott effect when a mixture of plasmas composed of several ion species are considered. Energetically, this can be achieved by inserting energy levels of other ion species into the gap of Ar plasma. Indeed, as pointed out by chemical reaction models4 K. Our first-principles investigation supports the traditional understanding of Mott effect, i.e., the Mott effect happens at the density where atomic distance is comparable with the diameter of the outer-most electronic orbitals. Although the detailed mechanism of Mott effect is still an open question, this investigation would help to have a better understanding of Mott effect as well as its relationship to SBSL experiments.In summary, we show from the perspective of electronic structures that the strong ionization observed in the SBSL experiments is unlikely the result of Mott effect in a pure Ar plasma. Instead, a mixture of plasmas composed of Ar and water energetically favors the occurrence of strong ionization around T = 102O and Ar, a total number of 36 atoms are used. The time step varies from 0.4–1 fs depending on the density and temperature. Each simulation lasts for 8000–20000 time steps, and the results presented are averaged over the last 2000 steps with a time interval of 200 steps. Degree of ionization is calculated following ref. T = 0. Note that the Kohn-Sham energy level is interpreted as an approximation to the quasiparticle energy following the convention of electronic structure calculation for extended systemsFTDFT+MD simulations are carried out by the Vienna ab initio simulation package (VASP)−8 in each step.TDDFT+MD calculations are carried out using the OCTOPUS codeα(t) is calculated as the charge excited from the ground state per atom. By projecting the time-dependent wave functions onto the ground-state Kohn-Sham wave functions at that instant, α(t) can be evaluated asIn the framework of TDDFT, the degree of ionization Na is the number of atoms, nex is the average number of excited electrons, wk is the weight of each k-point in the summation of Brillouin zone, t, and where How to cite this article: Kang, W. et al. First-Principles Investigation to Ionization of Argon Under Conditions Close to Typical Sonoluminescence Experiments. Sci. Rep.6, 20623; doi: 10.1038/srep20623 (2016).


Dietary-derived agents, such as the flavonoids, are of particular interest for prostate cancer (PCa) chemoprevention as they may offer a favourable safety and side-effect profile. An agent that demonstrates action on the androgen receptor (AR) axis may have value for preventing or treating castrate-resistant PCa. Four main flavonols – quercetin, myricetin, kaempferol, and fisetin – have been demonstrated in laboratory studies to have chemopreventive action in both castrate-resistant and castrate-sensitive PCa models. Mechanisms of flavonol action on the AR axis in PCa have been proposed to be inhibition of the 5α-reductase enzymes, direct androgen competition, suppression of the AR complex and transactivation by coregulators such as c-Jun, Sp1, and the PI3K/Akt pathway. It is, however, still unclear with current levels of evidence whether AR axis-mediated effects can fully account for the flavonols’ chemopreventive action. Prostate cancer (PCa) is a leading cause of morbidity and mortality within the male population. It was the cancer with the highest incidence rate in men in the United Kingdom in 2013, a total of 40,372 new cases were diagnosed, accounting for 27% of all new male cancer diagnoses . Recentlin vitro and in vivo [The flavonols, a class of phenolic phytochemicals exemplified by quercetin, myricetin, kaempferol, and fisetin  can be f in vivo –20. A USTheir structural similarity to androgens (testosterone is shown) is evident, and hence, AR receptor interaction has been postulated as a mechanism for their anti-carcinogenic activity in PCa.Development of PCa is thought to be a multistep process from the normal prostate epithelium to intraepithelial neoplasia (benign prostatic hyperplasia is not currently considered as a precursor to PCa), then localised carcinoma before progression to invasive or metastatic disease. Treatment options for advanced or metastatic disease are limited to androgen ablation and chemotherapy. Initially, most cancers are responsive to this; however, after a period of time, recurrences are common as the disease progresses to a castrate-resistant phenotype (CRPCa), for which there is a very poor prognosis . TransitARs are present in most human tissues; however, they are most important for driving the development of sexual characteristics. The AR-signalling pathway is essential for maintaining normal metabolic function, cell proliferation, and homeostasis  and is aThe AR axis has previously been a target for chemopreventive agents in PCa. The 5α-reductase inhibitors finasteride , 26 and Flavonols are related structurally to oestrogens and this similarity has led to the hypothesis that they exert their action either by competing with androgens for AR binding sites or by affecting endogenous androgen levels.Myricetin, quercetin, and fisetin have a catechol or pyrogallo configuration in the B-ring which may allow them to inhibit 5α-reductase isoenzyme 1, while kaempferol has been shown to inhibit isoenzyme 2 in transfected Rat 1A cells . This maQuercetin has been shown to compete with testosterone at high concentrations. However, the concentrations are higher than needed for cell growth inhibition, suggesting that this is unlikely to be its main cytostatic mechanism . Anotheret al observed that fisetin suppresses the growth of CWR22rv1 and LNCaP cells in vitro and competes with a labelled high-affinity androgen for the ligand-binding domain of the AR and in addition inhibits DHT-driven stabilisation of AR, leaving it vulnerable to proteasome degradation [in vitro; however, there is a lack of other studies to support this hypothesis. It is also possible that fisetin was acting through a separate pathway, and the effects on the AR pathway are a consequence, rather than a cause of this action.Khan radation . This woMany studies have focused on the effect of flavonols on the expression of the AR itself and its activity in the form of AR-dependant genes. AR luciferase reporter genes have been used to demonstrate that quercetin  and KaemIn terms of AR expression itself, quercetin seems to have varying effects. A reduction in AR expression in quercetin-treated CWR22Rv1 cells was observed in serum-free cultures, yet an increase in expression was seen when 10% foetal bovine serum was added . In the et al were able to demonstrate a less ambiguous effect of fisetin in their study. In the CWR22rv1 and LNCaP cell lines, treatment with fisetin suppressed AR expression, inhibited protein and mRNA expression of AR target genes, and reduced PSA levels in a mouse xenograft (32).The AR-dependant genes PSA, 5α-reductase and human kallikrein 2 (KLK2) serve as markers of AR activity. Quercetin was shown to suppress the gene expression of PSA and KLK2 at the transcriptional level , 38, whiThese studies show that there is likely to be a suppressive effect of flavonols on the AR expression and activity. They are able to achieve this in the androgen-dependant LNCaP cell line and the more insensitive CW22Rv1 line. This may be a sign that flavonols have potential for the prevention or treatment of CRPCa via action on the AR axis, independent of direct ligand-receptor stimulation.et al have looked extensively into the activity of quercetin on AR transactivation by coregulators, hypothesising that these may the main mechanism of action of its effects on the AR rather than a direct interaction. An increased expression of the coregulator c-Jun was observed in LNCaP and LAPC4 PCa cells treated with quercetin. It is thought that c-Jun works to inhibit AR expression, and in this study, an inhibition of AR at the transcriptional level was observed [Yuan observed . Sp1 is observed , suggestobserved .et al were able to demonstrate that quercetin induces apoptosis in the largely androgen-independent PC-3 and DU145 PCa cell lines as a direct result of their inhibition of heat shock protein 90 [Aalinkeel otein 90 , a key rotein 90 , 41. WheThe phosphatidylinositol 3-kinase/Akt (PI3K/Akt)-signalling axis serves as an AR transactivator, with Akt-dependant phosphorylation of the AR increasing its transcriptional activity , 42. FlaThe hormone-refractory nature of advanced PCa has meant that the AR has been a target of much research, especially into dietary-derived potential chemopreventive agents, such as the flavonols. Quercetin has been the most extensively studied of this group, with evidence lacking as to the other compounds’ effect on the AR axis.Whether or not flavonols exert their main effect through androgen competition remains ambiguous. All four main flavonols seem to have an inhibitory action on the 5α-reductases, hypothetically reducing DHT levels and blunting androgenic stimulation of growth in PCa. It is unclear, however, whether this would translate into a clinically relevant effect that is similar to finasteride and dutasteride. There is little evidence to show that flavonols directly compete with androgen binding to the AR. Fisetin demonstrated this ability in one study whilst quercetin was only able to compete at concentrations higher than needed to achieve its cytostatic effect. As the flavonols seem to exhibit an AR-mediated effect on the largely androgen-independent cell lines CW22Rv1 and PC-3  without et al hypothesised that quercetin’s action was mediated through the co-regulators c-Jun and Sp1; however, it remains to be seen whether kaempferol and fisetin act in a similar way.While it is undeniable that these flavonols exhibit action on the AR, it remains unclear, and in some places unlikely that their main effect is AR axis mediated. Indeed, flavonols have shown efficacy in many cancer types, such as lung and colon, that are not reliant on androgenic stimuli for progression , 48. It If the AR-signalling pathway is indeed the key to preventing or treating CRPCa, then methods to enhance flavonols’ effect on it may be beneficial. Structural modification of the flavonols, for example the quercetin-based PI3K inhibitor mentioned previously, is a potential avenue for increasing their action on the AR pathway. Another potential solution is using them in combination with other agents, for example other flavonoids , 49, 50 In conclusion, flavonols exhibit a modest effect on the AR-signalling axis, but their value as chemopreventives or adjuncts to existing or novel treatments for CRPCa is promising.The author declares that they have no conflict of interest.PCaProstate cancerCRPCaCastrate-resistant prostate cancerARAndrogen receptorDHTDihydrotestosteronePSAProstate specific antigenKLK2Human kallikrein 2