Saccharomyces cerevisiae) (SIRT1) that ultimately may control FoxOs and determine the fate and function of cells in the nervous system that control memory and cognition. Future work that can further elucidate the complex relationship FoxOs hold over cell fate and cognitive function could yield exciting prospects for the treatment of a number of neurodegenerative disorders including AD.Life expectancy of individuals in both developed and undeveloped nations continues to rise at an unprecedented rate. Coupled to this increase in longevity for individuals is the rise in the incidence of chronic neurodegenerative disorders that includes Alzheimer’s disease (AD). Currently, almost ten percent of the population over the age of 65 suffers from AD, a disorder that is presently without definitive therapy to prevent the onset or progression of cognitive loss. Yet, it is estimated that AD will continue to significantly increase throughout the world to impact millions of individuals and foster the escalation of healthcare costs. One potential target for the development of novel strategies against AD and other cognitive disorders involves the mammalian forkhead transcription factors of the O class (FoxOs). FoxOs are present in “cognitive centers” of the brain to include the hippocampus, the amygdala, and the nucleus accumbens and may be required for memory formation and consolidation. FoxOs play a critical role in determining survival of multiple cell types in the nervous system, drive pathways of apoptosis and autophagy, and control stem cell proliferation and differentiation. FoxOs also interface with multiple cellular pathways that include growth factors, Wnt signaling, Wnt1 inducible signaling pathway protein 1 (WISP1), and silent mating type information regulation 2 homolog 1 ( Life expectancy throughout the globe continues to rise in both developed nations as well as in large nations that are under development . For exaWith the advancing age of the global population and the progressive increase observed in life span, it is estimated that the incidence of sporadic AD will significantly increase throughout the world –9. In adFOXA to FOXS are now known to exist since the discovery of the Drosophila melanogaster gene forkhead [More than one hundred forkhead genes and 19 human subgroups that range from forkhead . Mammaliforkhead . In relaforkhead . For humforkhead –34. FoxOforkhead , 36. It forkhead –40. Follforkhead . In addiforkhead .Phosphorylation of forkhead transcription factors can be controlled by the serine-threonine kinase protein kinase B (Akt) , 43–48. Saccharomyces cerevisiae) (SIRT1) [Ubiquitylation and acetylation also control post-translational modification and activity of FoxO proteins , 55. For (SIRT1) , 58. In  (SIRT1) –63.FoxO proteins are acetylated by histone acetyltransferases that include p300, the CREB-binding protein (CBP), and the CBP-associated factor. Once acetylated, FoxO proteins translocate to the cell nucleus but have diminished activity since acetylation of lysine residues on FoxO proteins has been shown to limit the ability of FoxO proteins to bind to DNA . FurtherFoxO proteins may play multiple roles in the nervous system , 69. For+) precursors [FoxO proteins oversee cell survival in the nervous system through the programmed cell death pathways of apoptosis and autophagy , 80. Undecursors , and proecursors , 70, 74 ecursors –86. Otheecursors  with Wntecursors . Wnt1 inecursors , 89, alsecursors . Neuroprecursors , 37, 70,ecursors , suggestecursors , 92.During the induction of autophagy , 94, FoxFoxO proteins can influence neuronal precursor development and the maintenance of neurons , 99. LosIn differentiated cells of the nervous system, FoxO activation may impair cellular survival . Toxin eOn the flip-side, FoxO protein activity is sometimes necessary for neuronal protection. FoxO proteins such as FoxO3 may be important for the control of autophagic flux in Parkinson’s disease . A smallFoxO proteins are deacetylated by histone deacetylases, such as SIRT1 , 129–131Sirtuins and FoxO proteins also may function synergistically to promote neuronal cell survival , 61. In In other studies, FoxO proteins may assist with cell injury during Alzheimer’s disease. Nuclear translocation of FoxO3 is associated with DNA damage  and Aβ tMammalian forkhead transcription factors of the FoxO family offer exciting prospects for the treatment of cognitive neurodegenerative disorders and AD. FoxOs are present in multiple regions of the brain that can influence cognition and memory to include the hippocampus, the amygdala, and the nucleus accumbens. In addition, FoxOs may be necessary for memory formation and consolidation. In regards to cell survival, FoxOs control both apoptotic and autophagic pathways. For the most part, limiting FoxO activity is necessary to block apoptotic cell death. However, the degree that specific FoxO protein activity is reduced appears to be critical, since under some circumstances FoxO activity is necessary to protect against oxidative stress. In addition, fostering the induction of autophagy by FoxOs is required for conditions that involve neurogenesis and memory development. FoxOs are intricately involved with multiple pathways that include growth factors, such as EPO and neurotrophins, Wnt signaling, WISP1, and SIRT1. With each of these pathways, post-translational modification of FoxO proteins and subsequent FoxO cellular activity can influence how FoxOs drive cellular survival and potentially affect cognitive function. Future work that continues to tease apart the complex relationship that FoxOs hold for cognitive function should bear significant fruits for the development of new strategies to treat neurodegenerative disorders such as AD.


The methods used for the motor rehabilitation of patients with neurological disorders include a number of different rehabilitation exercises. For patients who have been diagnosed with multiple sclerosis (MS), the performance of motor rehabilitation exercises is essential. Nevertheless, this rehabilitation may be tedious, negatively influencing patients’ motivation and adherence to treatment.We present RemoviEM, a system based on Kinect that uses virtual reality (VR) and natural user interfaces (NUI) to offer patients with MS an intuitive and motivating way to perform several motor rehabilitation exercises. It offers therapists a new motor rehabilitation tool for the rehabilitation process, providing feedback on the patient’s progress. Moreover, it is a low-cost system, a feature that can facilitate its integration in clinical rehabilitation centers.A randomized and controlled single blinded study was carried out to assess the influence of a Kinect-based virtual rehabilitation system on the balance rehabilitation of patients with MS. This study describes RemoviEM and evaluates its effectiveness compared to standard rehabilitation. To achieve this objective, a clinical trial was carried out. Eleven patients from a MS association participated in the clinical trial. The mean age was 44.82 (SD 10.44) and the mean time from diagnosis (years) was 9.77 (SD 10.40). Clinical effectiveness was evaluated using clinical balance scales.P=.011) and the Anterior Reach Test in standing position (P=.011). Post-hoc analysis showed greater improvement in the experimental group for these variables than in the control group for these variables. The Suitability Evaluation Questionnaire (SEQ) showed good results in usability, acceptance, security, and safety for the evaluated system.Significant group-by-time interaction was detected in the scores of the Berg Balance Scale (The results obtained suggest that RemoviEM represents a motivational and effective alternative to traditional motor rehabilitation for MS patients. These results have encouraged us to improve the system with new exercises, which are currently being developed. Multiple sclerosis (MS) is an inflammatory disease in which the insulating cover of nerve cells in the brain and spinal cord are damaged. At present, there are approximately 3 million people who are affected with MS in the world, with rates varying widely in different regions and among different populations. Normally, the disease begins between the ages of 20 and 50, and it is twice as common in women as in men. There is no known cure for MS, but there are several therapies focused to improve function after an attack, preventing new attacks, and preventing disability. Therapies including medication and neurorehabilitation improve some symptoms, but do not change the course of the disease.The methods used for the motor rehabilitation of patients with neurological problems require the performance of several rehabilitation exercises. These methods have two fundamental problems. First, they propose exercising motor skills by performing the exercises in an insistent and repetitive way. This is not very motivating and decreases the interest of patients to perform them, affecting their adherence to the treatment. Second, these methods require the patients to be in specific centers with the supervision of qualified personnel to ensure correct performance.The use of new technologies, like virtual reality (VR) and natural user interfaces (NUI), in motor rehabilitation of patients with neurological disorders is well documented . The advFurthermore, several studies have demonstrated that by offering virtual rehabilitation exercises as games, greater efficiency is obtained in the rehabilitation process, and the patients are motivated to perform the rehabilitation exercises and their adherence to the treatment is also greater -4. The aSome studies focus on the use of technical solutions for the rehabilitation of patients with MS. For instance, the use of VR and augmented reality (AR) for the rehabilitation of the gait , or the In the case of patients with MS, the performance of motor rehabilitation exercises in the rehabilitation process is even more important, particularly if patients still have good motor skills. MS patients could begin a rehabilitation process by performing virtual rehabilitation exercises using games and interacting with them in an easy and intuitive way, . In this article, we present a Kinect-based virtual rehabilitation system (RemoviEM) that includes all of these features in one motor rehabilitation process. The specific features of the RemoviEM system were previously introduced by the authors in . In thisClinical collaborators determined that a public trial registry was not necessary because the features of the system do not create a potential risk for patients. Instead, before patient enrollment, an authorization was given by the clinical center staff. The patients were then informed about the study and all of the participants provided informed consent.There were 56 patients with MS who were potential candidates for this study. The inclusion criteria were: (1) men and women between 18 and 65 years old, (2) patients have relapsing-remitting and secondary-progressive MS, (3) patients have a minimum score of 6 on all items of the domain of the Functional Independence Measure (FIM), (4) patients do not need assistive devices for ambulation or at most a cane, and (5) patients do not have cognitive impairments. The exclusion criteria were: (1) patients with flare-up symptoms, or (2) patients that cannot physically complete all rehabilitation sessions. With these criteria, a final sample of 12 patients was selected and randomly assigned to either the control group  or the trial group (RemoviEM therapy). All patients were on a similar baseline, with a level and type of impairment constant within groups. The randomization schedule was computer-generated using a basic random number generator. One patient in the control group dropped out of the clinical trial and that data is not included our analysis. Thus, the final sample consisted of 11 patients (7 men and 4 women ranging from 28 to 60 years old). The time since diagnosis for the sample ranged from 2 to 32 years. Age, gender, and chronicity did not affect the performance of each group. The participants had no previous experience with virtual rehabilitation systems. The software and hardware of RemoviEM will be discussed in detail below. In brief, the program consists of several virtual environments that are designed and developed to allow the patient to perform several motor rehabilitation exercises. The well-developed software allows the therapist to select and configure the exercises to be performed by the patient. The therapist can also follow the patient’s progress in the system. The hardware is composed of low-cost devices that allow virtual environments to be displayed. Interaction with these devices is very intuitive. The low cost of the system facilitates its integration in clinical rehabilitation centers.RemoviEM has three motor rehabilitation exercises: TouchBall, TakeBall, and StepBall.The objective is to work on the balance and weight transfer of the patient, and to perform lateral movements of the trunk. Both issues are essential in order to achieve an improvement in gait and, consequently, to walk correctly.In this virtual environment, the patient can interact in a standing or sitting position. Virtual objects appear at different heights on both sides of the patient. The aim of the exercise is to touch virtual objects with your hands, before they disappear, keeping your feet in the predefined zone . The patThe objective is to work the Diagonals of Kabat MMSS. This is very important in neurological rehabilitation because MS patients work on complete movements of the upper limbs, requiring good coordination for their implementation.In this virtual environment, the patient (in standing or sitting position) must move virtual objects from an initial position to a final position using both hands . The patThe objective is to work balance and weight transfer and to perform lateral movements with monopodal load.In this virtual environment, virtual objects appear at ground level on both sides of the patient. The patient must step on the virtual objects before they disappear . To makeRemoviEM uses a LCD/LED TV (42"-47") to show the visual and auditory cues of the virtual environments. In order to interact with the virtual environments, the patient uses Microsoft Kinect. The system runs on a conventional PC.An AR setup is used for the system. The system shows the real image provided by the kinect, where the patient is included. Virtual objects are displayed over this image, allowing the patient to play with the system. The system identifies the different parts of the patient using the depth camera of the kinect. This type of AR allows increases the sense of immersion of the patient to perform exercises.The workflow is the same for all virtual environments. Initially, there is a screen that allows the therapist to setup the rehabilitation exercise taking into account the needs and features of the patient. The patient then watches a sequence of explanatory images about interaction with the virtual environment. The goal of these images is for the patient to learn how to interact with the system to be able to perform the rehabilitation exercise. Next, there is a new screen in which the patient can see how the system detects the skeleton of the patient’s body, and also whether or not the patient is correctly positioned to perform the rehabilitation exercise. When the system detects that the body is properly positioned, a countdown allows the patient to prepare for the start of the exercise. Finally, at the end of the rehabilitation exercise, the system shows the patient and the therapist a final screen with a summary of the results obtained.The clinical trial was carried out in the neurorehabilitation service of the Multiple Sclerosis Association of Castellon . Each patient participated in 10 one-hour sessions of rehabilitation and completed one session per week.In each session, the patients in the control group (n=5) performed standard balance and gait rehabilitation exercises. The patients belonging to the experimental group (n=6) spent forty-five minutes performing the same exercises, and during the last fifteen minutes of the session, they performed the virtual rehabilitation exercises.Before and after the rehabilitation program, all the patients were assessed by a specialist. This clinical assessment used several scales. Balance in static condition was assessed by the Berg Balance Scale (BBS) , the TinP value less than .05 was considered to be significant in each case.Repeated measure analyses of variance (ANOVAs) with time (before and after rehabilitation), the within-subjects factor and treatment option , and the between-subjects factor were performed for each one of the balance measures. The main effects of time, treatment option, and the time-treatment option interaction effects were evaluated. Simple contrasts were conducted for each significant time main effect to determine the source of the significant difference. A P=.240, P=.353 and P=.147 for age, gender, and chronicity, respectively).No significant differences in demographical (age and gender) or clinical (chronicity) variables at inclusion were detected between groups (P=.014), Tinetti (P=.003), SLB test right foot (P=.041), and 10MW (P<.001). Nevertheless, there were no significant time effects for SLB left foot (P=.052) or TUG (P=.346). No group effect was detected for any outcome, except for TUG (P=0.027). Finally, significant group-by-time interaction was detected in the scores for BBS (P=.030) and SLB test right foot (P=.033)  . In addiThe score of the SEQ for Virtual Rehabilitation systems  was 55.5The study presented in this paper assessed the influence of a KINECT-based virtual rehabilitation system (RemoviEM) on the rehabilitation of patients with MS.In relation with the static and dynamic balance abilities of the patients, measured with the Berg Balance Scale, there was a significant improvement over time on this scale. While the control group remained virtually stagnant over time, the experimental group showed a clear improvement. The results provide evidence of significant improvement in the experimental group compared to the control group over time.Regarding the Tinetti Balance Scale, other common clinical test used to assess the static and dynamic balance abilities of the patients, both groups showed a similar trend. There was a significant improvement over time, although there were no significant differences over time between the two groups. The Tinetti Balance Scale measures balance as well as gait, but our system is focused only in balance; this partially explains why there were no significant differences between the groups overtime.P=.033).The Single Leg Balance test shows that the improvement in the experimental group was greater than the control group for both the right and left feet. This was statistically significant in the right foot .Nevertheless, more rehabilitation exercises to train specific movements like Sit-To-Stand, and other generic movements like gait and running should be incorporated in RemoviEM. Thus, it would be possible to study whether these improvements would also be significant on Tinetti and 10MW.RemoviEM has helped patients with MS become more motivated and involved in the rehabilitation process. In addition, the exercises included in the system have allowed patients to obtain significant improvements in the rehabilitation of those movements for which they were designed. We think that the inclusion of new exercises that are designed for the treatment of other movements but that are not currently addressed can also give satisfactory results. Currently, a second stage of the RemoviEM system is being designed to include these new rehabilitation exercises, taking into account the suggestions of clinical specialists and MS patients.


The extent of epithelial damage in allergic (AR) and nonallergic rhinitis (NAR) and its association to inflammatory changes in nasal washes (NW) are not fully understood.Investigate the relationship of inflammatory cells in NW and the level of epithelial damage and basement membrane thickening (BMT) of the upper airway mucosaD.pteronyssinus (35/97%) and L.perenne (18/50%) extracts from Hollister-Stier. Total and differential cell counts were evaluated by a quantitative method of nasal cytology; albumin and IL-8 concentrations were determined in the supernatant of NW. The epithelium damage and BMT were assessed on H&E-stained sections by staging system. Statistical analysis was performed by nonparametric Mann-Whitney U test for comparison between cell counts and differences in frequencies by Fisher exact test.Total nasal symptom score (TNSS), NW and turbinate biopsy specimens were obtained from 36 AR and 20 NAR patients. Atopic patients had positive skin prick test to The median age was 24.5ys. (14-58). TNSS was higher in AR (9 [1-18]) as compared to NAR (6.5 [0-12]) (p=0.01). Total cell and neutrophil counts, as well as albumin and IL-8 levels were not different in NW of AR and NAR patients. Median eosinophil count in nasal fluid (ECNF) was higher in AR (3% [0-66]) than in NAR (1% [0-21]) (p<0.01). ROC curve analysis and AUC for ECNF accuracy in distinguishing AR from NAR, has showed a cut-off value of 4%, AUC=0.71. ECNF was ≥4% in 44% of AR and 20% of NAR patients; at this point the probability of atopy was 80%, with 44% sensitivity and 90% specificity. Epithelial damage was more frequent in AR (94%) than in NAR (65%) . According to the presence of BMT, NW of AR patients without BMT had higher median eosinophil (3% [1-7]) and neutrophils (47.5% [0-87]) counts compared to eosinophils (1% [0-4]) and neutrophils (12% [0-23]) counts in NAR. On the other hand, in the presence of BMT, there were no differences in the NW of AR and NAR patients. The intragroup analysis showed that neutrophil count was higher in NW of NAR patients with BMT (45% [12-83]) than without BMT (12% [0-23]) (p<0.01).The best cut-off value of ECNF to discriminate atopic patients was 4%. Despite of differences in mechanisms of inflammatory reactions in rhinitis, airway remodeling assessed by BMT is associated with similar cellularity in NW of both AR and NAR.


MYC amplification, an event linked to hormone escape. Interesting, we showed that both clones acquired AR gene amplification by deploying different evolutionary paths. Overall, these data demonstrate the timeframe of tumor evolution in response to therapy and provide a framework for the multi-scale analysis of fluid biopsies to quantify and monitor disease evolution in individual patients.Timely characterization of a cancer's evolution is required to predict treatment efficacy and to detect resistance early. High content analysis of single Circulating Tumor Cells (CTCs) enables sequential characterization of genotypic, morphometric and protein expression alterations in real time over the course of cancer treatment. This concept was investigated in a patient with castrate-resistant prostate cancer progressing through both chemotherapy and targeted therapy. In this case study, we integrate across four timepoints 41 genome-wide copy number variation (CNV) profiles plus morphometric parameters and androgen receptor (AR) protein levels. Remarkably, little change was observed in response to standard chemotherapy, evidenced by the fact that a unique clone (A), exhibiting highly rearranged CNV profiles and AR+ phenotype was found circulating before and after treatment. However, clinical response and subsequent progression after targeted therapy was associated with the drastic depletion of clone A, followed by the sequential emergence of two distinct CTC sub-populations that differed in both AR genotype and expression phenotype. While AR- cells with flat or pseudo-diploid CNV profiles (clone B) were identified at the time of response, a new tumor lineage of AR+ cells (clone C) with CNV altered profiles was detected during relapse. We showed that clone C, despite phylogenetically related to clone A, possessed a unique set of somatic CNV alterations, including  The androgen-androgen receptor (AR) signaling pathway is essential for the development and progression of prostate cancer and is a key target of many therapeutic agents Circulating tumor cells (CTCs) have the potential to provide a non-invasive means of assessing progressive cancers in real time during therapy, and further, to help direct therapy by monitoring phenotypic physiological and genetic changes that occur in response to therapy. In most CRPC patients, the primary tumor has been removed, and CTCs are expected to consist of cells shed from metastases, providing a ‘fluid biopsy’. Currently, the only method approved for CTC enumeration  is based on an immune enrichment approach that pre-selects for cells that express Epithelial Cell Adhesion Molecule (EpCAM), an epithelial cell surface marker et al.et al.Here, we demonstrate the power of two newly developed technologies in order to provide a more comprehensive portrait of the molecular changes occurring, at the single cell level, in a CRPC patient under the treatment pressure in both ADT and chemotherapy settings. The High Definition-CTC (HD-CTC) method was used for the longitudinal identification and enumeration of CTCs The study was approved by the institutional review board (IRB) of University of Southern California Comprehensive Cancer Center. The patient provided written informed consent.153Sm) lexidronam  were employed with limited palliative benefit. At 12 months after diagnosis, treatment with abiraterone acetate, a highly-selective androgen synthesis inhibitor, was initiated. Blood was drawn prior to starting abiraterone (Draw 2), at 3 weeks of continuous treatment coinciding with a clinical response represented by decreased pain and PSA level (Draw 3), and at 9 weeks coinciding with clinical progression represented by increasing pain and PSA levels (Draw 4). Following abiraterone, treatment was changed to cabazitaxel without clinical response followed by a rapid clinical deterioration. The patient died of widely metastatic prostate cancer 4 months following Draw 4 (17 months after diagnosis).The patient presented with PCa metastatic to a lumbar vertebrae at diagnosis for which the primary biopsy represents the first specimen in this study. Initial treatment consisted of androgen deprivation therapy (leuprolide acetate). After 5 months, there was clinical progression to CRPC and the patient was enrolled in a clinical trial of docetaxel combined with bevacizumab and everolimus . Before chemotherapy was initiated, a baseline blood draw was taken (Draw 1) according to the sample collection protocol. Clinical progression was noted after 4 months of protocol-specified chemotherapy. Over the next 3 months, additional doses of docetaxel as well as external-beam radiotherapy and samarium  status within the cytokeratin (CK) positive CTC population +) or absence (AR−) of AR staining, and (2) AR subcellular localization (nuclear AR versus cytoplasmic staining or both). The threshold for AR positivity was defined as a signal more than 6 standard deviations over the mean signal intensity (SDOM) observed in the surroundings leukocytes (background). Subcellular localization was measured using the relative pixel density of AR staining over the nucleus and cytoplasm.The slides were imaged and putative CTCs were recorded using a computerized high-throughput fluorescence microscope at 10× magnification. CTCs were identified by a hematology technician using the previously published criteria of having a DAPI+ nucleus plus cytokeratin positivity and CD45 negativity 2 = 0.9997 on linearity testing as previously reported. These experiments were performed using SK-BR-3 cell lines and 0 to 3×102 cells per mL of normal donor control blood. The coefficient of variation is 16% and inter-processor correlation is R2 = 0.979. Sample preparation process adhered to standard operating procedures for patient samples through a bar coded system for all consumables and instrumentation. All off-the-shelf instrumentation was calibrated according to the technical validation protocols established during the commissioning The HD-CTC assay was technically validated with cell line spiking experiments to reach an RAs a standard procedure, aimed at minimizing DNA fragmentation cells were picked within 5 days of the initial staining procedure. The experimental protocol for HD-CTC fluid phase capture was divided into three discrete sequential steps: (1) CTC relocation, (2) cell extraction and (3) isolation and manipulation of single CTCs for downstream molecular analyses.HD-CTCs were relocated (step 1) using a transformation matrix from the initial data acquisition for HD-CTC identification. After calibration and relocation, each candidate cell was re-imaged at 40× resolution for the detailed morphometric analysis. For the cell extraction (step 2) an Eppendorf Transfer Man NK2 micromanipulator was used to capture the cell of interest inside a 25° jagged micropipette  by applying fluid suction. Once the cell of interest was captured inside the micropipette (step 3), the cell was rinsed with PBS and deposited inside a 0.2 mL PCR tube containing 2 µL of lysis buffer . The sample was then and immediately frozen and stored at −80°C until further processing. All instruments and consumables were decontaminated using a DNAase solution and exposure to UV light for 30 min prior to the experiment.The cell containing vials were transferred in dry ice to the sequencing laboratory. Briefly, the lysed cell mixture was thawed and subjected to WGA and sequencing library construction as previously reported In addition, detailed methods used to analyze sequencing data were published recently by our group The hierarchical clustering was performed in R Using median centered CNV profiles, cutoff ratios versus the median of 0.8 and 1.25 were used to define deletions and amplifications, respectively. These cutoffs were used both to color the heatmap and to do the frequency analysis.cell roundness) was analyzed by tracing the cell cytoplasm contour in the composite image of each CTC. The traced cell image was imported into R, and an ellipsis was fitted to the shape using a least squares fitting algorithm described by Halir and Flusser c) is estimated as the fraction of the de facto cell area (A) and the area of a circle with the radius (r) set to the cell's major axis.The cell shape  AR protein expression phenotype, (2) AR subcellular localization and (3) CNV genomic profiling were performed in the CTCs identified in the blood samples collected across four different intervals representing decision points in the standard care of CRPC including: (Draw 1) immediately prior to initiation of docetaxel based chemotherapy, (Draw 2) immediately prior to abiraterone acetate (a highly-selective androgen synthesis inhibitor), (Draw 3) after three weeks, and (Draw 4) after nine weeks of continuous abiraterone treatment. The specific data for all profiled cells is presented in the supporting information. In addition, a similar sequencing based method was used to obtain the CNV profile of one metastatic site from the patient using a bone biopsy taken at the time of diagnosis (5 months prior to draw 1) prior to receiving any cancer-specific therapy. As shown in declined .+ cells from Draws 1 and 2 were of two types (+AR−) while the majority (16/19) showed high levels of AR protein (CK+AR+). One AR− and one AR+ cell had near normal CNV profiles comparable to those obtained from single CK−CD45+ leukocytes (+AR+ cells exhibited a complex pattern of genomic rearrangements that were similar to the genomic profile obtained retrospectively from the patient's bone metastasis (hormone naïve tissue sample) obtained at diagnosis  profiles  of phenotypically ARprofiles  placing  lineage .+ with a cell roundness value of 0.81 typical of cells from the first two draws  cells formed the majority of a new, apparently clonal, subpopulation  . This fiAlthough our study is based on longitudinal study of a single patient, our findings are consistent with previous studies involving genomic analysis from either CTCs or circulating cell-free DNA isolated from patients with metastatic prostate cancer Clonal evolution of cancer is a well-established principle that has been validated in multiple published studies The bulk metastatic biopsy taken prior to initiation of therapy provides the root CNV profile to from which the subsequent time course CTC profiles have evolved. It exhibits a backbone of CNV elements that defines a lineage, based on CNV breakpoints, that is carried forward in the circulating cells from blood draws taken during later treatment. The first two of these draws were taken after an initial course of androgen deprivation therapy (ADT) (leuprolide acetate). One population in Draws 1 and 2 (Clone A) was a clearly a direct descendant of the met biopsy profile with the exception that all cells showed high-copy AR amplification and strong AR protein expression. We interpret these cells to be the products of metastatic deposits that had evolved to amplify the AR gene locus and overexpress androgen receptor protein as a result of genetic selection for resistance to the initial round of ADT. It is noteworthy that in addition to the AR amplified cells, both draws contained a significant fraction of cytokeratin positive, AR negative cells with near-normal (pseudodiploid) genomes forming a separate CNV cluster . It is aIn contrast to the similarity of cell phenotype and genotype in Draws 1 and 2, the selective effects of abiraterone acetate were very evident in Draws 3 and 4. After three weeks of treatment the androgen dependent, AR positive cells in Draws 1 and 2 were nearly absent and the CK+ population consisted almost entirely of AR negative pseudodiploid cells. The clone (Clone C) that would become dominant at the nine-week timepoint (Draw 4) was first seen as a single incidence in Draw 3. By Draw 4, AR+ cells had once again become a substantial fraction of the population, albeit with a significantly altered CNV profile . We thus+ AR− cells Through this selective process, the population of AR negative, pseudodiploid cells remained a significant fraction of cytokeratin positive cells. The presence of these phenotypically  and genoIn an era of clinical oncology that is progressively moving towards targeted cancer therapy, approaches that allow for non-invasive monitoring of therapeutic response at both phenotypic and genetic levels are essential. We have chosen to approach this goal through a combined phenotypic and genetic analysis of non-leukocyte circulating nucleated cells, without a pre-selection step that may bias the CTC population. Our method allows us to correlate genomic events with complex phenotypes based on protein expression and cell morphology. Alternative methods, such as sequencing of free DNA from plasma (ctDNA) are also powerful tools and can yield both mutation and copy number information, but only for an admixture of the various cellular components Figure S1Representative gallery of 40× high resolution immunofluorescence images of the two phenotypically distinct CTCs subpopulations identified. A and B, Composite and non-merged images of an AR+ and AR− HD-CTC isolated from pre docetaxel (A) and pre abiraterone (B) treatment timepoints. C and D, Two different AR− and AR+ HD-CTCs, the predominant tumor cell phenotypes found in 3 (C) and 9 weeks post abiraterone (D). Panel D, CTCs with different pattern of AR subcellular localization. Nuclear and cytoplasmic AR is shown in the top panel and nuclear AR in the bottom panel. Composite and non-merged images for the individual immunofluorescence channels were colored as followed: DAPI (blue); cytokeratin-CK (red), androgen receptor-AR (white) and CD45 (green).(DOCX)Click here for additional data file.Figure S2Complete collection of single CTC CNV profiles. The genome wide copy number fingerprints for all successfully profiled cells at each of different treatment timepoint.(DOCX)Click here for additional data file.Figure S3Examples of cell roundness estimation. The cell shape was analyzed by tracing the cell cytoplasm contour in the composite image of each CTC. The traced cell image was imported into R, and an ellipsis was fitted to the shape using a least squares fitting algorithm described by Halir and Flusser. Black line represents the manually drawn cell outline, red line the fitted ellipse. The cell roundness is estimated as the fraction of the de facto cell area and the area of a circle with the radius set to the cell's major axis. The cell roundness calculated to be 0.62 for the oval-shaped cell (left) and 0.96 for the more rounded cell (right). The p-value used in the comparison of the roundness between the CTCs isolated between the different draws was calculated using the Wilcoxon rank-sum test.(DOCX)Click here for additional data file.Table S1Summary of the different phenotypic and genotypic traits analyzed in the 41 individual cells profiled for copy number alterations. Concordance between AR phenotype-genotype was determined by comparison of the AR amplification status with the AR staining phenotype (Negative or Positive) for each individual cell. In red are cells that exhibited discordant AR phenotype-genotype.(DOCX)Click here for additional data file.


There is currently no gold standard rehabilitation outcome measure for Critical Care in the UK. Given the complex nature of these patients, it has been suggested that a 'battery' of outcome measures maybe more appropriate for this setting . Prior To assess the feasibility and usefulness of implementing a battery of rehabilitation outcome measures on an Adult Liver Intensive Care Unit (LITU).A prospective service improvement project. Adult patients with a length of stay greater than 5 days on LITU were included. Three outcome measures were selected for use on the LITU; Rehabilitation Complexity Scale (RCS), ICU Mobility Scale (ICUMS) and The Chelsea Critical Care Physical Assessment tool (CPAx). These outcomes were measured on day 5 of admission, start of rehabilitation and on discharge. Start of rehabilitation versus discharge scores for each outcome measure were evaluated using the paired sample t test and Pearson's correlation analysis.Between August 2014 and January 2015, 105 patients were admitted to LITU with a length of stay greater than 5 days and included in this project. The RCS was measured in all patients on day 5 of admission. From the start of rehabilitation to discharge from LITU repeated measures were obtained for RCS = 85 /87 (97%), ICU Mobility Scale = 65/78 (83%) and CPAx = 65/ 78 (83%) of eligible patients.Reasons for missing data include; RCS , ICUMS and CPAx .All measures demonstrated significant improvements with mean SD differences of; RCS = 2.37 (2.78), ICU Mobility Scale = 1.58 (2.13) and CPAx 6.6 (9.26) p= < 0.001. RCS and CPAx at start of rehab were correlated with length of stay .All measures were feasible to use with the majority of appropriate patients being scored throughout the critical care rehabilitation pathway. All measures demonstrated significant improvements from start of rehabilitation to discharge from LITU showing they are useful measures to use within this setting.


We use the self-tuning Experience Weighted Attraction model with repeated-game strategies as a computer testbed to examine the relative frequency, speed of convergence and progression of a set of repeated-game strategies in four symmetric 2 × 2 games: Prisoner's Dilemma, Battle of the Sexes, Stag-Hunt, and Chicken. In the Prisoner's Dilemma game, we find that the strategy with the most occurrences is the “Grim-Trigger.” In the Battle of the Sexes game, a cooperative pair that alternates between the two pure-strategy Nash equilibria emerges as the one with the most occurrences. In the Stag-Hunt and Chicken games, the “Win-Stay, Lose-Shift” and “Grim-Trigger” strategies are the ones with the most occurrences. Overall, the pairs that converged quickly ended up at the cooperative outcomes, whereas the ones that were extremely slow to reach convergence ended up at non-cooperative outcomes. Robert Axelrod pioneered the area of computational simulations with the tournaments in which game-playing algorithms were submitted to determine the best strategy in the repeated Prisoner's Dilemma game Axelrod, . AxelrodIn Ioannou and Romero , we propstrategies, unless there is a risk of confusion). First, identifying empirically relevant strategies can help future theoretical work to identify refinements or conditions that lead to these strategies. The literature on repeated games has made little progress toward this target thus far. Second, pursuing an understanding of the strategies that emerge may also help identify in which environments cooperation is more likely to be sustained. Third, identifying the set of strategies used to support cooperation can provide a tighter test of the theory. For instance, we could test whether the strategies that emerge coincide with the ones that the theory predicts.In this study, our goal is to use the self-tuning EWA model with repeated game strategies as a computer testbed to examine the relative frequency, speed of convergence and progression of a set of repeated-game strategies in the four aforementioned symmetric 2 × 2 games. Learning with repeated-game strategies is important on many levels , whereas the ones that were slow to reach convergence ended up at non-cooperative outcomes.I = {1, …, n}. Each player i ∈ I has an action set denoted by i. An action profile a =  consists of the action of player i and the actions of the other players, denoted by ai− =  ∈ i−. In addition, each player i has a real-valued, stage-game, payoff function gi:  → ℝ, which maps every action profile a ∈  into a payoff for i, where  denotes the cartesian product of the action spaces i, written as  In the infinitely-repeated game with perfect monitoring, the stage game in each time period t = 0, 1, … is played with the action profile chosen in period t publicly observed at the end of that period. The history of play at time t is denoted by th =  ∈ t, where ra =  denotes the actions taken in period r. The set of histories is given byTo simplify exposition, we start with some notation. The stage game is represented in standard strategic  form. The set of players is denoted by 0 = {∅}. A istrategy s ∈ iS for player i is, then, a function  where the strategy space of i consists of iK discrete strategies; that is, iS = {s1i, s2i, …, iKis}. Furthermore, denote a strategy combination of the n players except i by si− = . The set of joint-strategy profiles is denoted by S = S1 × … × nS. Each player i has a payoff function πti: S → ℝ, which represents the average payoff per period when the joint-strategy profile is played for t periods.where we define the initial history to the null set iN(χ), which is interpreted as the number of observation-equivalents of past experience in block χ of player ijiA(χ), indicates player i's attraction to strategy j after the χth block of periods. The variables iN(χ) and jiA(χ) begin with some prior values, iN(0) and jiA(0). These prior values can be thought of as reflecting pre-game experience, either due to learning transferred from different games or due to pre-play analysis. In addition, we use an indicator function 𝕀 that equals 1 if x = y and 0 otherwise. The evolution of learning over the χth block with χ ≥ 1 is governed by the following rules:Players have attractions, or propensities, associated with each of their strategies, and these attractions determine the probabilities with which strategies are chosen when players experiment. Initially, all strategies have an equal attraction and hence an equal probability of being chosen. The learning process evolves through the strategies' attractions that are periodically updated. Similar to its predecessors, the self-tuning EWA model consists of two variables that are updated once an agent switches strategies. The first variable is andiR(χ) is the reinforcement payoff and (χ) is the expected forgone payoff to player i for strategy j.where iR(χ), is defined as the average payoff obtained by player i over the χth block,The reinforcement payoff, h(χ) is the sequence of action profiles played in the χth block, and iT(χ)is the χth block's length for player i. In addition, the forgone payoffs in the self-tuning EWA model with repeated-game strategies are not as simple as in the case of the self-tuning EWA model with actions, where the opponent's action is publicly observed in each period. To calculate the forgone payoff (χ) players need to form beliefs about the current repeated-game strategy of their opponent. In particular, the expected forgone payoff for player i of repeated-game strategy j over the χth block is the payoff player i would have earned had he chosen some other repeated-game strategy j given his beliefs about player −i's current repeated-game strategy.where h =  for t1 ≤ t2 be the truncated history between periods t1 and t2 . Also, let h  = ∅ be the empty history. Let  be the total number of periods at the end of block χ. Then, repeated-game strategy si− is consistent with  for the last t′ periods ifWe indicate next how beliefs are specified. To determine the beliefs, let  asDefine the fitness function       for the last t′ periods}. asDefine the belief function i's belief that the other player was using repeated-game strategy si− at the end of block χ. Therefore, the expected foregone payoff for player i of strategy j over the χth block is given bywhich can be interpreted as player si|h− is the continuation strategy induced by history h andwhere −is is consistent with is the longest history such that In the original EWA model of Camerer and Ho , the atti to reinforce only unchosen strategies with weakly better payoffs. On the other hand, the decay rate function ϕ (·) weighs lagged attractions. When a player senses that the other player is changing behavior, a self-tuning ϕi(·) decreases so as to allocate less weight to the distant past. The core of the ϕi(·) is a “surprise index,” which indicates the difference between the other player's most recent strategy and the strategies he chose in the previous blocks. First, define the averaged belief function σ : Si− × ℕ → ,Thus, the attention function enables player si−. The surprise index  simply sums up the squared deviations between each averaged belief σ and the immediate belief  that is,which averages the beliefs, over the χ blocks, that the other player chose strategy Thus, the surprise index captures the degree of change of the most recent beliefs from the historical average of beliefs. Note that it varies from zero (when there is belief persistence) to two (when a player is certain that the opponent just switched to a new strategy after playing a specific strategy from the beginning). The change-detecting decay rate of the χth block is theni's beliefs are not changing, ϕi(χ) = 1; that is, the player weighs previous attractions fully. Alternatively, when player i's beliefs are changing, then ϕi(χ) = 0; that is, the player puts no weight on previous attractions.Therefore, when player j. Thus, the probability of a player i choosing strategy j when he updates his strategy at the beginning of block χ + 1 isAttractions determine probabilities of choosing strategies. We use the logit specification to calculate the choice probability of strategy The parameter λ ≥ 0 measures the sensitivity of players to attractions. Thus, if λ = 0, all strategies are equally likely to be chosen regardless of their attractions. As λ increases, strategies with higher attractions become disproportionately more likely to be chosen. In the limiting case where λ → ∞, the strategy with the highest attraction is chosen with probability 1.i updates his strategy set in period t,  is determined endogenously via the expected length of the block term, , which is updated recursively; that is,The probability that player t(χ) is the first period of block χ, and χ(t) is the block corresponding to period t. In addition, g = maxa1, a2, jgj is the highest stage-game payoff attainable by either player, and g = mina1, a2, jjg is the lowest stage-game payoff attainable to either player. The normalization by  begins with an initial value . This prior value can be thought of as reflecting pre-game experience, either, due to learning transferred from other games, or due to (publicly) available information. The law of motion of the expected block length depends on the absolute difference between the actual average payoff thus far in the block and the expected payoff of strategy is. The expected payoff for player i,  is the average payoff that player i expects (anticipates) to receive during block χ(t) and is calculated at the beginning of the block. The difference between actual and expected payoff is thus a proxy for (outcome-based) surprise. As Erev and Haruvy  = 1.5 for each strategy j in iSiN (0) = 1. Players are matched in fixed pairs and update their attractions at the end of each block. The play ends when the average payoff of a given pair converges. More specifically, each simulation is broken up into epochs of 100 periods. The simulation runs until the average epoch payoff of the pair has not changed by more than 0.01 from the previous epoch (in terms of Euclidean distance) in 20 consecutive epochs is set to 100 periodsIn the simulations, players engage in a lengthy process of learning among strategies. At the beginning of the simulations, each agent is endowed with initial attractions B. Figure A, A) is sustained in a pair consisting of Grim-Trigger automata. This finding is confirmed in Figure averaged over the last 1000 periods. The blue solid line represents the average payoff of the automaton pair g1 − g2|). Points on the blue solid and the green dashed line are sorted according to the corresponding point on the red dotted line. About 20% of the simulations converged quite quickly in less than 3000 periods. At this point in time, the blue solid line signifies that the average payoff of the automaton pairs was 3. Given the convergence criterion, we can deduce that about 20% of the automaton pairs started off by cooperating and maintained cooperation until convergence. The next 70% of the simulations were (roughly) uniformly distributed across the range of 3000−27,000 periods. The last 10% of the simulations converged in the range of 27,000−34,000 periods. Looking at the green and blue lines, we observe that the pairs that were converging in less than 27,000 periods ended up at the cooperative outcome, while the pairs that converged at 31,000 periods and beyond converged to the defecting outcome. After 31,000 periods, the automaton pairs that did not attain cooperation experienced short expected block lengths, which prompted them to constantly update the strategies in a manner similar to action-learning models hence converged to the defecting outcome. Pairs that converged between 27,000 and 31,000 periods ended up in either the cooperating or the defecting outcome.The payoff matrix of the Prisoner's Dilemma game is indicated in Figure A, B) and . Figure B in the previous period. Automaton 18 switches actions every period unless both players choose A in the previous period. Therefore, a pair consisting of Automaton 12 and Automaton 18 would end up alternating between the two pure-strategy Nash equilibria of the stage game. Each automaton would thus earn an average payoff of 3. This is shown in Figure The payoff matrix of the Battle of the Sexes game is indicated in Figure A, A) and . However, outcome  is the Pareto dominant equilibrium. Figure A, A) repeatedly. Automaton 26 paired with Automaton 26 corresponds to alternating between the two pure-strategy Nash equilibria, which yields an average payoff of 2. Figure A repeatedly. Note that there is also a small number of pairs that converged to . Figure The payoff matrix of the Stag-Hunt game is indicated in Figure A, B) and . Recall that in the Chicken game, the mutual conciliation outcome of  yields higher payoffs than the average payoffs for each of the players when alternating between the pure-strategy Nash equilibria. Figure The payoff matrix of the Chicken game is indicated in Figure anticipation of punishments and triggers. Such sophisticated behavior is instrumental in capturing cooperative behavior in the Prisoner's Dilemma game and mutual conciliation in the Chicken game, precisely, because the threat of punishment may drive a selfish player to conform to cooperation and conciliation in the two games. An alternative approach could be to assume a mixture of adaptive and sophisticated players. An adaptive player responds to either the payoffs earned or the history of play, but does not anticipate how others are learning, whereas a sophisticated player responds to his forecasts using a more sophisticated forward-looking expected payoff function and a mental model of an opponent's behavior  around 70% of the time before convergence; eventually, the pairs learn to play the cooperative outcome .Figures B. Eventually pairs learn to play automata that alternate between the two pure-strategy Nash equilibria. Figure B, B) and half of the time pairs are playing one of the two pure-strategy Nash equilibria. Pairs rarely ever play the  outcomeFigure Figure B. It continues to do so as long as the co-player plays A; otherwise, it switches to A. A pair of such automata are quite infrequent, whereas the relative frequency of the other three action profiles is about the same. This is what is observed in Figure Figure Recently, Rabin  proposedRecently, Dal Bó and Fréchette  requiredThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Forced expression of let-7e suppressed β1-AR expression at the protein level, without affecting β1-AR mRNA level, in neonatal rat ventricular cells (NRVCs). Silencing of let-7e by let-7e antisense inhibitor (AMO-let-7e) enhanced β1-AR expression at the protein level in NRVCs. Administration of the lentivirus vector containing precursor let-7e (len-pre-let-7e) significantly inhibited β1-AR expression in rats, whereas len-AMO-let-7e up-regulated β1-AR relative to the baseline control level, presumably as a result of depression of tonic inhibition of β1-AR by endogenous let-7e. Len-negative control (len-NC) did not produce significant influence on β1-AR expression. Len-pre-let-7e also profoundly reduced the up-regulation of β1-AR induced by AMI and this effect was abolished by len-AMO-let-7e. Importantly, len-pre-let-7e application significantly reduced arrhythmia incidence after AMI in rats and its anti-arrhythmic effect was cancelled by len-AMO-let-7e. Notably, anti-arrhythmic efficacy of len-pre-let-7e was similar to propranolol, a non-selective β-AR blocker and metoprolol, a selective β1-AR blocker. Down-regulation of let-7e contributes to the adverse increase in β1-AR expression in AMI and let-7e supplement may be a new therapeutic approach for preventing adverse β1-AR up-regulation and treating AMI-induced arrhythmia.Beta-adrenoceptor (β-AR) exerts critical regulation of cardiac function. MicroRNAs (miRNAs) are potentially involved in a variety of biological and pathological processes. This study aimed to investigate the role of miRNA let-7e in the up-regulation of β Acute myocardial infarction (AMI) or heart attack as a result of blockage of a coronary artery in the clinical setting is a common cause of mortality and morbidity worldwide. It occurs after a prolonged period of myocardial ischemia, which can result in cardiac electrophysiological disturbance, haemodynamic disorder, metabolic disorders, necrosis and apoptosis of cardiomyocytes ,2, accom1-AR, β2-AR and β3-AR. β1-AR and β2-AR are the major subtypes that modulate cardiac contractility and HR by stimulating the G-protein/adenylate cyclase/protein kinase A pathway [3-AR produces negative inotropic effects by coupling to inhibitory G proteins [1-AR and β2-AR subtypes are expressed at a ratio of 70:30 [β-adrenoceptor plays a pivotal role in regulating cardiac function and heart rate (HR). And β-AR blocker is commonly used to treat patients with arrhythmia and heart failure ,9. To da pathway ,11; howeproteins . In physof 70:30 ,14. Howeof 70:30 ; in contof 70:30 .et al. reported that subtype-selective increase in β1-AR induced by AMI is associated with a transcriptional up-regulation, and occupation or activation of β1-AR is not involved in this subtype-specific up-regulation of β1-AR [et al. reported that a protein synthesis inhibitor cycloheximide regulates β1-AR gene expression by acting on repressive elements in the promoter region of the gene in brown adipocytes [Dynamic expression regulation of β-AR and functional desensitization are considered adaptive or protective mechanisms in the heart. Regulation of β-AR expression involves a variety of factors and pathways in cardiac cells. Ihl-Vahl of β1-AR . Bengtssipocytes . Calciumipocytes .etc. [MicroRNAs (miRNAs) are a family of single-stranded non-coding RNAs that post-transcriptionally regulate the translation of protein-coding genes by targeting to the 3′ untranslated regions (3′UTRs) of mRNAs. Recently, accumulating evidence has demonstrated that miRNAs play a critical role in pathophysiology of cardiovascular diseases, such as hypertrophy, heart failure and arrhythmias, etc. . Inhibitetc. . Overexpetc.  or cardietc.  respectietc. , atrio vetc.  and afteetc. ; while metc. . Studiesetc. , miR-26 etc. , miR-328etc.  particip1-AR in AMI in rats, which provides new insight into the mechanisms for regulation of β1-AR expression and overexpression of miRNA let-7e potentially inhibited AMI-induced arrhythmia in rat. This indicates that targeting miRNA let-7e may be a promising therapeutic strategy for modulating β1-AR.In this study, we, for the first time, displayed the involvement of let-7, a conserved and abundant miRNA in the heart , in the ad libitum for 1 week before experimental interventions. All experimental procedures were in accordance with, and approved by the Institutional Animal Care and Use Committee of the Harbin Medical University.Healthy male Wistar rats  used in this study were kept under standard animal room conditions  with food and water Rats were anaesthetized with ketamine (60 mg/kg) and xylazine (6 mg/kg). Tracheal cannula was performed with a polyethylene tube and ventilated with the TOPO small animal Ventilato , and then the chest was opened through the fourth intercostal space and propped ribs by a rib spreader. The pericardium was opened carefully to expose the heart. The left anterior descending coronary artery (LAD) was ligated using a 5/0 silk thread to create infarction of the LV free wall. Cardiac infarction was confirmed by apparent S-T segment elevation in ECG and cyanosis of the myocardium. The sham procedure consisted of a superficial suture in the epicardium of the LV.We performed miRNA expression profiling with the heart samples from with or without AMI (6 hrs) rats. RNA samples 5 μg were labelled with the Exiqon miRCURY Hy3/Hy5 power labelling kit and hybridized on the miRCURY LNA Array (version 11.0) station. Scanning was performed with the Axon GenePix 4000B microarray scanner. GenePix pro version 6.0 was used to read image and analyse raw intensity. The threshold value for significance used to define up-regulation or down-regulation of miRNAs was a fold change >1.5 or <0.5.1-AR , or to β2-AR , or to GAPDH , or to β-actin  in 5% non-fat milk, and incubated at 4°C overnight. The membranes were washed with PBS-T and PBS, and then incubated with secondary antibody  for 1 hr at room temperature. Finally, western blot bands were collected by Imaging System (LI-COR Biosciences) and quantified with odyssey v1.2 software by measuring intensity in each group with GAPDH as an internal control, but for the ischaemia tissues using β-actin as an internal control [Total protein was extracted with RIPA Lysis Buffer  mixed with 1% proteinase inhibitors, and degenerated by admixing with 5× loading buffer (Beyotime) at 100°C for 5 min. Extracted protein samples (120 μg from NRVCs and 60 μg from tissues) were separated in 10% SDS-PAGE and blotted to nitrocellulose membrane. The blots were blocked with 5% non-fat milk overnight at 4°C, probed with a primary antibody to β control . The res1-AR mRNA were determined using SYBR Green I incorporation methods on ABI 7500 fast Real Time PCR system (Applied Biosystems), with U6 as an internal control of miRNA or GAPDH as an internal control of β1-AR mRNA. The sequences of primers used in the qRT-PCR experiments  were listed in After experimental treatment, total RNA samples were isolated from cultured NRVCs and cardiac tissues using Trizol reagent  according to manufacturer's protocol. RNA (0.5 μg) was then reverse transcribed using High-Capacity cDNA Reverse Transcription Kit  to obtain first-strand cDNA. Levels of let-7a/c/d/e/i, miR-1 and β1-AR, which is highly conserved among mammals. A segment containing the let-7 miRNA binding sites flanked by the Hand lll and Sac I restriction sites and a scramble sequence as a negative control (NC) were synthesized by Invitrogen. The sequences were inserted separately into the pMD18T-simple vector (Invitrogen), and then transferred into the pMIR-REPORT™ Luciferase miRNA Expression Reporter Vector . pRL Renilla Luciferase Reporter vector  was used as an internal control. The plasmids and miRNAs were transfected grouply with X-tremeGENE siRNA transfection reagent  into HEK293 cells. The dual luciferase reporter assay kit (Promega) and the GlouMax biological fluorescent tester were used to detect the activity of the luciferase 36 hrs after transfection. The results were expressed as fold changes by normalizing the scaled data. The sequence of human and rat ADRB1 3′UTR inserted into the vectors were as follows: The human β1-AR 3′UTR : 5′-CGAGCTCTTAAGCTCTTCTTGGAACAAGCCCCACCTTGCTTTCCTTGTGTAGGGCAAACCCGCTGTCCCCCGCGCGCCTGGGTGGTCAGGCTGAGGGATTTCTACCTCACCTGTGCATTTGCACAGCAGATAGAAAGACTTGTTTATATTAAGCTTGGG-3′. The rat β1-AR 3′UTR : 5′-CGAGCTCCTCGGTGGTCCTGCTGTGGGTCCTCTACCTCACTCTGTGCATATTGCACAGCAAGATAGAAAGACTTGTTTATATTAAACAGCTTATTTATGTATCAATATTAGTTGGAAGGACCAGGCGCTGAAGCTTGGG-3′.Targetscan predicts the presence of a putative binding site for let-7 in the 3′UTR of ADRB1 mRNA, the gene encoding β6 per well, and incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air.The neonatal Wistar rats of 1–3 days old were disinfected by 75% ethanol. The chest was opened to expose the heart. The heart was isolated and sheared into 1- to 3-mm pieces. The cardiac tissues were digested into single cardiomyocytes by 0.25% trypsin. Dispersed cells were placed in high glucose DMEM containing 10% fetal bovine serum (FBS) and 1% benzylpenicillin-streptomycin to terminate the digestion procedure. Pooled cell suspensions were centrifuged and resuspended in DMEM supplemented with 10% fetal bovine serum, and 100 U/ml penicillin and 100 μg/ml streptomycin. The resuspension was transferred onto a 100-mm culture dish for 30 min., then changed to a new dish, and repeated for three times. This procedure allowed for preferential attachment of fibroblasts to the bottom of the culture dish and left non-adherent cardiomyocytes in cell suspension. The cells were plated into a 6-well plate at a density of 1 × 10Rno-let-7c/d/e and their antisense oligonucleotides (AMO-let-7c/d/e) were synthesized by RiboBio . In addition, a scrambled RNA was used as a NC (sequence: 5′-UUCUCCGAACGUGUCACGU-3′) that designed according to the Blast search of the human/rat/mouse genomic by RiboBio. let-7c: 5′-UGAGGUAGUAGGUUGUAUGGUU-3′; AMO-let-7c: 5′-AACCAUACAACCUACUACCUCA-3′; let-7d: 5′-AGAGGUAGUAGGUUGCAUAGUU-3′; AMO-let-7d: 5′-AACUAUGCAACCUACUACCUCU-3′; let-7e: UGAGGUAGGAGGUUGUAUAGUU; AMO-let-7e: 5′-AACUAUACAACCUCCUACCUCA-3′. All pyrimidine nucleotides in the NC or miRNA mimics were substituted by their 2′-O-methyl analogues to improve RNA stability. Transfection of synthesized RNAs was accomplished by using X-tremeGENE siRNA Transfection Reagent (Roche). Thirty-six hours after transfection of let-7e mimic (100 nM) or AMO-let-7e (200 nM), the NRVCs were used for qRT-PCR and Western blot analysis.Lentivirus vectors expressing mature let-7e, anti-miRNA-oligo of let-7e (AMO-let-7e) or NC sequence were constructed by Invitrogen (Invitrogen). Briefly, the precursor sequence of let-7e and its antisense fragment were synthesized by Invitrogen. The synthesized fragments were annealed and inserted into the pcDNA™ 6.2-GW/EmGFP-miR vector (Invitrogen). Then, lentivirus plasmid was constructed by BP and LR recombination into pLenti6.3/TO/V5-DEST vector (Invitrogen) through the Gateway recombination technology. The constructed pLenti6.3/TO/V5-DEST plasmid and an optimized mix of the three packaging plasmid  were cotransfected into 293FT procedure cell line by liposome reagent to package lentivirus. The virus liquid was collected and concentrated 48 hrs after cotransfection, and the titre of the lentivirus liquid was determined.8 TU) including len-NC, precursor let-7e (len-pre-let-7e), AMO-let-7e (len-AMO-let-7e) or len-pre-let-7e and len-AMO-let-7e was injected using an insulin syringe into LV wall of rat heart. Rats in the sham and MI groups underwent the same procedures but received the same volume of saline (20 μl) with the constructs. Rats in β-blocker groups received non-specific β-AR blocker propranolol  and specific β1-AR blocker metoprolol  by intragastric administration for 7 days. Ligation of LAD was performed at day 7 after injection and administration. All surgery equipments were sterilized to ensure the minimum level of infection. Rats were given penicillin  for 7 days. After surgery, the rats were given food and water ad libitum.Thoracotomy was performed in the fourth left intercostal space to expose the heart of rats. Virus-containing solution . Haemodynamic measurements were performed by the Vevo®2100 High-Resolution Imaging system  in rats 6 hrs after ligation or 7 days after treatments with len-pre-let-7e or len-AMO-let-7e. Rats were positioned on a RatPad  equipped with integrated heater. Body temperature was maintained at 37°C. Pre-warmed Aquasonic Clear® Ultrasound Gel  was used as a coupling agent between the ultrasound scan-head and the skin. Two-dimensional targeted M-mode traces were obtained with the transducer (MicroScan MS 250-0206) held immobilized. LV end-diastolic and end-systolic wall thickness and LV internal diameter were measured from at least three consecutive cardiac cycles. Ejection fraction (EF) and fractional shortening (FS) were calculated based on the Vevo®2100 High-Resolution Imaging system .As described above, rats were infected with len-NC, precursor let-7e (len-pre-let-7e), AMO-let-7e (len-AMO-let-7e) or len-pre-let-7e + len-AMO-let-7e, or administrated with either β-AR blocker propranolol or metoprolol. Ligation of LAD was performed on day 7 after infection and administration. Then, standard lead II digital ECG tracings were recorded for evaluation of arrhythmias using the BL-420F bio-function experiments system . Arrhythmia score was evaluated from an ECG recorded during 30 min. period after the LAD ligation using Score F as described by Curtis and Walker ,33, baseanova followed by Turkey's multiple comparison test. Data of arrhythmia score were statistically analysed by using Wilcoxon signed-rank test. Differences were considered as statistically significant when P < 0.05. Data are presented as mean ± SD or mean ± SEM.All data were statistically analysed by using one-way 1-AR between the infarcted and non-infarcted LV tissues in a rat model of AMI. β1-AR expression was up-regulated by 3.0 ± 0.8-fold (P < 0.01) at the protein level 6 hrs after AMI  and 24 hrs (P < 0.01) after AMI -induced β1-AR overexpression, and which was abolished by co-treated with len-AMO-let-7e . By comparison, rats with treatment of propranolol (10 mg/kg/day), a non-selective β-AR blocker had an arrhythmia score of 2.1 ± 0.3 (P < 0.05), and those treated with metoprolol (80 mg/kg/day), a selective β1-AR blocker had a score of 2.4 ± 0.2 (P < 0.05). All three groups showed significant anti-arrhythmic effects in AMI rats. In contrast, len-AMO-let-7e increased arrhythmia score from 3.3 ± 0.4 to 5.8 ± 0.5 (P < 0.05). Len-NC had no effect on the score  and FS from 46.8 ± 5.5 to 56.0 ± 5.2 (P < 0.05). Both metoprolol and propranolol significantly lowered HR from 355 ± 16 to 280 ± 24 (P < 0.05) and from 355 ± 16 to 264 ± 27 (P < 0.05) in AMI rats respectively. However, len-pre-let-7e had no significant effect on HR of AMI rats  possibly via cAMP/PKA/CREB pathway [1-AR expression cannot be ruled out, such as post-transcription regulation of the receptor.βan heart , β1-AR ud in AMI ,5 and β1 failure . Acute mpression ,5, but ngulation , possiblgulation ,36. Abov pathway ,36. HoweIn silico prediction with Targetscan and miRanda showed that ADRB1 is a target of let-7; the seed sequence is conserved among species, such as rat, mouse and human beings. We also demonstrated that β1-AR is a target gene for let-7 revealed by luciferase reporter assay and Western blot analysis. We further verified that let-7a, c, d, e, and i were down-regulated in the acute ischemic tissue and forced overexpression of let-7e inhibited β1-AR expression and knockdown of this miRNA by AMO-let-7e increased β1-AR expression in neonatal rat cardiomyocytes. However, let-7e did not inhibit the mRNA level of ADRB1 gene, indicating that let-7e regulates β1-AR expression by disrupting mRNA translation, not by degrading the mRNA. Importantly in vivo study, we demonstrated that β1-AR expression was regulated by let-7e via locally applying this miRNA to rat heart. Notably, len-AMO-let-7e caused an overshoot of β1-AR expression relative to the baseline control level, strongly suggesting that this cardiac-enriched miRNA exerts important tonic inhibition of β1-AR in the heart.A variety of studies have demonstrated that let-7, an abundant and conserved miRNA, participates in various pathophysiological processes, such as cancer growth and formation  and axonlet-7 family shares the same seed sequence (5′ GAGGUAG 3′) and are highly conserved across species in both their sequences and functions . Study a1-AR blocker, and that are commonly used to treat a variety of cardiac arrhythmia, including ischemic arrhythmia. Cardiac function and HR were not influenced by either len-pre-let-7e or len-AMO-let-7e in normal rats. However, in AMI rats, len-pre-let-7e reduced cardiac function but not affected HR. However, both propranolol and metoprolol inhibited cardiac function and slowed HR. These may suggest that local intramuscular injection of let-7e to LV wall is unable to affect β1-AR level in the sinoatrial node that controls HR. The present data indicate that like β-AR blocking agent, let-7e has pronounced antiarrhythmic effect in the setting of AMI.The most important finding is that len-pre-let-7e significantly lowered the incidence of arrhythmia induced by AMI in rats; as expected, len-AMO-let-7e increased the incidence of AMI-induced arrhythmia. Our results also showed that efficiency of its antiarrhythmic effects was very similar with that of classic β-AR blocker propranolol, a non-selective β-blocker, and metoprolol, a selective β1-AR expression is up-regulated in AMI. However, the mechanisms involved in subtype-selective regulation of β1-AR are not fully understood. Several studies showed that adenylyl cyclase and cAMP participate in up-regulation of β1-AR in AMI by enhancing transcription of the receptors [et al. [2-AR, a predominant subtype [2-AR in addition to up-regulation of β1-AR. However, it is known that AMI only causes β1-AR up-regulation, without altering β2-AR. Clearly, mechanisms other than let-7 may also participate in the regulation of β2-AR expression in the heart. This study does not provide explanation of this issue. More importantly, our study also showed that let-7e presented a potential antiarrhythmic efficacy in AMI rats as β-AR blocker did. let-7e exerted its beneficial effect mainly through inhibiting the up-regulated β1-AR induced by ischemia. However, we definitely could not rule out other molecular targets of let-7, which are involved in its antiarrhythmic effect in the rats with AMI. Our study reveals a new mechanism of β1-AR regulated by let-7e and find that let-7e exerts a potential antiarrhythmic effect by targeting β1-AR in AMI rats. let-7e might be a promising target for intervention of β1-AR in the pathological condition, and let-7e supplement may be a new therapeutic approach for preventing and treating ischaemia-induced arrhythmia.Previous study demonstrated that only βeceptors ,36,41,42 [et al.  reported subtype , in lung


The androgen receptor (AR) plays a central role in the oncogenesis of different tumors, as is the case in prostate cancer. In triple negative breast cancer (TNBC) a gene expression classification has described different subgroups including a luminal androgen subtype. The AR can be controlled by several mechanisms like the activation of membrane tyrosine kinases and downstream signaling pathways. However little is known in TNBC about how the AR is modulated by these mechanisms and the potential therapeutic strategists to inhibit its expression.We used human samples to evaluate the expression of AR by western-blot and phospho-proteomic kinase arrays that recognize membrane tyrosine kinase receptors and downstream mediators. Western-blots in human cell lines were carried out to analyze the expression and activation of individual proteins. Drugs against these kinases in different conditions were used to measure the expression of the androgen receptor. PCR experiments were performed to assess changes in the AR gene after therapeutic modulation of these pathways.AR is present in a subset of TNBC and its expression correlates with activated membrane receptor kinases-EGFR and PDGFRβ in human samples and cell lines. Inhibition of the PI3K/mTOR pathway in TNBC cell lines decreased notably the expression of the AR. Concomitant administration of the anti-androgen bicalutamide with the EGFR, PDGFRβ and Erk1/2 inhibitors, decreased the amount of AR compared to each agent given alone, and had an additive anti-proliferative effect. Administration of dihydrotestosterone augmented the expression of AR that was not modified by the inhibition of the PI3K/mTOR or Erk1/2 pathways. AR expression was posttranscriptionally regulated by PI3K or Erk1/2 inhibition.Our results describe the expression of the AR in TNBC as a druggable target and further suggest the combination of bicalutamide with inhibitors of EGFR, PDGFRβ or Erk1/2 for future development. The understanding of the heterogeneity of cancer is of great importance for the development of effective therapeutic strategies in specific subgroups of patients. Indeed, genomic studies have classified breast cancer into different subtypes . At a clThe influence of sex hormones in the development of breast and prostate cancer is known. Estrogens and androgens act through their receptors by a direct transcriptional regulation of genes involved in cell growth and survival . In breaTestosterone and particularly dihydrotestosterone (DHT) are the main activators of the androgen receptor . Upon liThe control of AR is mediated at different levels, but signalling pathways play a key role by stabilizing or enhancing its transcriptional activity. In prostate cancer, membrane receptor tyrosine kinases (RTK) have been shown to modulate the AR expression. For instance, overexpression of HER2 in cell lines of prostate cancer results in increase AR activity and stability . FurtherIn TNBC, expression of the epidermal growth factor receptor (EGFR) or Platelet-derived Growth Factor Receptor (PDGFRβ) has been described using immunohistochemical techniques . A frequThe mechanism of action of androgens in TNBC remains controversial and the relationship between activated RTKs and the expression of AR has not been fully explored, although some recent reports have suggested the AR as a potential therapeutic target .In order to contribute to a better understanding of the role of AR in TNBC, the aim of this study is to evaluate the expression of the AR in TNBC and its relationship to the activation status of RTKs and its downstream routes.Cell culture media and supplements , glutamine, penicillin/streptomycin) were purchased from Invitrogen . The anti-pErk1/2, anti-Erk2, anti-PDGFR β, anti-pPDGFR β, anti-EGFR and anti-pEGFR antibodies were purchased from Santa Cruz Biotechnology . The anti-Akt and anti-AR antibodies were from Cell Signalling Technologies . The anti-phosphorylated Akt (Serine 473) antibody was generated against the sequence RPHFPQFpS473YSAS (p = phosphorylated) .2–95% air. Cells were grown in DMEM or in RPMI medium containing a high glucose concentration  supplemented with antibiotics , glutamine 2 mM and 10% FBS. Cells were treated with different drugs: PD98059 (50 μM), BEZ235 (0.5 μM), BIC (20 μM), Imatinib (10 μM), Lapatinib (10 μM) and DHT (50nM).All cell lines were cultured at 37°C in a humidified atmosphere in the presence of 5% COHuman samples were obtained from the tumor bank of the Salamanca University Hospital and Albacete University Hospital following institutional and ethical guidelines. All patients signed the study consent form. TNBC samples were defined as those with HER2 negative by inmunohistochemistry (IHC), HER2 of 0 or 1+ or a negative fluorescence in situ hybridization (FISH). HER2 amplification  was defined as a HER2-chromosome 17 ratio of < .2.0, as required by guidelines. Hormone receptor (HR) negative was defined as follow <10% positive cells by IHC for both estrogen receptor (ER) and progesterone receptor (PR) following recommendations before 2011.Frozen human samples were inspected by haematoxylin-eosin staining for epithelial tumor content by analysis of two slices at each end of the tumor. Only samples containing 70% epithelial tumoral cells were selected for Western analyses. The tumors were minced, washed with phosphate-buffered saline (PBS), and homogenized in ice-cold lysis buffer  with a tight-fitting Dounce homogenizer. This homogenate was centrifuged at 10,000 g for 20 minutes at 4 uC, and the supernatants were transferred to new tubes.Two commercial arrays were used for the studies; the human phospho-RTK array kit  and the PathScan RTK Signaling Antibody Array Kit . The Image J 1.44 software  was used for the quantification of the different RTKs in the human phospho-RTK array kit, and the Odyssey V3.0 program for the quantification of the cell signalling intermediates.Cells were washed with phosphate-buffered saline and lysed in ice-cold lysis buffer , and 1 mM sodium orthovanadate) . LysatesCells were plated in 24-well plates at 15,000–20,000 cells/well and cultured overnight in DMEM or RPMI + 10% FBS. The next day medium was replaced with DMEM or RPMI containing the different drugs. Cell proliferation was analyzed days later by an MTT-based assay as described . Unless Total RNA was extracted with RNeasy Mini Kit (Quiagen) as recommended by the supplier. cDNAs were synthesized from 5 μg of total RNA by using RevertAid H Minus First Stand cDNA Synthesis Kit (Fermentas) in a total volume of 20 μl. Reverse transcription was performed at 65° for 5 min followed by 60 min at 42°. PCR reactions were performed with 2 μl cDNA in a 50 μl volume and using Taq Polymerase (Biotools) and the following primer pairs (fragments size indicated in brackets): AR (247 bp) forward 5′-CTCACCAAGCTCCTGGACTC-3′ and reverse 5′-CAGGCAGAAGACATCTGAAAG -3′, β-actin (661 bp), forward 5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′ and reverse 5′-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3′. PCR cycle conditions were 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C for AR and β-actin. The number of cycles was 35 and the products were resolved on a 1% agarose gel and visualized with ethidium bromide.The design of this study is approved by the Committee of Ethics in Research of the University hospital of Albacete (Reference PI-2010/017).We used a panel of eight TNBC cell lines to evaluate the expression of the AR. Five out of eight cell lines expressed the AR, except for HBL100, HCC3153, and MDA-MB231. To corroborate these findings at a genetic level we extracted gene expression data from public libraries and evaluated the expression of the AR gene in a panel of cell lines. BT549 and HS578T (claudin-low subtype) had the highest level, HCC3153 and HCC70  the lowest, and MDA-MB231 (claudin-low subtype) and MDA-MB435 (claudin-low subtype), had low to moderate levels , and their activation status correlated at a significant level with the expression of the AR measured by western-blot (Figure We then studied the expression of RTKs and downstream pathways in the panel of TNBC cell lines. EGFR was activated in HCC1187, BT 549, HCC 70 and HCC 3153; and PDGFR was activated in HS578 and HCC 1187. All these cell lines described, except HCC3153, expressed the AR Figure C.in vitro model. However, the increased existence of activated AKT and Erk1/2 in most of these cell lines made difficult to identify any association between the expression of the AR and the activation of these pathways.In addition we studied downstream activated pathways including AKT-mTOR and Erk1/2. All cell lines had activation of pAKT except MDA-MB231. pS6 and pErk1/2 were expressed in all cell lines at different amount levels (Figure Given the association observed between some RTKs and downstream pathways with the expression of the AR in human samples and cell lines, we evaluated if the pharmacological inhibition of these receptors could modify the expression of the AR.For this purpose we used two cell lines; BT549, with constitutive activation of EGFR; and HS578T, with activation of PDGFRβ. Both cell lines have activation of AKT, S6 and Erk1/2, being HS578T a cell line with more activation of Erk1/2. Treatment with imatinib mesylate, a PDGFβ inhibitor, do not decreased the amount of the AR in HS578T; and a similar effect was observed for lapatinib, an EGFR inhibitor, in BT549 Figure A.As EGFR and PDGFR signal through downstream pathways, mainly the PI3k-mTOR and the Erk1/2 pathway, and these routes have also been implicated in the androgen-independent control of the AR in prostate cancer, we evaluated if the inhibition of these central nodes could have more effect on the expression of the AR than individual inhibition of RTKs. Using the same two models, we observed that the administration of PD98059, a MEK inhibitor that inhibits Erk1/2, did not reduce the amount of the AR (Figure We next explored the action of the anti-androgen bicalutamide when combined with inhibitors of EGFR, PDGFRβ and the PI3K-mTOR and Erk1/2 pathways. Interestingly we observed that the concomitant administration of bicalutamide with EGFR, PDGFRβ and MEK inhibitors reduced the amount of the AR compared to each agent alone (Figure We decided next to evaluate the effect on proliferation of pharmacological inhibitors of these receptors and pathways alone or in combination with antiandrogens. Bicalutamide alone reduced the MTT uptake in both cell lines Figure B. IntereWe then evaluated the action of DHT and bicalutamide on the expression of AR and how the inhibition of these kinases can modulate its expression. Interestingly administration of DHT increased the amount of the AR in HS578T and BT549 Figure . In the In the present work, we describe the expression of the AR in TNBC and its relation to RTKs and their downstream signal routes. It is also interesting to remark that the expression of the AR was studied in cell lines not representative of a luminal androgen receptor gene expression subtype .In prostate cancer evidence support the link between the activation of RTKs, like the insulin-like growth factor, keratinocyte growth factor, EGFR or HER2, and the transactivation of the AR in the absence of androgens ,23,24. PIn our study we show a correlation between activated membrane tyrosine kinase receptors and the expression of the AR in TNBC. It is known that in triple negative tumors EGFR and PDGFRβ are phosphorylated and present in a significant number of tumors ,27. If eIt was interesting to find how the concomitant administration of an Erk1/2 inhibitor with bicalutamide reduced the amount of the AR in a more profound manner than when each agent was given alone. This effect was also observed when using inhibitors of EGFR and PDGFRβ.We then studied the effect on proliferation of these combinations. Although an important effect was noted with BEZ235 given alone, no effect on proliferation was found when associated with an anti-androgen. Furthermore, the concomitant administration of PD98059 plus bicalutamide produced a profound arrest in proliferation compared to each agent given alone. This effect suggests that the inhibition of the MAPK pathway facilitates the anti-proliferative effect of anti-androgens. A synergistic interaction between anti-androgens and Erk1/2 inhibitors has been described in other models including apocrine breast cancer, but not in TNBC . A similStimulation with DHT increased the amount of the AR, and this was not prevented by the administration of either an inhibitor of PI3K or Erk1/2; showing that the mechanism associated with the expression of the AR by androgens is independent of a kinase control. Furthermore, effects on the expression of AR after stimulation with DHT or when administering kinase inhibitors were independent of the expression of the AR gene, suggesting a posttranscriptional mechanism.Our findings have some potential therapeutic translations. It has been suggested that the AR is a potential druggable target in TNBC and indeed, clinical studies are currently evaluating the role of androgen deprivation in the treatment of patients with breast cancer . In our We observed a positive correlation between the activation of EGFR and PDGFRβ and the expression of the AR. Inhibition of PI3K pathway decreased the total amount of the AR. Inhibition of Erk1/2, PDGFRβ and EGFR decreased the expression of the AR when given concomitantly with bicalutamide and had an additive anti-proliferative effect, suggesting that administration of anti-androgens in TNBC should be given concomitantly with inhibitors of these kinases. Modification of AR expression by these kinase inhibitors was posttranscriptionally regulated. In conclusion, our results describe mechanisms associated with the control of the AR expression in TNBC, and demonstrate a potential therapeutic opportunity for the combination of anti-androgens with RTK inhibitors and its downstream routes.The authors declare that they have no competing interests.AO carried out the design and drafting the manuscript. AP participates in the design of the manuscript. MDC-L carried out the molecular studies, participated in the design and drafting manuscript. JC Montero, JC Morales and AP participate in the molecular studies. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/302/prepubHuman Phospho Kinase Array kit including Receptor Tysosine Kinases and downstream proteins in human samples from triple negative breast cancer patients. Lower pannels describe the exact possition of each protein in duplicate. For more details see the Methods section.Click here for fileRelationship between pAKTS473, pAKTS308, pErk1/2 and AR expression in human samples using data from the kinase arrays. AR expression was measured by western blot (as described in Figure Click here for file


SPOP E3 ubiquitin ligase gene is frequently mutated in human prostate cancers. Here, we demonstrate that SPOP recognizes a Ser/Thr-rich degron in the hinge domain of androgen receptor (AR)and induces degradation of full-length AR and inhibition of AR-mediated gene transcription and prostate cancer cell growth. AR splicing variants, most of which lack the hinge domain, escape SPOP-mediated degradation. Prostate-cancer-associated mutants of SPOP cannot bind to and promote AR destruction. Furthermore, androgens antagonize SPOP-mediated degradation of AR, whereas antiandrogens promote this process. This study identifies AR as a bona fide substrate of SPOP and elucidates a role of SPOP mutations in prostate cancer, thus implying the importance of this pathway in resistance to antiandrogen therapy of prostate cancer.The  Prostate cancer is one of the most common malignancies, with over 913,000 new cases and over 261,000 deaths worldwide each year . AlthougTMPRSS2 in approximately 50% of all human prostate cancers (Androgen receptor (AR) is a pivotal transcription factor that is essential for normal prostate cell growth and survival. AR is also important for initiation and progression of prostate cancer. The role of AR in prostate cancer initiation is accentuated by the seminal discovery that the oncogenic ETS family transcription factors, such as ERG and ETV1, are translocated to the loci of androgen regulated genes including  cancers . Develop cancers . The imp cancers .Drosophila and human, such as Puc, Ci/Gli, MacroH2A, Daxx, and SRC-3  constitutes a fundamental mechanism that promotes either protein turnover or signaling transduction. Ubiquitin ligases, or E3s, selectively bind to and target substrates for ubiq-uitination and subsequent proteasome degradation. The largest E3 ligase subfamily consists of Cullin-RING ligases (CRLs), which are multisubunit enzymes, consisting of hundreds of distinct CRL complexes with the capacity to recruit numerous substrates . Human cnd SRC-3 .SPOP  and one imperfectly matched (203EGSSS207) SPOP-binding motif . The BTB deletion mutant of SPOP (SPOP-ΔBTB) cannot form a complex with CUL3 and RBX1 to function as an enzymatically active E3 ligase . As showTo determine whether SPOP regulates AR polyubiquitination, HA-Ub and pCMV5-AR were coexpressed in 293T cells with different doses of wild-type SPOP (SPOP WT) or enzymatic dead mutant (SPOP-ΔBTB). AR protein was robustly polyubi-quitinated by coexpression of SPOP WT, but not SPOP-ΔBTB, in a dose-dependent manner . AccordiPSA and TMPRSS2, two well-studied AR transcriptional targets  assay. As shown in 645ASSTT649) and one imperfectly matched (203EGSSS207) SPOP-binding motif  and the 645ASSTT649 motif were deleted individually or together. CoIP assays demonstrated that AR-WT and the ΔEGSSS mutant were coimmunoprecipitated by Myc-SPOP. In contrast, ΔASSTT and the double mutant (2Δ) totally lost the SPOP binding capability  to determine how SPOP regulates degradation of these mutants. Overexpression of SPOP decreased the protein level of AR-WT, S646A, and T649A mutants, but had little or no effect on the level of AR-S647A or T648A mutants  were cotrans-fected with or without SPOP in 293T cells. In vivo, ubiquitination assays demonstrated that SPOP robustly enhanced polyubiquitination of AR-WT and the ΔEGSSS mutant, but not the ΔASSTT and 2Δ mutants  in AR. Deletion of the 645ASSTT649 motif completely abolished the interaction between SPOP and AR, as well as SPOP-mediated polyubiquitination of AR. Thus, like other canonical SPOP substrates, AR contains a functional SPOP-binding consensus motif. SRC-3, a coactivator of AR that is often upregulated in prostate cancer  that mediates SPOP binding . This moe cancer , is anote cancer . A simile cancer . Importae cancer . Thus, i645ASSTT649 is located in the hinge domain of the AR, we demonstrated that none of the hinge domain null AR variants could be bound by SPOP, thereby escaping SPOP-mediated protein degradation in prostate cancer cells. In contrast, we showed that the v567es variant , in which the hinge domain remains, can be bound and degraded by SPOP. Because the majority of the AR variants identified thus far do not harbor the hinge domain , wild-type AR (AR-WT), and AR variants V2, V4, V5, and V7 were described previously . The AR Immunoprecipitations were performed as described previously . For wesExperiments were carried out with three or more replicates unless otherwise stated. Statistical analyses were performed by Student’s t test for most studies. Values with p < 0.05 are considered statistically significant.Supplement I