Introduction. This study aimed to assess rehabilitation professionals' attitude toward implementation and application of telerehabilitation technology as a novel study in rehabilitation academic centers and affiliated clinics in Tehran. Methods. It was a descriptive cross-sectional study. To collect data, a researcher-designed questionnaire was developed. 141 rehabilitation experts participated in the study. Results. A majority of faculty members (78%) and clinicians (89.7%) either were in “definite agreement” or “somewhat agreed” with implementation and application of this technology, which demonstrates an overall positive attitude. Discussion. Based on the positive attitudes of the majority of participants toward implementation and application of this technology and their preferences in offering different telerehabilitation services, it seems that there is an appropriate and desirable acceptance and administrative culture to implement this technology among rehabilitation experts in Tehran. It is thus expected that implementation and application of this technology will be a promising experience in rehabilitation academic centers and affiliate clinics in Tehran. Today, new technologies have come into existence in different occupations and work areas, one of which is information and communication technology . Due to Today, healthcare organizations are faced with a new technology called telehealth. Telehealth is the use of information and communication technologies to provide remote healthcare services . The WorTelerehabilitation is a relatively new area which was first defined by the National Institute of Disability and Rehabilitation in the United Sates in 1997 . It encoTelerehabilitation has the potential to benefit both the consumers and providers of rehabilitation services by lessening the inconvenience and/or cost of patient transfers and reducing unnecessary travel time . The manAttitude plays an important role in our lives, thoughts, and our individual and social behaviors. In fact our views on various issues determine our actions and reactions and justify our motivation or reluctance to show certain behaviors . AttitudIn the past few years in Iran, there have been feasibility or review studies which have addressed different aspects of telemedicine and e-health including telelaparoscopy , telepsyDue to the very newness of this technology in our country and considering the importance and advantages of this technology, this study aims to assess rehabilitation professionals' attitude towards implementation and application of telerehabilitation technology in rehabilitation academic centers in the city of Tehran. In the present study, attitude toward telerehabilitation implementation was defined as the respondents' views on the efficacy and the potential benefits of application of this technology in therapy and therapeutic systems.Since rehabilitation professionals working in academic environments are expected to have more realistic attitudes towards this issue based on their more update knowledge in the rehabilitation domain and the fact that they are the most familiar ones with the current position and status of rehabilitation in the country, thus they were considered as a desirable population for conducting this study.This was a descriptive cross-sectional study conducted on faculty members of four universities and clinicians of affiliated rehabilitation centers in Tehran. A preliminary draft of an attitude questionnaire was developed by integration and cultural adaptation of different tools collected in the field of telehealth implementation. The face and content validity of the questionnaire was evaluated by a panel of rehabilitation faculty members in the University of Social Welfare and Rehabilitation Sciences using Lawshe's method and the reliability was assessed by test-retest correlation and Cronbach's alpha determinations. Finally, a researcher-designed questionnaire with favorable validity and reliability was obtained.The designed questionnaire consisted of 51 questions scored by a Likert scale of 5, ranging from “definitely agree” with a score of 5 to “definitely disagree” with a score of 1. The higher the score, the more positive the attitude it reflected. By using 4 cut-off points for the total attainable score, 5 levels of total attitude were defined.The questions in the questionnaire were categorized in 4 different groups, including questions that dealt with the possible positive or negative impacts of telerehabilitation implementation on (1) the experts' own work domain, (2) the work domain of colleagues, (3) the quality of therapeutic procedures and service delivery for the clients, and (4) the national health and ICT system. Overall, the questions were meant to determine what attitude the experts expressed regarding each of the suggested positive or negative outcomes or impacts of telerehabilitation implementation and application. Some examples of the questionnaire's content are stated in The study received an approval from the IRB and Ethical Committee of the University of Social Welfare and Rehabilitation Sciences. The study population included rehabilitation experts (faculty members and clinicians) in the University of Social Welfare and Rehabilitation Sciences and Tehran, Iran, and Shahid Beheshti Universities of Medical Sciences. Stratified sampling was used. The universities were considered as the main classes and the experts' positions as a faculty member or a clinician constituted the subclasses. The samples from each class and subclass were selected by simple random sampling. The questionnaire then was distributed manually by the researcher and participants were asked to complete the questionnaire in a few days, after which the researcher returned to collect them. Totally, 150 rehabilitation experts were recruited of which 141 participated by completing them.Regarding the implementation and application of telerehabilitation technology, as the results show both faculty members and clinicians expressed a high percentage of positive attitude toward the implementation of telerehabilitation technology. This finding is somewhat in concordance with studies conducted by Mirhosseini et al. in the Kerman University of Medical Sciences  and AlizIn our study clinicians surpassed the faculty members in their positive attitude toward this technology. It may be that since the majority of clinicians practice in clinical centers and are thus more familiar with the present obstacles of patient treatment they have a better understanding of the possible advantages of this technology. On the other hand, probably because faculty members in this study had higher seniority and thus more experience see , they arBased on the results, with increasing age a predominantly decreasing trend of definite positive attitude has emerged. It seems that since people less than 30 years of age have more interest in and are in more contact with a variety of modern technologies they thus have a more positive view towards implementation of this modern technology. Also, our results showed that the percentage of “definite agreement” in participants with 40 to 50 years and above 50 years of age was 0%. It seems that aging has a considerable impact on the participants' cautious views, since older participants may have a better and deeper understanding about the possible challenges of implementation of this technology in our current situation, based on their higher experience. However, in a study conducted on medical students' points of view toward establishment of telemedicine in Mazandaran University of Medical Sciences, no significant correlation was found between age and attitude . EvidentIf “overall positive attitude” is defined as the sum of the total percentage of “definite agreement” and the total percentage of persons who responded with “somewhat agree,” our results showed that participants with a Ph.D. degree expressed more positive attitude than other degree holders. However, participants with a Ph.D. degree were the only group among whom no “definite agreement” was obtained. The findings of a study which was conducted by Alizadeh et al. showed that in Mazandaran University of Medical Sciences medical students with more years of education had more positive attitude than those with lower levels of education toward telemedicine .The findings show that the University of Social Welfare and Rehabilitation Sciences was the only university in which some participants showed “definite agreement” with implementation of telerehabilitation. This may be due to the fact that this university is the only specialized university in the field of rehabilitation in Iran, with exclusive focus on welfare and rehabilitation sciences. However, if “overall positive attitude” is defined as the sum of the total percentage of “definite agreement” and the total percentage of persons who responded with “somewhat agree,” it is noteworthy that the highest percent of positive attitude belonged to the Tehran University of Medical Sciences and the lowest belonged to the Shahid Beheshti University of Medical Sciences.As the findings show teleconferences were the most popular telerehabilitation service, followed by tele-experts consultation and tele-patient consultation, in all of which it is possible to establish a live connection between the therapist and his colleague or patient and it is also possible to benefit from both sound and image in interactions.Overall, based on the results of the present study it can be anticipated that, in case of implementation of this technology in the field of rehabilitation, an overall positive trend in its acceptance and application by experts can be expected.One of the limitations the authors faced was that to their knowledge, no study of any kind had ever been conducted before regarding telerehabilitation in Iran or even in other countries of the region. All similar studies including feasibility studies or review articles, have been conducted on different domains of telemedicine, e- helath and tele-health implementation. Thus we have been faced with lack of adequate national or regional research benefits from similar experiences in this field for further comparison and discussion of the results. It seems that further feasibility studies are required in this field in Iran and similar developing countries to enhance rehabilitation service quality by implementing telerehabilitation technology.


Asymmetric cell division, whereby a parent cell generates two sibling cells with unequal content and thereby distinct fates, is central to cell differentiation, organism development and ageing. Unequal partitioning of the macromolecular content of the parent cell — which includes proteins, DNA, RNA, large proteinaceous assemblies and organelles — can be achieved by both passive  and active (e.g. motor-driven transport) processes operating in the presence of external polarity cues, internal asymmetries, spontaneous symmetry breaking, or stochastic effects. However, the quantitative contribution of different processes to the partitioning of macromolecular content is difficult to evaluate.Saccharomyces cerevisiae. This model exposes quantitative degeneracies among the physical parameters that govern macromolecular partitioning, and reveals regions of the solution space where diffusion is sufficient to drive asymmetric partitioning and regions where asymmetric partitioning can only be achieved through additional processes such as motor-driven transport. Application of the model to different macromolecular assemblies suggests that partitioning of protein aggregates and episomes, but not prions, is diffusion-limited in yeast, consistent with previous reports.Here we developed an analytical model that allows rapid quantitative assessment of partitioning as a function of various parameters in the budding yeast In contrast to computationally intensive stochastic simulations of particular scenarios, our analytical model provides an efficient and comprehensive overview of partitioning as a function of global and macromolecule-specific parameters. Identification of quantitative degeneracies among these parameters highlights the importance of their careful measurement for a given macromolecular species in order to understand the dominant processes responsible for its observed partitioning. Saccharomyces cerevisiae by budding is a classical example of asymmetric cell division. The future daughter yeast cell grows as a bud on the surface of the mother cell, receiving macromolecules and organelles that “escape” from the mother compartment through the narrow opening of the bud neck  bodies or RNA granules -6]. Pro. Pro6]. Asymmetric partitioning of oxidatively damaged protein aggregates is linked to yeast replicative aging ,14]. Th. Th14]. In early studies, the binding of episomes to structures retained in the mother cell was hypothesized to explain their exclusion from the bud , thus coHere we argue that passive diffusion should always be considered when analyzing the partitioning of macromolecular assemblies during cell division. To facilitate the analysis of the role of diffusion, we developed an analytical model of diffusion-driven transfer of macromolecules between the mother and bud compartments of budding yeast. The model is based on a description of diffusive transfer according to the “narrow escape” approximation, which gives the mean time required for a diffusing particle to escape from a compartment through a narrow opening -32]. Ap. Ap32]. D from a spherical volume V (radius R) through a narrow circular opening of diameter d can be asymptotically described by an exponential decay with a mean escape time of:Ke corresponds to the first-order rate constant for escape . Th. ThD frope . Ap. ApD froDm = Db = D), for which kr = kf = Kf = dD/Vm;(I) passive diffusion with equal compartmental viscosities (Dm ≠ Db), for which kf = Kf = dDm/Vm and kr = KfDb/Dm = dDb/Vm;(II) passive diffusion with different compartmental viscosities (kf and kr (as defined with respect to the mother) are determined by directional trafficking processes and can differ in magnitude.(III) active transport, for which te and the now anomalous diffusion constant, with this relationship dependent on the exact mathematical definition of anomalous diffusion.Anomalous diffusion, ubiquitously observed in biological systems ,36], ca, ca36], Transport by convection (bulk flow or cytoplasmic streaming) can also influence macromolecular partitioning ,38]. Th. Th38]. σ of the mother volume at the cell division time T .In the above, we have implicitly considered a static mother-bud geometry. Assuming a linear growth of the bud to a final fraction Bm the binding site concentration and Kd the dissociation constant. Similarly, for target sites, the unbound fraction is:α = ϕmkfT and β = ϕbkrT/σ. In addition to binding sites, transient trapping of macromolecules on membranes or within organelles can similarly be accounted for through the reduction in their compartmental free fractions.Binding sites in the bud (target sites) or in the mother (retention sites) can bias macromolecular partitioning. In the simplest scenario, binding sites are assumed far from saturation with on/off binding fast compared to the transfer timescales. For retention sites, the unbound fraction is then:σ = 0.61, R = 2.5 μm, d = 1.25 μm, D = 0.001 μm2 s‐ 1, T = 90 min), we obtain 8.4%. However, Zhou et al.  in  in 32] iα and β for which partitioning does not reach equilibrium. Examples of temporal trajectories for different values of α and β are displayed in Figure α and β roughly less than 1, defining a critical diffusion constant Dc ≈ Vm/(dT). Significant retention of molecular species i in the mother cell can be expected for Di < Dc  such that bidirectional transfer is efficiently equilibrated at each time point. Figure α and β in the phase plot.To explore the model, we calculated the bud fraction of macromolecules at the end of the division process s Figure a. Note tyeq = 0.5 upon complete equilibration). To illustrate the scope of our model, consider the shift from the solution at α = 1, β = 1 (blue circle) to that at α = 0.1, β = 0.1 (blue square). This displacement could be achieved in several different ways: a ten-fold increase in the mother volume; or a ten-fold reduction in bud neck diameter, diffusion constant, or division time. Ten-fold reductions of both compartmental free fractions due to efficient retention/target site binding would have the same effect. Our model therefore reveals the exact quantitative degeneracy among these various parameters, obviating much of the need for laborious stochastic simulations of different scenarios  is, of course, not obtainable with unbiased passive diffusion. Solutions above or below the diagonal, the latter including the possibility of bud enrichment, are obtained with different compartmental diffusion constants (Dm ≠ Db), asymmetric distribution of binding sites (leading to ϕm ≠ ϕb) or, more generally, with different transfer rate constants (kf ≠ kr) for the case of active transport between the compartments.The blue diagonal in these plots Figure  gives thu et al.  and Gehlu et al. ). Note tS. cerevisiae, in which the final bud volume is 2/3 the mother volume (here equilibration corresponds to yeq = 0.4). Again, only solutions below the red diagonal cover the possibility of particle enrichment in the bud (y > yeq). Due to parameter degeneracy, an increase in the bud neck opening, an extension of the cell division duration, or a corresponding decrease in the cell size will move the partitioning solutions globally along the diagonal towards equilibration. Molecule-specific equilibration can, for example, be achieved by decreasing the number of binding sites in both mother and bud compartments.The offset red diagonal in Figure σ, fast diffusing particles are partitioned between mother and bud proportional to compartmental volumes, whereas particles with lower diffusion constants are progressively more retained in the mother  . In. Inσ, fagregates , 0.004 μfor ERCs ), consisfor ERCs ,28]  may explain their exclusion from the bud. Further characterization of aggregate dynamics should help to test this hypothesis and to investigate the alternative possibility of active transport mechanisms.Prions present another interesting case where diffusion could in principle limit partitioning. These infectious proteins can misfold and aggregate into β-sheet-rich amyloid fibers that catalyze the conversion of newly made proteins into the prion form. Prion propagation by self-templated fiber growth, chaperone-mediated fiber fragmentation and transmission of infectious aggregates to the bud during cell division can generate heritable phenotypes ,49] whe whe49] wkf > kr) can obviously lead to bud enrichment. Indeed, most organelles in S. cerevisiae are actively transported into the bud by myosin motors acting along actin cables [As diffusion can limit the partitioning of macromolecular content in the context of narrow escape, how does the dividing yeast cell ensure equal partitioning of structures such as large membranous organelles? Decreasing the reverse transfer efficiency (e.g. by increasing the number of binding sites in the bud) can allow enrichment of fast diffusing particles in the bud but has little effect on the partitioning of slow diffusing particles . Consequently, precise measurements of all parameters are clearly needed to understand how the partitioning of each macromolecular species is accomplished, and to determine whether passive diffusion is sufficient to account for experimentally observed partitioning or whether other processes such as active transport or convection should also be considered. Using fluorescent protein fusions, the mobility of labeled macromolecular species can be investigated with photoconversion, photobleaching or fluorescence correlation spectroscopy, which permit investigation of average mobilities on different time scales, whereas single particle tracking can provide access to the mobilities of individual molecules . DisturbThe authors declare that they have no competing interests.All authors conceived the research. AKi developed the model with input from AKh and MK. All authors discussed the results and wrote the manuscript. All authors read and approved the final manuscript.


The α2-adrenergic receptor (α2-AR) is widely distributed in the brain with distinct roles for α2-AR subtypes . In this article, data are provided on Activity Regulated Cytoskeleton Associated Protein (Arc) and Zif268 expression in the brain of the α2A-AR knockout (α2A-AR KO) mouse. These data are supplemental to an original research article examining Arc and Zif268 expression in rats injected with the α2-AR antagonist, RX821002 ( Specifications tableValue of the data•These data may stimulate research into the role of specific α2-AR subtypes in regulating cortical and hippocampal plasticity.•These data may stimulate research into the brain activity of α2A-AR KO mice in behavioral models of stress and anxiety •These data may stimulate research into learning and memory processes of mice with deletions of α2-AR subtypes.1These data show Arc and Zif268 mRNA levels in the brains of saline injected WT mice compared to saline injected α2A-AR KO mice. , Fig. 3.2n=5 per group) were injected i.p. with 100 μL of saline. Brains were harvested 1 h later, frozen on dry ice and stored at −80 °C. All animal use procedures were in strict accordance with The National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of Nebraska Medical Center Animal Care and Use Committee.Male C57 Bl/6J mice were purchased from Charles River Laboratories , and are designated as wild type (WT) mice in this data report. Male α2A-AR KO mice were purchased from Jackson Labs. WT and α2A-AR KO mice  using terminal deoxyribonucleotidyl transferase . Hybridization buffer containing 1×106 cpm of labeled probe was applied to each slide. Slides were coverslipped, sealed with D.P.X.  and placed overnight in a 1XSSC humidified sealed Tupperware container at 42 °C. The next day coverslips were removed in 55 °C 1XSSC and slides were washed 4×15 min in 1XSSC at 55 °C. Slides were apposed to Biomax film  for 2–3 weeks. Films were developed using standard techniques and analyzed using the MCID-M7 image analysis system .2.235S Arc and Zif268 mRNA levels in saline injected WT and α2A-AR KO mice were quantified with image analysis. Autoradiographic densities were quantified using commercial tritium standards  that were previously calibrated to 3t-test in each cortical layer and in the hippocampus.Arc and Zif268 mRNA levels were compared in saline injected WT and α2A-AR KO mice with a Student׳s


Patients with chronic aortic valve regurgitation (AR) causing left ventricular (LV) volume overload can remain asymptomatic for many years despite having a severely dilated heart. The sudden development of heart failure is not well understood but alterations of myocardial energy metabolism may be contributive. We studied the evolution of LV energy metabolism in experimental AR.in vivo by positron emission tomography (microPET) scanning of 6-month AR rats. Sham-operated or AR rats  were evaluated 3, 6 or 9 months post-surgery. We also tested treatment intervention in order to evaluate their impact on metabolism. AR rats  were trained on a treadmill 5 times a week for 9 months and another group of rats received a beta-blockade treatment (carvedilol) for 6 months.LV glucose utilization was evaluated MicroPET revealed an abnormal increase in glucose consumption in the LV free wall of AR rats at 6 months. On the other hand, fatty acid beta-oxidation was significantly reduced compared to sham control rats 6 months post AR induction. A significant decrease in citrate synthase and complex 1 activity suggested that mitochondrial oxidative phosphorylation was also affected maybe as soon as 3 months post-AR.Moderate intensity endurance training starting 2 weeks post-AR was able to partially normalize the activity of various myocardial enzymes implicated in energy metabolism. The same was true for the AR rats treated with carvedilol (30 mg/kg/d). Responses to these interventions were different at the level of gene expression. We measured mRNA levels of a number of genes implicated in the transport of energy substrates and we observed that training did not reverse the general down-regulation of these genes in AR rats whereas carvedilol normalized the expression of most of them.This study shows that myocardial energy metabolism remodeling taking place in the dilated left ventricle submitted to severe volume overload from AR can be partially avoided by exercise or beta-blockade in rats.The online version of this article (doi:10.1186/1471-2261-14-190) contains supplementary material, which is available to authorized users. The role of impaired myocardial energetics in the development and progression of heart failure (HF) seems to be central . The eneAlterations of myocardial metabolism caused by chronic valve disease such as aortic regurgitation (AR) are unclear and have not been studied like the ones caused by pressure overload or ischemia –19. ChroWe suggested that AR left ventricles with severe eccentric hypertrophy suffer from significant myocardial metabolic impairment even before systolic dysfunction becomes apparent and observed that as early as 8 weeks post-AR myocardial energy substrate preference was altered and a switch toward increased glucose utilization was observed .Here, we studied the long-term alteration in LV energy metabolism associated with chronic volume overload caused by severe AR in Wistar rats. We show that treatments (training and beta-blockade) that reduce LV dilatation and help maintain function are also associated with a normalization of the energy metabolism.Six groups of Wistar male rats (350–375 g) were studied for either 90, 180 or 270 days. For each end-point time, the animals were divided in two groups: sham-operated animals (sham) or surgically induced AR. All groups consisted of 15 animals with the exception of the 270-day AR group consisting of 30 animals. An additional group (n = 10) of young healthy rats served as controls. For the μPET study, eight additional animals (4 shams and 4 AR) were studied 6 months after surgery. For endurance training protocol, a group of 20 animals were exercised 5 days/week for 270 days on a motorized treadmill with a slope of 10°. The duration and the intensity increased progressively during the first 8 weeks until the animals were running for 30 minutes at 20 m/min as previously described . The infA complete M-Mode, 2D and Doppler echocardiogram was performed on the animals under 1.5% inhaled isoflurane anesthesia using a 12 MHz probe with a Sonos 5500 echograph  immediately before and during surgery, after 2 weeks, 3, 6 and 9 months as previously described .18F]-fluorodeoxyglucose ([18F]-FDG)  was injected via the caudal vein over 30 s. A 45-min dynamic PET data acquisition followed by a 15-min static acquisition was done to determine glucose utilization [myocardial metabolic rate of glucose (MMRG)] using multicompartmental analysis as previously described -FDG after intravenous bolus injection using μPET. As illustrated in Figure Micro-PET imaging was used to investigate how glucose consumption was altered We measured enzymatic activity levels in LV crude homogenates Figure . The HADIn order to evaluate if some alterations of the myocardial energy metabolism could be reversed, we tested the impact of moderate endurance training we previously showed to improve the condition of chronic AR rats. AR rats were thus submitted to moderate intensity endurance training on a treadmill (up to 20 m/s for 30 minutes) for a period of 9 months. Of the 20 animals, 14 survived the entire protocol. As illustrated in Figure The results of the 9 month AR gene expression levels of various enzymes and transporters related to fatty acid and glucose metabolism in the myocardium compared to age-matched sham animals as well as the effects of training are summarized in Figure A six-month carvedilol treatment improves the energy metabolism enzyme activity levels as well as the expression profile of metabolic genes in AR rats Figures  and 8.FiAt the end of the 6-month protocol, all sham-operated treated or not with carvedilol were alive while 9/15 and 12/15 rats were still present in the AR-Veh and AR-Carv groups, respectively. LV hypertrophy was present in both AR groups but significantly less in the animals treated with carvedilol Figure . This waAs illustrated in Figure The same was true for the LV mRNA levels of a number of genes associated with energy metabolism where the general down-regulation was mostly reversed by carvedilol.Factors influencing the development and evolution of LV remodelling in AR are poorly understood. Here, we provide a longitudinal study focusing on myocardial energy metabolism in the LV of rats with chronic severe AR.The heart is in a constant need of energy substrates since it does not maintain significant reserve . The myoOur experimental model causes severe LV dilatation. Despite the presence of important hypertrophy in this rat model, HF remains a late occurring event as seen in humans . As we hAs reported by others in models of LV concentric hypertrophy and of HF, we too observed a shift in the ratio of the gene expression of myosin heavy chains α and β in AR rats. This also occurs but much less, with normal aging . This shIn this study, we showed in vivo using μPET that the LV myocardium of AR rats increased its glucose consumption. This increase seems to be more pronounced in the LV free wall mostly in the lateral and anterior portions. It could be suggested that dilation may not be homogeneous through the LV was and that the observed metabolic changes in the LV myocardium may reflect this. We already observed the opposite situation in the AR rat where fatty acid uptake was reduced in the same LV region where we now observe an increase in glucose uptake . ConcentWe also described the impact of normal aging on the levels of enzymatic activity related to myocardial metabolism. We detected a significant loss of myocardial activity for three central metabolic enzymes  due to normal aging. The activity levels of these enzymes decreased by at least 25% in the last six months of the protocol. It is possible though that these changes reflect a progression from a stage of global body growth at a younger age to the more stable adult stage. Adding AR amplified this effect on HADH and CS activities. This suggests that fatty acid oxidation is further impaired in the late stage of AR and that the total mitochondrial oxidative capacity of the myocardium may then be less than normal . On the This study also clearly shows that regular exercise has beneficial effects on the myocardial energetic machinery in this animal model of volume overload cardiomyopathy even before systolic heart failure occurs. These effects were detectable on enzymes and pathways related to fatty acid oxidation and glycolytic capacity as well as to mitochondrial efficiency. The benefits of exercise on LV remodeling, diastolic function and survival we have recently reported could therefore be in part related to improvement in myocardial energetics . One posWe also observed an improvement of myocardial energetics in AR animals treated with the beta-blocker, carvedilol. We had reported that beta-blockade using either metoprolol or carvedilol can reduce the extent of LV hypertrophy development in the rat AR model , 36. TheIn this study, we used a range of techniques to evaluate myocardial metabolism in AR rats to demonstrate that substrate preference as well as general energy metabolism is modified in this model and that endurance training and beta-blockade can partially reverse these changes. Obviously, our study can only offer an incomplete portrait of the complex metabolic changes taken place in the myocardium submitted to severe and chronic volume overload. Enzyme activity determinations and gene expression studies made here cannot encompass the wide array of modification in energetics in the hypertrophied myocardium. More thorough studies using μPET in vivo, isolated heart or mitochondria studies could offer supplementary information to better describe these changes.Our results clearly show that the myocardium with chronic VO suffers from a significant metabolic stress and develops over time important metabolic abnormalities.These findings provide for the first time new longitudinal data which may improve our view of the dilated hearts of patients with severe AR. Clinicians currently feel comfortable to follow those patients without any intervention for many years, simply waiting for the LV to become too dilated, for the occurrence of symptoms or until systolic function begins to fall. Based on our findings, we suggest that those volume overloaded hearts develop severe metabolic abnormalities even when systolic function seems preserved. Focusing on myocardial metabolism by various interventions such as targeted drugs, specific diets or exercise may help this metabolically stressed myocardium to improve energy production and maybe prolong the pre-heart failure state significantly. Further studies will be needed to confirm this hypothesis.Supplemental methods and data. This file contains more detailed methods for the enzymatic assays as well as references. In addition, Figure S1. is a complement of data for the carvedilol study of Figures Additional file 1:


Minoxidil also suppressed AR-responsive reporter activity and decreased AR protein stability in human hair dermal papilla cells. The current findings provide evidence that minoxidil could be used to treat both cancer and age-related disease, and open a new avenue for applications of minoxidil in treating androgen-AR pathway-related diseases.Although minoxidil has been used for more than two decades to treat androgenetic alopecia (AGA), an androgen-androgen receptor (AR) pathway-dominant disease, its precise mechanism of action remains elusive. We hypothesized that minoxidil may influence the AR or its downstream signaling. These tests revealed that minoxidil suppressed AR-related functions, decreasing AR transcriptional activity in reporter assays, reducing expression of AR targets at the protein level, and suppressing AR-positive LNCaP cell growth. Dissecting the underlying mechanisms, we found that minoxidil interfered with AR-peptide, AR-coregulator, and AR N/C-terminal interactions, as well as AR protein stability. Furthermore, a crystallographic analysis using the AR ligand-binding domain (LBD) revealed direct binding of minoxidil to the AR in a minoxidil-AR-LBD co-crystal model, and surface plasmon resonance assays demonstrated that minoxidil directly bound the AR with a  The androgen-androgen receptor (AR) pathway is involved in a wide range of physiological development and disease processes. Notable in this latter context is androgenetic alopecia (AGA). In AGA, the androgen-AR pathway may affect hair growth in a paradoxical pattern at puberty, inhibiting hair growth in the scalp of genetically predisposed individuals but enhancing hair growth at most other body sites outside the scalp area. AGA is the most frequent cause of hair loss in men . BaldingThe AR is a transcription factor that belongs to the steroid hormone receptor superfamily. Prior to androgen binding, the AR is associated with the chaperone proteins HSP70 and HSP90. Upon androgen binding, the androgen-AR complex undergoes a conformational change and translocates to nucleus, where it forms a dimer on target gene promoters and turns on target gene transcription. This process is assisted by an array of coregulators .Testosterone, the major androgen in the circulation, is converted to the more potent androgen, dihydrotestosterone (DHT), via 5α-reductase in tissue. Two types of 5α-reductase isoenzymes have been described, type I and type II, the latter of which is more highly expressed in AGA follicles than in follicles of normal controls . The AR Minoxidil was introduced clinically in the early 1970s as an anti-hypertension medication, but was found to have the side effect of hypertrichosis, an abnormal amount of hair growth over the body . CurrentPrevious studies have consistently concluded that minoxidil does not act directly through an androgen effect , 20. Howin vitro organ culture or animal studies, high concentrations, ranging from 1-100 mM, have been reported in skin tissue [Minoxidil has been used to treat the AR-related disease, AGA, for more than two decades, suggesting that it may interfere with AR-related functions. To investigate this possibility, we first tested minoxidil in an AR transcription reporter assay using LNCaP prostate cancer cells, which harbor an endogenous AR with a T877A mutation. Cells were transiently transfected with an AR-responsive MMTV -Luc construct and treated with different concentrations of minoxidil. Minoxidil concentrations were chosen based on most common reports in the literature: 0.1 to 10 μM for oral intake and about 1 mM for topical application of 5% minoxidil (100 mM) in skin tissue , assuming 1.7% absorption. In n tissue , 32. AftTo further clarify whether minoxidil suppresses human AR target gene transcription, we co-transfected PC-3 cells with PSG5-AR and a PSA-Luc reporter construct containing 1.5 kb of the prostate-specific antigen (PSA) promoter linked to the luciferase gene. As shown in Fig. Clinically, anti-androgens are used to treat prostate cancer. To further test minoxidil suppression of AR-related function, we tested the effects of minoxidil on the growth of LNCaP cells, an androgen-sensitive prostate cancer cell line that, as noted above, endogenously expresses AR. As shown in Fig. To assess possible nonspecific effects of minoxidil at high concentrations, we performed control studies, testing different concentrations of minoxidil (1-100 μM) on glucocorticoid receptor (GR) transcriptional activity. As shown in Fig. We next investigated the mechanism underlying the ability of minoxidil to suppress AR-related functions. Full transcriptional activity of the AR requires the assistance of coregulators. Phage display and real AR coregulator analyses have shown that an FxxLF-like motif in coregulators mediates coregulator-AR interactions. To determine whether disruption of these interactions contributes to the suppression of AR-related function by minoxidil, we first examined the effect of minoxidil on the interaction between an FxxLF-containing peptide and the AR. In these experiments, PC-3 cells were co-transfected with an FxxLF-containing peptide (GAL4-DBD-3-18), an expression plasmid for full-length AR (VP16-AR), and a pG5-Luc construct. After treating with DHT and different concentrations of minoxidil, cell extracts were assayed for luciferase activity. As shown in Fig. Formation of an AR dimer, which is required for transcriptional activity, involves an interaction between AR N- and C-termini mediated by the AR N-terminal FxxLF motif. Co-expressing truncated AR N- and C-termini recapitulated this AR N-C interaction, turning on target gene expression , as a function of time (s). As shown in Fig. in vitro studies. To extend these findings to skin cells, we tested whether minoxidil has similar effects in HHDPCs  using an AR reporter assay. AR expression in HHDPCs was confirmed at mRNA . The cell lines PC-3, LNCaP, LAPC4, and HepG2 were purchased from American Type Culture Collection . Human hair dermal papillae cells (HHDPCs) were purchased from ScienCell Research Laboratories . The plasmids pSG5-AR, pSG5-GR, Gal-4-ARA54C, Gal-4-peptide, pCDNA3-AR-N, pCDNA3-AR-C, pCMX-VP16-AR, MMTV-Luc, PSA-Luc, and pG5-Luc were constructed as described previously , 22. AntcDNA for the human AR ligand-binding domain  was amplified from pSG5-AR by polymerase chain reaction (PCR) and inserted into the pET28c vector . Protein expression and purification were as described previously . The DHTP212121, with unit cell parameters of a = 55.43 Å, b = 65.54 Å, c = 70.38 Å, and α = β = γ = 90°. The structures were determined by the molecular replacement method AMoRe, using the unliganded AR-LBD structure (PDB code: 1T7T) as the search model [The 2.4 Å dataset was collected at the BL13B at the National Synchrotron Radiation Research Center in Hsinchu, Taiwan, using an ADSC Quantum 315r CCD detector. Indexing and processing of the measured intensity data were performed using the HKL2000 software package . The crych model . Refinemch model  and coupch model . The refch model . Data coThe PC-3 human prostate cancer cell line was grown in Dulbecco's minimal essential medium containing 10% fetal bovine serum, 2 mM L-glutamine, and 10 U/ml penicillin; the LNCaP cell line and HHDPCs were maintained in RPMI-1640 medium containing 10% fetal bovine serum and antibiotics. Culture media for all cell lines were phenol red-free, and charcoal-treated fetal bovine serum was used in DHT-related assay. For transfection, cells were plated in 24-well plates and transfected with plasmids using a Superfect kit , as described by the manufacturer. After 16 h of incubation, the cells were treated with ethanol (vehicle control), steroid hormones, or different concentrations of minoxidil for an additional 16 h and then harvested for luciferase assays. Luciferase activity in whole-cell extracts was measured using a luciferase assay kit, as described by the manufacturer  and normalized to the protein concentration in the respective extract. The results were presented as means ± standard deviations (SD) of triplicate determinations, expressed relative to untreated control cells (defined as 1); three independent transfections were performed.4 cell/well in 24-well plates and incubated with or without 1 nM DHT and different concentrations of minoxidil for 2 to 6 d. Cell growth was measured by MTT [LNCaP cells were plated at 5 × 10d by MTT . BrieflyWestern blot analyses were performed as previously described in detail  using anThe effect of minoxidil on AR protein stability was assessed by first seeding and incubating LNCaP cells for 24 h. The culture medium was then replaced with medium containing 10 μg/ml cyclohexamide alone or with 10 nM DHT or different concentrations of minoxidil, and plates were incubated for different lengths of time. Thereafter, cell lysates were analyzed for AR and β-actin by Western blotting.SPR experiments were carried out on a Biacore 3000 instrument . All experiments were performed at 25°C as described previously [ATP) channel subunits, Kir6.1 and SUR2B, in HHDPCs and cancer cell lines. mRNA was converted to cDNA using the avian myeloblastosis virus (AMV) reverse transcription (RT) system , as described by the manufacturer. PCR amplification was performed under standard conditions using Taq DNA polymerase  and the following primer pairs: AR (M20132.1), 5'-CAG GAA TTC CTG TGC ATG AAA GCA CTG C-3' (forward) and 5'-TCA CTG GGT GTG GAA ATA GAT GGG C-3' (reverse); SUR2B (NM_005691), 5'-GCT GAA GAA TAT GGT CAA ATC TC-3' (forward) and 5'-TGG AGT GTC ATA TTC TAA AAT A-3' (reverse); Kir6.1 (NM_004982), 5'-CAT CTT TAC CAT GTC CTT CC-3' (forward) and 5'-GTG AGC CTG AGC TGT TTT CA-3' (reverse). pSG5-AR served as a positive control in AR studies, and HHDPCs served as a positive control in KATP channel studies. The target bands were sequenced and compared with published sequences [RT-PCR was used to investigate the expression of mRNA for the ATP-sensitive potassium (Kequences .t-test.Data are expressed as means ± SEM. Differences between two groups were assessed using an unpaired two-tailed Student's


Gastrointestinal nematodes (GINs) are one of the main constraints to ruminant production worldwide. Anthelmintic resistance (AR) has been reported in goats throughout Europe, yet little is known about the AR status in Italy. The aims of the study were: i) determine the frequency of AR in GINs in goat flocks in Northern Italy, Italy, ii) survey goat farmers on the current practices of parasite control, iii) update the species composition of the gastrointestinal helminthofauna. Thirty three flocks were enrolled and 1288 individual fecal samples were collected. Based on the egg per gram (EPG), 15 flocks were selected to evaluate the presence of AR in GINs with the Fecal Egg Count Reduction Test (FECRT). A questionnaire surveyed 110 dairy goat farmers to acquire information about farm management and drenching practices against GINs. Further, the gastrointestinal tracts of 42 goats were analyzed.Teladorsagia/Trichostrongylus L3 were isolated from the post-treatment coprocultures of all flocks with resistance but not from the flock with suspected oxfendazole resistance. Treatments against helminths were performed once annually in 73.63% of the flocks, but 20.00% of farmers declared not regularly treating their goats every year. Annual treatments usually occurred in autumn or winter at dose rate for sheep. Te. circumcincta, H. contortus, Tr. colubriformis, Skrjabinema caprae and Oesophagostomum venulosum were the most abundant and prevalent species of the gastrointestinal tract.The FECRs indicated that five of the 15 flocks had problems of AR, which was identified in all two of the anthelmintic classes tested. Resistance and suspected resistance was found in 40% of the flocks selected for AR testing that were treated with benzimidazoles while 20% of the flocks treated with eprinomectin had resistant GINs. Strategies to prevent the development of AR should be widely adopted in Northern Italy. Further, farmers and practitioners should be educated about the importance of using the correct dose rates in goats. In addition, the presence of highly pathogenic GINs coupled with high worm burden in all sector of gastrointestinal tract and the prevalence values further suggest that improved diagnosis and active surveillance of GINs infection is needed. Gastrointestinal nematodes (GINs) remain one of the main constraints to ruminant production, since they can causing reduction in skeletal growth, live-weight gain and in milk yield,2. GINs A survey was performed with a questionnaire from November 2010 to June 2011 in Lombardy, a region of Northern Italy . Members of SATA, a regional association offering zootechnical and veterinarian support to breeders, were involved in the study. The majority of surveyed farms were in the areas of Northern Italy most suitable for goat breeding.All animal procedures used in this study were approved by the Milan University Institutional Animal Care and Use Committee.Individual fecal samples were collected from the rectums of 1288 dairy goats from 33 flocks. All animals were older than two years of age. The parasitological analyses used a modified McMaster method,10, withBenzimidazoles and probenzimidazoles (BZs) and macrocyclic lactones (MLs) were the classes of anthelmintic selected for evaluation in this survey based on the information obtained from the questionnaire. Albendazole (ABZ) (7.5 mg/kg body weight (bw)), oxfendazole (OFZ) (10 mg/kg bw), febantel (10 mg/kg bw) and netobimin (15 mg/kg bw) were tested in ten flocks. Ivermectin (IVM) and moxidectin (MOX) at 0.4 mg/kg were administrated to goats of one and two flocks, respectively. Finally, eprinomectin (EPM) at an extra-label dose of 1 mg/kg bw was tested in two flocks. All drugs were orally dosed except EPM, which was applied with a pour-on formulation. The dose rates of the anthelmintics were twice as high as those for sheep or cattle. The body weight of the visually heaviest goat of each flock was estimated to determine the anthelmintic dose rate. The number of dosed animals in each flock, based on the recommendations provided by Coles et al., varied Individual fecal samples were collected on the day of treatment (T1). T1 mean EPG and standard deviation were calculated for each flock. FECRTs were performed only on goats with EPGs ≥ 150 at T1. Individual post-treatment fecal samples (T2) were collected after 8–10 days from goats treated with BZs and after 14–17 days from goats treated with MLs. The fecal egg count reduction (FECR) was calculated with the formula proposed by Cabaret and Berrag, in whicTi2 is post-treatment and Ti1 is pre-treatment EPG in host i from a total of n hosts. The tested population of nematodes was classified as resistant when the FECR was <95% and the lower limit of the 95% confidence interval (CI) was <90% or as “suspected resistant” when the FECR was <95% or the lower limit of the 95% CI was <90%[where was <90%.Teladorsagia/Trichostrongylus, Oesophagostomum/Chabertia, Haemonchus, Nematodirus, Bunostomum and Cooperia, according to MAFF[Pre- and post-treatment larval cultures were performed from pooled fecal samples for each flock; pools were composed with feces collected from each animal of the group. The feces sampled at T1 and T2 were incubated for 10 days at 25°C in a large vessel, ensuring adequate moisture (50-80%), and third-stage larvae (L3) were recovered from the coprocultures by a Baermann technique. The fir to MAFF and van  to MAFF. When feA questionnaire surveyed 110 dairy goat farmers from 51, 43, 10 and six farms from eastern (Bergamo (BG) and Brescia (BS) provinces), western (Como (CO), Lecco (LC) and Varese (VA) provinces), northern (Sondrio (SO) province) and southern (Pavia (PV) province) areas of Lombardy, respectively. Each farmer was asked about their farm management, including the presence and dimensions of pastures, demography and breed of goats, the presence of other species on the farm (e.g. cattle) and drenching practices against GINs, such as treatment times, frequency, products and dosages of anthelmintic drugs. Farm veterinarians contributed to answering these last questions.The gastrointestinal tracts of two or three goats from the 15 flocks previously selected to evaluate the presence of AR were collected at abattoirs. The species composition and abundance of abomasal and intestinal nematodes of 42 goats were analyzed. Abomasa and small and large intestines were examined separately; they were opened and the contents were filtered through metallic sieves to collect adult helminths. Ten perTeladorsagia/Trichostrongylus were the most common GINs in the study area: Teladorsagia/Trichostrongylus L3 were recovered from 13 of the 15 flocks (86.6%) and were the most prevalent GINs in eight of the 13 flocks (61.5%) infected with these worms. Oesophagostomum/Chabertia and Haemonchus L3 were recovered from 11 (73.3%) and nine (60%) flocks, respectively, and Haemonchus was the most prevalent GIN in six of the nine flocks (66.6%) infected with this species. Bunostomum, isolated from five flocks (33.3%), was the most prevalent GIN in flock 11. According to Table 1Nematodirus L3 were isolated only from flock 3, and Cooperia L3 were not found in any flock.Adult goats of the 15 flocks selected for testing AR had a mean pre-treatment EPG of 426.93 (±249.11)  were isolated from the coproculture of the flock with EPM resistance  of the goat farms practiced grazing, and the majority of farms with pastures (60.5%) were in the western area. Pastures had a mean area of 11.63 ha  and altitudes from 200 to 1500 m a.s.l. Goats usually grazed from March or May to October or November during the day (or at night in the hottest months) and were kept in the fold at night (or day in the hottest months), depending on the region. Intensive breeding was the most common system in farms from southern (100%) or eastern (90.2%) areas, whereas only 55.8% or 50% of farms from western or northern areas, respectively, had this type of farming. Farms in the eastern and northern areas had the largest mean flock sizes ; these farms also had the highest number of does  of the flocks, but 20.00% (22/110) of farmers declared not regularly treating their goats every year. No differences in frequency of treatments were detected depending on the breeding system and reared breeds. Annual treatments usually occurred in autumn or winter (November to January), depending on the farm and particularly on the dry status of does. A few flocks received an additional treatment in summer when necessary, and a low percentage of farms (2.72%) regularly administered two treatments. The choice of anthelmintic varied among the farms but was dominated by BZs (87.27%); only 2.73% (3/110) of farmers treated their flocks with MLs. Anthelmintics were not rotated on any of the farms. In 89.09% (98/110) of the flocks, anthelmintic dose rates were calculated on the basis of the visually estimated body weight of the goat that appeared heaviest. The dose rate for sheep was used to treat the goats in all these flocks. Eleven of the 110 farmers (10%) did not indicate the dose rate used in their flocks. Annual treatments were usually done without any parasitological analyses, which were only performed on particular occasions Table .T. circumcincta was the most abundant (mean A = 558.62) and most frequent (P = 73%) abomasal parasite in all studied goats, followed by H. contortus . Other abomasal species belonging to the subfamily Ostertagiinae  were also present but with low abundances and prevalences  were the dominant species in the small intestine, whereas Skrjabinema caprae and Oesophagostomum venulosum had the highest epidemiological parameters in the large intestine (Table All necropsied goats were infected by GINs (P = 100%). mean A = 58.62 andThe questionnaire indicated that the anthelmintic classes BZ and ML were used against GINs in Northern Italy and the FECRTs performed in accordance with WAAVP guidelines demonstrated that AR involved all two anthelmintic classes, with an overall prevalence of 33.33% (5/15 flocks selected for AR testing). Resistance and suspected resistance was found in 40% (4/10) of the flocks selected for AR testing that were treated with BZs, while 20% (1/5) of the flocks treated with MLs had resistant GINs. The absence of L3 in the post-treatment coproculture from the flock with suspected resistance to OFZ, however, did not permit an accurate assessment of sensitivity to this drug. Excluding this flock would lower the percentage of flocks presenting AR to BZs to 30% (3/10) and the overall prevalence of AR resistance to 26.67% (4/15).Tr. colubriformis was recently demonstrated in southern Italy[Very little data is available on AR on Italian goat farms. Resistance to BZs in rn Italy. A generrn Italy, and AR rn Italy,25,26. Trn Italy found thrn Italy. AR has rn Italy, Germanyrn Italy and Slovrn Italy. BZs arern Italy,28,32.Teladorsagia/Trichostrongylus group was resistant to this anthelmintic class. Reports of AR to ML on European goat farms are rare. AR in Teladorsagia, Trichostrongylus and Haemonchus has been reported in Denmark, Switzerland, Germany and the UK[Teladorsagia/Trichostrongylus and Haemonchus. The T1 and T2 coprocultures from this flock showed that AR probably involved primarily Teladorsagia/Trichostrongylus. Haemonchus L3 were the most abundant (39%) in T1 coprocultures, followed by Teladorsagia/Trichostrongylus L3 (32%), while the proportion was inverted in T2 coprocultures (75% Teladorsagia/Trichostrongylus L3 and 25% Haemonchus L3). Nevertheless, AR to EPM in flock 15 appeared to involve both Teladorsagia/Trichostrongylus and Haemonchus. Even though AR to EPM in goats had already been described in Switzerland and Germany, the results for this flock were quite unexpected. EPM is only registered for use in cattle in Italy, and the questionnaire indicated that its extra-label use occurred in only 0.91% (1/110) of Northern Italy farms. Flock 15 had never been treated with EPM, but the goats had been dewormed once a year for four years with IVM at the dose rate indicated for sheep. This practice in flock 15 may have caused the appearance of AR to the subclass of avermectins or to the entire ML anthelmintic class, despite the low frequency of treatment. The development of AR to the entire ML anthelmintic class  due to the repeated use of ivermectin is reasonably still uncommon in goats; a treatment with MOX in goats that failed against IVM-resistant Ostertagia (Teladorsagia) species was described by Leathwick[AR to BZs in Northern Italy also appears to be more widespread than AR to MLs; this difference is probably due to the long-term use of BZs and the lack of rotation with other drugs on dairy-goat farms in this region. The isolation of L3 from post-treatment coprocultures from flocks with resistance to BZs indicated that only the d the UK,33,34. Teathwick in New Zeathwick were reseathwick. MOX, ifThe questionnaire submitted to goat farmers of Northern Italy indicated that the annual number of treatments is very low respect to other countries,37. In fTe. circumcincta, H. contortus, Tr. colubriformis, Sk. caprae and Oesophagostomum venulosum. were the most abundant and prevalent species of the gastrointestinal tract. Most of the species were previously reported in naturally infected goats in Northern Italy but N. lanceolatus is described for the first time in Italian goats[N. lanceolatus, placed in synonymy with N. oiratianus by Samson[Sp. spiculoptera morph spiculoptera, Sp. spiculoptera morph mathevossiani and Os. leptospicularis are common parasites in Cervus elaphus and Capreolus capreolus[Te. circumcincta is one of the most dominant species in the Alpine chamois, Rupicapra rupicapra[H. contortus), and the presence of Os. ostertagi in goats is probably due to its presence in cattle in the same pasture zones. Further, the analysis of helminthic community structure shows that these species, particularly Sp. spiculoptera morph spiculoptera, give an important contribution to composition of the community even if they have been classified into the categories of codominant or subordinate species by the importance values. In addition, the detection of a few species typical of cervids emphasizes that the goats may incur other parasitic risks resulting from interaction with these hosts on pastures[Te. circumcincta. It may result from the few ecological niches offered by that environment to parasites life cycle rather the frequent use of anthelminthics[Te. circumcincta and Tr. colubriformis showed both high prevalence and worm burden in agreement especially with data of Chartier and Reche[Tr. colubriformis. As suggested by the authors, it may depend on the scarce ability to overwintering on pastures by this parasite. Further, H. contortus showed higher prevalence rates than has been previously found in goats from the same area or from different areas with temperate climate[H. contortus has a wide distribution being also found in regions characterized by a very severe climate due its ability to survive inside the hosts[Sk. caprae in the large intestine is remarkable; this parasite is generally considered with a very low pathogenicity but it can cause in dairy goats restlessness, itching and lesions in the perianal region[The survey on the composition of gastrointestinal helminthofaunas by the necroscopy of goats showed that an goats,42. Accoan goats, N. lancy Samson, showed capreolus, and Te.rupicapra,44. The pastures. Overallpastures. In thisminthics,47. By qnd Reche but disand Reche that in  climate,46,49. The hosts. At lastl region. In addil region,49. ThesThe established presence of AR in 30% of the tested flocks in Northern Italy suggests that strategies to prevent the development of AR should be widely adopted in this region. The answers to questionnaire show that farmers and practitioners should be firstly educated about the importance of using the correct dose rates in goats and about the rotation of different anthelmintic classes, because AR can develop even at low frequencies of treatment. The management of resistance on the farms presenting AR should include the use of targeted selective treatments to sustain susceptible GINs population in refugia. The queThe authors declare that they have no competing interests.The study idea was conceived by SAZ and MTM. ADC and MV participated in the design of the study; ADC helped administering the questionnaire. SAZ and AG participated in the acquisition of the laboratory data. SAZ and MTM carried out the statistical analysis. Data interpretation was done by all authors. SAZ, MTM and MV drafted the manuscript. All authors contributed to the critical revision of the manuscript for important intellectual content and have seen and approved the final draft.


AR cryptic exon 3b inclusion. Here, we explore the role of the RNA-binding protein (RBP) Sam68 (encoded by KHDRBS1), which is over-expressed in clinical PCa, on AR-V7 expression and transcription function. Using a minigene reporter, we show that Sam68 controls expression of exon 3b resulting in an increase in endogenous AR-V7 mRNA and protein expression in RNA-binding-dependent manner. We identify a novel protein-protein interaction between Sam68 and AR-V7 mediated by a common domain shared with full-length AR, and observe these proteins in the cell nucleoplasm. Using a luciferase reporter, we demonstrate that Sam68 co-activates ligand-independent AR-V7 transcriptional activity in an RNA-binding-independent manner, and controls expression of the endogenous AR-V7-specific gene target UBE2C. Our data suggest that Sam68 has separable effects on the regulation of AR-V7 expression and transcriptional activity, through its RNA-binding capacity. Sam68 and other RBPs may control expression of AR-V7 and other splice variants as well as their downstream functions in CRPCa.Castration-resistant (CR) prostate cancer (PCa) partly arises due to persistence of androgen receptor (AR) transcriptional activity in the absence of cognate ligand. An emerging mechanism underlying the CRPCa phenotype and predicting response to therapy is the expression of the constitutively-active AR-V7 splice variant generated by  Prostate cancer (PCa) onset and progression is driven by androgen steroid hormones binding to their cognate androgen receptor (AR) nuclear hormone receptor transcription factor. Androgen deprivation therapy (ADT) is the mainstay of treatment for locally-advanced/metastatic PCaAn evolving mechanism for the persistence of AR signaling in CRPCa is the generation of constitutively-active AR variants sharing a common amine (N)-terminus domain (NTD) and DNA-binding domain (DBD) but lacking a ligand-binding domain (LBD)trans-acting RNA-binding proteins (RBPs) SRSF1 (encoded by SRSF1) and U2AF65 (encoded by U2AF2) were found to be involved in an ADT-mediated increase in AR-V7 expression. We have previously identified the RBP Sam68 (encoded by KHDRBS1) as an AR-interacting protein partner, and co-regulator of AR-dependent transcription and splicingAlthough ligand-activated AR can affect overall expression of both full-length AR and AR-V77in vitro and co-activates AR-dependent transcription and splicingAR cryptic exon 3b as well as constitutive exons 3 and 4 under the transcriptional control of the constitutively-active cytomegalovirus (CMV) promoter in a minigene reporter (V229F mutantHNRNPA2B1)Since Sam68 interacts with AR reporter 8. HEK293p = 0.0003) (p = 0.04). Ectopic expression of the Sam68-interacting hnRNPA2 protein did not have a statistically-significant effect on exon 3b (or exon 4) expression (p > 0.05), suggesting that exon 3b expression may be directly controlled by Sam68 itself rather than by other Sam68-interacting RBP partners.Ectopic expression of Sam68 protein resulted in a ~3.5-fold increase in cryptic exon 3b expression compared with the empty vector control ( 0.0003) . ConsistV229F mutantV229F mutant was much less efficient at stimulating expression of exon 3b (~2-fold increase above controls), and did not have an effect on expression of mRNAs containing exon 4 (p > 0.05)  .cis-acting exonic splicing enhancer (ESE) close to the 3′ splice site (SS)p = 0.14)  . We alsoin vivo, we employed the full-length AR- and AR-V7-expressing CWR22 and VCaP PCa cell linesKHDRBS1 mRNA or Sam68 protein at similarly high levels to other common PCa cell lines (8To determine whether the Sam68-dependent increase in exon 3b expression translated into an increase in endogenous AR-V7 mRNA and protein expression ll lines . ADT/antl lines 8.p > 0.05)  and absence of DHT (p = 0.002) (p < 0.001) (p > 0.05)  .p = 0.04) without an effect on full-length AR mRNA expression (p > 0.05)  . HoweverConsistent with published findings in LNCaP cellsTo test this hypothesis, both Sam68 and AR-V7 proteins were separately immunopreciptated in whole cell extracts from CWR22 cells cultured in full medium using their cognate antibodies prior to Western blotting . The antTo verify this novel Sam68-AR-V7 protein-protein interaction, HEK293 cells cultured in full medium were transiently transfected with expression vectors for Sam68 and AR-V7 proteins . Consistp = 0.0001).The above findings identify a novel protein-protein interaction between Sam68 and AR-V7. Since both full-length AR and AR-V7 share a common NTD , we hypoin vivo, we sought to determine if these proteins also co-localise in vivo. Using indirect immunofluorescence and confocal microscopy, we compared the intracellular distributions of endogenous Sam68 protein with full-length AR in LNCaP cells  from the core promoter region of the KLK3 gene . In keeping with published findings3 reporter in an RNA-binding-independent manner  (3 reporter activity in the absence of the AR (data not shown).Consistent with the absence of the LBD, AR-V7-mediated reporter activity was not affected by the addition of DHT . In the e of DHT . In the  = 0.02) . Ectopicp = 0.04)  . Westernin vivo. To test this hypothesis, we used qRT-PCR to examine the effect of ectopic Sam68 protein expression on canonical AR  or AR-V7-specific (UBE2C) target gene expression in CWR22 cells grown in steroid-depleted medium   (UBE2C (p = 0.02) (UBE2C gene expression following ectopic expression of the Sam68V229F mutant (p = 0.06).AR splice variants, including AR-V7, have been shown to regulate a transcriptional programme distinct to full-length AR in PCa cells14d medium . Ectopic = 0.03) . However > 0.05) . Ectopic = 0.02) . We alsoKHDRBS1) has been shown to be over-expressed9KHDRBS1 as well as SRSF1 and U2AF2, which encode the RBPs SRSF1 and U2AF65, respectively, and have been implicated in AR-V7 splicingKHDRBS1 or SRSF1 gene expression between primary localised PCa (n = 59) and CRPCa (n = 35)  (U2AF2 gene expression in CRPCa as compared with primary PCa (p < 0.001)  correlated with expression of the AR-V7 mRNA in clinical PCa we used The Cancer Genome Atlas (TCGA) dataset (n = 417). We tested for correlations in expression of AR-V7 (ENST00000504326) and the major Ensembl transcripts encoding Sam68 (ENST00000327300), SRSF1 (ENST00000582730), and U2AF65 (ENST00000450554) (KHDRBS1 (encoding Sam68) appeared to be expressed at higher levels than U2AF2 and SRSF1 in primary PCa (To determine whether expression of genes encoding RBPs involved in AR-V7 expression (0450554) . Althoug3 reporter in an RNA-independent manner. Finally, we show that Sam68 controls expression of canonical AR and AR-V7 target genes in PCa cells via both RNA-binding-dependent and –independent mechanisms.In this study, we demonstrate for the first time that the RBP Sam68 preferentially increases AR cryptic exon 3b expression, and controls mRNA and protein levels of the AR splice variant AR-V7 in PCa cells largely via an RNA-binding-dependent mechanism. Consistent with our previous findings for full-length ARThe emergence of constitutively-active AR splice variants in CRPCa, particularly AR-V7, poses a major clinical challenge as these variants not only seem to mediate resistance to novel inhibitors of androgen biosysthesis and ligand-dependent AR activity in PCa models13trans-acting complexes of several RBPs binding cis-acting regulatory pre-mRNA elements, alternative splicing can affect the processes underlying the hallmarks of cancercis-acting effect mediating AR-V7 expression may be due to an intragenic intron 1 deletiontrans-acting RBPs SRSF1 and U2AF65 appear to mediate an increase in AR-V7 expression in response to ADT in vitro through recruitment and binding to an intronic splicing enhancer (ISE) and ESE, respectively, close to the cryptic exon 3b 3′ SSControlled by V229F mutant to partially regulate exon 3b expression may be due to recruitment of endogenous wild-type protein, as Sam68 is thought to bind RNA as a homodimerSRSF1 mRNAOur data suggest that Sam68 also appears, at least in part, to have an effect on exon 3b expression via the SRSF1-responsive ESE . This efHowever, the trend towards a Sam68-regulated increase in exon 3b expression from the ESEm AR-V7 minigene suggests the effect of Sam68 may not be completely mediated by the ESE. An alternative mechanism, in keeping with its ability to control transcript stability via 3′-untranslated region (UTR)-bindingAR-V7 mRNA expression, our data suggest that this function is not required to co-activate AR-V7-dependent transcriptional activity. However, high total levels of Sam68 protein may be required for it to function as a co-activator of AR-V7-dependent transcriptional activity of the luciferase reporter  gene expression in CRPCa and a correlation with AR-V7 expression in primary PCa, although we did reveal up-regulation of U2AF2 gene expression in CRPCa and correlations between SRSF1 (encoding SRSF1) or U2AF2 (encoding U2AF65) and AR-V7 mRNA expression in primary PCa. In the absence of any existing large datasets from CRPCa patients that allow similar transcript correlations, our observations in localised PCa suggest that absolute expression levels of U2AF2 and possibly SRSF1 may be important for regulation of AR-V7 mRNA expression in CRPCa. Since KHDRBS1 is already expressed at higher levels in primary PCa as compared with SRSF1 and U2AF2, post-translational modifications of Sam68 protein, rather than absolute expression levels, may be of greater importance for AR-V7 expression. Phosphorylation of Sam68 has been reported in primary PCaBy knowledge-based validation, we were unable to identify up-regulation of cis-elements and other trans-acting RBPs (e.g. SRSF1 and U2AF65) appear to be involved, both directly and indirectly, Sam68 is the only RBP known to regulate AR-V7 expression and downstream transcription function that is over-expressed9Since AR-V7 protein appears to be able to bind to and regulate a distinct subset of genes in CRPCa cell line models14V229FEcoRI and SalI. cFlag AR-V7 was generated by cloning of a PCR-amplified cDNA fragment containing the AR-V7 coding sequence from the 22Rv1 PCa cell line  into c-Flag pcDNA3XhoI and BamHI. p(ARE)3Luc contains 3x repeats of the 1st androgen response element (ARE) plus TATA box from KLK3 promoter region cloned into pGL3basic . The identity and integrity of newly-generated clones was confirmed by Sanger sequencing. Sequences used to generate siRNA duplexes are as previously described8The following antibodies were used: anti-Sam68 , anti-AR G122-434) , anti-AR (N-20) , anti-AR-V7 , anti-SC35 , anti-α-tubulin , normal mouse IgG , anti-mouse IgG HRP-linked , anti-rabbit IgG HRP-linked , Alexa Fluor® 488 anti-Mouse IgG  and Alexa Fluor® 555 anti-Rabbit IgG . The following plasmids have been described previously or are commercially-available: pEGFP3-Sam68 , 22Rv1 , VCaP  and CWR22All cells were grown at 37 °C in 5% COhttp://rsb.info.nih.gov/ij/), and intensities used to calculate relative normalised fold-change in protein expression. Western blotting data shown are from at least two independent biological experimental replicates.Protein immunoprecipitation was performed as previously describedLNCaP and PC3-M cells were grown and transfected on glass coverslips , and stained with primary and secondary antibodies as previously describedTotal RNA extraction, quantitative RT-PCR (qRT-PCR), and relative gene expression analyses were performed as previously described5 cells/ml in full medium into 6-well plates (Asahi Techno Glass). Cells were transfected with DNA as detailed in figure legends. RNA was extracted using RNeasy (Qiagen) and subjected to qRT-PCR using oligonucleotides as previously describedHEK293 cells were seeded at a density of 1 × 104 cells/ml in full medium into 12-well plates (Asahi Techno Glass). Cells were transfected with DNA as detailed in the figure legends. Luciferase and β–Galactosidase assays were performed to give relative activity as previously described4 cells/well in steroid-depleted medium into 24-well plates (Asahi Techno Glass). Cells were transfected with DNA, and supplemented with 100 nM DHT as detailed in figure legends. Firefly and Renilla luciferase assays were carried out using the Dual Luciferase Reporter Assay system  as per manufacturer’s instructions to give relative luciferase activity. Data shown are from at least three independent biological experimental replicates with three technical replicates.For mammalian 2-hybrid assays, HEK293 cells were seeded at a density of 5 × 102 transformed probe signal, and plotted against the PCa cell lines as annotated in the sample information. For clinical samples, median-centered gene expression data were extracted from the Grasso datasetp < 0.05 taken to indicate statistical significance . FASTQ files containing pre-processed (Level 3) RNA-Seq data were downloaded from The Cancer Genome Atlas (TCGA) data portal (Accession number: 23700-2). Adaptors were trimmed using Skewerq-values reported using the false discovery rate (FDR) with FDR < 0.05 taken to indicate statistical significance.Pre-processed microarray data were downloaded from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO)p < 0.05 taken to indicate statistical significance.Graphical data shown represent the means ± standard error (SE) of independent experiments. The one-tailed independent sample T-Test was employed to identify differences in means between groups with How to cite this article: Stockley, J. et al. The RNA-binding protein Sam68 regulates expression and transcription function of the androgen receptor splice variant AR-V7. Sci. Rep.5, 13426; doi: 10.1038/srep13426 (2015).


Cardiac sympathetic nerve sprouting and the dysregulation of β-adrenergic receptor (β-AR) play a critical role in the deterioration of cardiac function after myocardial infarction (MI). Growing evidence indicates that exercise provides protection against MI. The aims of this study were to investigate whether aerobic exercise following MI could inhibit sympathetic nerve sprouting and restore the balance of β3-AR/β1-AR.Male Sprague-Dawley rats were divided into three groups: sham-operated control group (SC), MI group (MI), and MI with aerobic exercise group (ME). The rats in ME group were assigned to 8 weeks of exercise protocol . The expression of nerve growth factor (NGF), the sympathetic nerve marker-tyrosine hydroxylase (TH), the nerve sprouting marker-growth associated protein 43 (GAP43), and β1- and β2-AR expression in the peri-infarct area of the left ventricle (LV) were measured by Western blot and immunohistochemistry, while β3-AR expression was determined by Western blot and immunofluorescence. Endothelial nitric oxide synthase (NOS2), phospho-NOS2 (p-NOS2), and neuronal nitric oxide synthase (NOS1) were measured by Western blot.MI increased LV end-diastolic pressure (LVEDP), and decreased LV systolic pressure (LVSP). Compared with the MI group, aerobic exercise significantly decreased LVEDP and increased LVSP. The protein expression of TH, GAP43 and NGF was significantly increased after MI, which was normalized by exercise. Compared with the SC group, the ratios of β2-AR/β1-AR and β3-AR/β1-AR were elevated in the MI group, and the protein expression of β3-AR and NOS1 increased after MI. Compared with the MI group, the ratios of β2-AR/β1-AR and β3-AR/β1-AR were normalized in the ME group, while the protein expression of β3-AR and NOS1 significantly increased, and NOS2 was activated by exercise.Aerobic exercise inhibits cardiac sympathetic nerve sprouting, restores β3-AR/β1-AR balance and increases β3-AR expression through the activation of NOS2 and NOS1 after myocardial infarction. Myocardial infarction (MI) induces cardiac sympathetic nerve sprouting in humans In the failing heart, the sustained sympathetic activation results in downregulation of the β1-adrenergic receptor (β1-AR) Exercise is an important clinical intervention for the prevention and treatment of MI Male Sprague-Dawley rats  were provided by the Laboratory Animal Centre of Xi’an Jiaotong University. These studies were performed in accordance with the “Guiding principles for research involving animals and human beings” MI was induced by ligation of the left anterior descending coronary artery under anesthesia  as previously described O2max) Seven days after the surgery, MI rats (n = 24) were randomly divided into two groups: the MI group , and the MI with aerobic exercise group . Rats in the ME group were submitted to 8 weeks of aerobic exercise using a motorized rodent treadmill , while the other group remained sedentary throughout the experiment period. To allow a gradual adaptation to the exercise stress, training was initiated at 10 m/min at a 5°incline for 10 min per day. During the second week, the speed and duration were gradually increased to 16 m/min and 50 min per day (including a 5-min warm-up at 10 m/min), which was maintained constant throughout the experiment. The training intensity was approximately 55% of maximal oxygen uptake (Vmax), and the time constant of LV pressure decay (Tau). All rats were euthanized after hemodynamic measurements.At the end of the 8 weeks of training or sedentary behavior, rats were anesthetized as mentioned above. A pressure transducer was inserted retrograde from the right carotid artery to the LV cavity, and traditional intraventricular catheter recordings  were performed to evaluate cardiac function. The following hemodynamic parameters were measured: LV systolic pressure , LV end-diastolic pressure , maximal positive and negative first derivative of LV pressure  staining. Briefly, the heart was cut into six transverse slices, and incubated for 30 min in a 1% TTC solution to differentiate the infarcted  from viable (brick red) myocardial area. The total area of necrosis was calculated by planimetry using IMAGE-PRO PLUS 6.0  and expressed as percentage of the total LV area.Heart samples taken from the LV infarct border area were fixed in ice-cold 4% paraformaldehyde for 24–48 h, embedded in paraffin and sectioned (5 µm thick) for histopathologic examination. The slices were stained with Masson’s trichrome, and were used to observe the construction of cardiac tissue in the infarct area of the LV. To evaluate the degree of fibrosis, the collagen volume fraction (CVF) was measured in 10 fields for each LV section of Masson’s trichrome staining. CVF  values were calculated using IPP 6.0.2/mm2). The nerve density of each slide was determined by the mean density of nerves calculated from all three selected fields.Briefly, the paraffin sections were incubated with the following diluted primary antibodies overnight at 4°C: TH , GAP43 , NGF . β1-AR , and β2-AR . Following this incubation, sections were exposed to a secondary antibody for 30 min at room temperature. The stains were developed using diaminobenzidine as a chromogen. The sections were then counterstained with hematoxylin and examined by conventional light microscopy . Six sections from each group were scanned, with 10 fields per section viewed, and the value of mean optical density (OD) was calculated by IPP 6.0. The density of stained cardiac sympathetic nerves was determined using IPP 6.0 and expressed as the nerve area divided by the total area examined  overnight at 4°C. As a negative control, PBS was used in place of the primary antibody. Then TRITC-conjugated goat anti-rabbit IgG  was used as the secondary antibody to detect the primary antigen-antibody reaction. The nuclei was stained by 4′-6-diamidino-2-phenylindole (DAPI) dye . Immunofluorescent labeling of the sections were observed with a fluorescence microscope . Quantification of the β3-AR fluorescence density was determined by IPP 6.0.ser1177, 1∶500, Cell Signaling), NOS1 , at 4°C overnight. Following washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies . GAPDH was used as an internal control. Protein bands were subsequently detected with enhancedchemiluminescence and sections were exposed to X-ray film.The tissues from the LV infarct border area (5 mm) was homogenized. Total proteins were extracted with RIPA lysis buffer containing protease inhibitors . Protein samples were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes . The membrane was incubated with the following diluted primary antibodies: TH , GAP43 , NGF , β1-AR , β2-AR , β3-AR , NOS2 , phosphorylation of NOS2 at serine residue 1177  were included. P<0.05 was considered significant.All data in this study are expressed as the mean ± SD. Differences between mean values in the three groups were analyzed by one-way analysis of variance (ANOVA), followed by post-hoc testing using the Student-Newman-Keuls’ test when appropriate. The P<0.01) (The result of TTC staining showed that the infarct area was significantly reduced in the ME group (13.32±2.77%) compared with the MI group , an indicator of interstitial fibrosis, was significantly higher in the MI group  than in the SC group (5.79±0.52%). Aerobic exercise  significantly reduced CVF compared with the MI group  and Tau  (both P<0.01), and a decrease in LVSP  and ±dp/dtmax  (both P<0.01) compared with the SC group. And MI was recognized on an electrocardiogram by ST-segment elevation . However, 8 weeks of aerobic exercise resulted in a significant reduction in LVEDP and Tau (both P<0.01), and an increase in LVSP and LV±dp/dtmax (both P<0.01) compared with the MI group  and GAP43-  positive nerves were observed in the LV after MI (both P<0.01), but the densities of TH- and GAP43-positive nerves were significantly decreased (both P<0.01 vs. MI) by aerobic exercise  in the LV increased after MI (P<0.01). However, following 8 weeks of aerobic exercise, the value significantly decreased compared with the MI group (P<0.05) , GAP43  and NGF  in the LV was significantly increased after MI , which was normalized by aerobic exercise   in the LV was reduced after MI (P<0.05) (P<0.01 vs. SC)  was both unaffected after MI in IHC staining and Western blot analysis  was significantly increased after MI (P<0.01 vs. SC)  expression was up-regulated (P<0.05 vs. SC) following MI (P<0.01). This finding was supported by Western blot analysis of β1-AR, which indicated that the protein expression of β1-AR  was significantly up-regulated after aerobic exercise (P<0.01 vs. MI)  was unaltered after aerobic exercise (P<0.05). Furthermore, β3-AR protein expression was up-regulated (P<0.05 vs. MI) following 8 weeks of aerobic exercise   (both P<0.01 vs. SC) (P<0.05 vs. MI). The negative control images of IHC and IFC staining were provided  (P = 0.01)  in the LV was significantly up-regulated after MI (P<0.01), while the expression of total NOS2  and p-NOS2  remained constant in the MI group. Compared with the MI group, aerobic exercise significantly increased the expression of NOS1 (P<0.01). Furthermore, NOS2 was activated through Ser1177 phosphorylation (P<0.01 vs. MI) by exercise in the failing heart, whereas the expression of total NOS2 was unaltered (P = 0.048), and β3-AR and NOS1 expression  .In this experiment, we measured 2 of the 3 major NOS isoforms (NOS2 and NOS1). As shown in The major findings of the present study are: (i) 8 weeks of aerobic exercise inhibits cardiac sympathetic nerve sprouting and restores β3-AR/β1-AR balance after MI (ii) Aerobic exercise increases the expression of β3-AR through the activation of NOS2 and NOS1 following MI.Growing evidence indicates that exercise, started early after MI, can improve cardiac function The conception of “cardiac nerve sprouting” was well described by Zhou et al. Previous studies have suggested that exercise can increase β1-AR protein (48%) In contrast to β1- and β2-AR, β3-AR modulates a negative inotropic effect through inhibitory G-protein coupled NOS/NO signaling Our study shows that aerobic exercise inhibits cardiac sympathetic nerve sprouting and restores β3-AR/β1-AR balance after MI. However, it remains unclear if this contributes to a benefit for cardiac function. Although an association among these findings has been described, it does not provide a direct evidence of cause-effect.In summary, the current study demonstrated that 8 weeks of aerobic exercise can improve cardiac function after MI and that the underlying mechanisms may be related to the inhibition of sympathetic nerve sprouting, the restoring of β3-AR/β1-AR balance, and the upregulation of β3-AR.Figure S1Effects of aerobic exercise on electrocardiographic recording. MI was recognized on an electrocardiogram by ST-segment elevation. MI resulted in an elevated ST-segment, which was attenuated by aerobic exercise.(TIF)Click here for additional data file.Figure S2The negative staining control image of IFC. No staining was observed in the negative control.(TIF)Click here for additional data file.Figure S3Correlation between cardiac β3-adrenergic receptors and eNOS, nNOS expression. A. Positive correlation between cardiac β3-AR and eNOS expression, B. Positive correlation between cardiac β3-AR and nNOS expression. Coefficients of correlation (R) are indicated.(TIF)Click here for additional data file.