The first national study in Israel of post-acute rehabilitation service use for elderly patients with stroke and hip fracture reported regional variation in care receipt. Although lower likelihood of admission to inpatient rehabilitation was observed in districts with known shortages of rehabilitation beds, supply alone did not explain the findings. This commentary explores other potential contributing factors. It argues that greater uniformity in the process and documentation of discharge planning in combination with decision support would help to standardize provider behavior. Implementation of a system of functional status data collection that is linked to administrative data is recommended to enable examination of the impact of care receipt and variation. Additional research is needed to provide a clearer understanding of factors contributing to regional variation and to identify solutions to ensure equal access to post-acute rehabilitation services in Israel. In the current issue of the Israeli Journal of Health Policy and Research, Zucker and colleagues report the results of a nationwide study of post-acute rehabilitation service delivery to elderly persons after stroke and hip fracture . Their sAlthough prior studies show that availability of clinical services is a stronger predictor than patient characteristics when determining type of post-acute care , the datHowever, Zucker found that, according to health plan data, patients living in Jerusalem had a 24% likelihood of receiving inpatient rehabilitation after hip fracture, and patients living in the North had a 48% likelihood of receipt as compared to patients living in the Center district. Likelihood of receipt of inpatient rehabilitation after stroke was lower only for patients living in Jerusalem, but not for patients living in the North as compared to those from the Center district. These findings contrast with earlier reports of lower likelihood of inpatient rehabilitation receipt for patients living in the North district, but not in Jerusalem [Other explanations for regional variation include socioeconomics, religious and cultural differences between regions of the country. Jerusalem is known to have poverty rates that are nearly twice the national average, as well as high rates of unemployment. Jerusalem also has a higher proportion of Ultraorthodox Jews and Arab residents than do other districts. The association between these factors and receipt of post-acute rehabilitation services for the elderly should be explored.Only 11% of stroke and 14% of hip fracture patients had ambulatory rehabilitation services without prior inpatient rehabilitation. However, it is possible that additional patients were referred directly to ambulatory rehabilitation , but at four months post-stroke had not yet received them. Shortages and long waiting times at community based rehabilitation centers across Israel, especially for older persons with chronic disease have been reported [It is also possible that there is regional variation in the provider referral process or practice preference with clinicians in Jerusalem and the North less likely to evaluate the rehabilitation needs of elderly patients and/or less likely refer them for post-acute care. In an effort to promote equal access to rehabilitation services, the Israeli Ministry of Health developed and disseminated referral regulations to acute care hospitals. However, the impact of these regulations is unknown. It was not possible for the Zucker study to determine whether rehabilitation needs were being assessed because the recommendations for rehabilitation were not consistently present in hospital discharge letters. Thus, the authors were not able to uniformly discern when providers had failed to evaluate and consider rehabilitation needs, when rehabilitation services were ordered but not received, or which type of services were ordered.This information gap points to the need for re-engineering of hospital systems to ensure that discharge recommendations are uniformly documented using a standardized documentation template. Such a template should specify the provider’s evaluation of rehabilitation potential, and clearly indicate when a referral for services has been issued. This requirement, when combined with auditing and reporting of compliance, would drive uniformity in provider behavior.Further, automated decision support can be implemented within the electronic health system to help providers make appropriate rehabilitation referrals. Such a decision support system should draw upon standard data elements collected during physical and occupational therapy encounters during the acute hospital stay. When clinicians have access to organized patient data, they are much more likely to refer patients to rehabilitation . An examEarly rehabilitation is important to maximize functional gain after stroke. Patients who are functionally dependent and who are discharged home without needed rehabilitation services are more likely to be readmitted to acute hospitalization. These patients will almost certainly incur higher societal costs in terms of need for paid or unpaid personal care. Additional research should be conducted to study the impact of variation of care receipt on patient outcomes and costs of care. In order to conduct this type of research across the country of Israel an organized system of outcome assessment is needed. Such a system would track patients’ functional status in the hospital and reassess patients as they transition to post-acute care and beyond. This type of data, embedded in the electronic health record, and merged with administrative data on service use and health care costs can be used to examine short and long-term outcomes of care. Israel’s Maccabi Health Plan has begun integrating functional status data collection with the electronic health record in the outpatient setting and using this data for quality improvement in the physical therapy service . This tyI commend the authors for conducting this important study which is the first to describe national patterns of post-acute rehabilitation service use for elderly patients after stroke and hip fracture in Israel. Their findings suggest a need for system wide improvements in data collection, as well as additional research to provide a deeper understanding of determinants and impact of regional variation. This type of effort is also needed in other countries, where geographic variation in rehabilitation service delivery has been documented.While adding rehabilitation service capacity can be costly, the motivation to make this investment will be strengthened when there is evidence that links the receipt of inpatient and ambulatory rehabilitation to improved functional status, and lower long-term health care costs due to reductions in rehospitalization, and costs for personal care. This type of understanding will be needed to identify solutions to insure equal access to post-acute rehabilitation services in Israel.The author declares that she has no competing interests.Linda Resnik, PT, PhD is Associate Professor (Research) in the Department of Health Services, Policy and Practice, Brown University, Providence RI and a Research Health Scientist at the Providence VA Medical Center, Providence, RI.1*, Irit Laxer2, Iris Rasooli2, Shulamit Han2, Aaron Cohen2, and Tamar Shohat1.Regional gaps in the provision of inpatient rehabilitation services for the elderly in Israel: Results of a national survey by Inbar Zucker1Israel Center of Disease Control and the 2Geriatric Department of the Ministry of Health.


The androgen receptor plays a critical role throughout the progression of prostate cancer and is an important drug target for this disease. While chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-Seq) is becoming an essential tool for studying transcription and chromatin modification factors, it has rarely been employed in the context of drug discovery.Here we report changes in the genome-wide AR binding landscape due to dose-dependent inhibition by drug-like small molecules using ChIP-Seq. Integration of sequence analysis, transcriptome profiling, cell viability assays and xenograft tumor growth inhibition studies enabled us to establish a direct cistrome-activity relationship for two novel potent AR antagonists. By selectively occupying the strongest binding sites, AR signaling remains active even when androgen levels are low, as is characteristic of first-line androgen ablation therapy. Coupled cistrome and transcriptome profiling upon small molecule antagonism led to the identification of a core set of AR direct effector genes that are most likely to mediate the activities of targeted agents: unbiased pathway mapping revealed that AR is a key modulator of steroid metabolism by forming a tightly controlled feedback loop with other nuclear receptor family members and this oncogenic effect can be relieved by antagonist treatment. Furthermore, we found that AR also has an extensive role in negative gene regulation, with estrogen (related) receptor likely mediating its function as a transcriptional repressor.Our study provides a global and dynamic view of AR’s regulatory program upon antagonism, which may serve as a molecular basis for deciphering and developing AR therapeutics. I. I28]. Ide facto the protein target of these compounds, we compared the mRNA profiles of VCaP cells treated with Compound 30 and those treated with AR-siRNA using the Affymetrix HG-U133Plus2.0 GeneChip array. Differential expression analysis was conducted with Significance Analysis of Microarray (SAM) algorithm .VCaP human prostate cell lines were obtained from ATCC and grown in DMEM (GIBCO Cat# 11995) 10% FBS (SIGMA Cat# 12103 C-500 ml). Medium was supplemented with standard antibiotics .AR trans-activation assay was performed as previously described . BrieflyLNAR cells were starved for two days in phenol red free RPMI medium containing 5% charcoal-dextran stripped FBS (Omega Scientific) prior to the assay. For Nuclear Translocation (NT) assay, 3,000 cells per well (in 384 well plates) were treated with compounds and 100pM R1881. Following overnight incubation the cells were fixed with 10% Formalin and permeabilized with PBS containing 0.5% Triton X-100 and blocked with 1% BSA. Cells were then stained with anti-AR monoclonal antibody (Abcam) followed with alexa 488 conjugated anti-Mouse IgG secondary reagent (Invitrogen). Finally the cells were counter stained with Hoechst 33342 (Invitrogen) and Cell mask Deep Red (Invitrogen). Plates were sealed and imaged using an Evotec Opera high content imager. Images were analyzed using an Acapella (Evotec) algorithm customized by Pfizer to quantify the fluorescence associated with anti-AR in the cytoplasm and nuclear regions. The ratio of Nuclear to Cytoplasm fluorescence was calculated and used as to tract inhibition of AR translocation.Cells were seeded at a density of 15,000 cells/well (VCaP) or 1,000 cells/well (PC3 and DU145) in 96 well-plates and treated after attachment to the plate with test compounds. Medium and compounds were refreshed every 2–3 days. Number of live cells was analyzed at day 7 using the Resazurin assay (SIGMA Cat# R7017).2 and then processed.Cells were starved for 3 days in phenol red free DMEM containing 5% charcoal stripped FBS, and then seeded in 6-well plates at a density of 1 million cells per well in starvation medium. In the case of compound treatment (for ChIP-Seq study), cells were allowed to attach overnight and then were treated with various doses of the AR antagonists or vehicle alone (0.1% DMSO) in the absence or presence of 1nM R1881. Cells were incubated for 30 min at 37°C, 5% CO2 for 48 h and then processed.For siRNA treatment, cells were seeded in DMEM containing 10% FBS and transfected the day after seeding with 25nM AR-siRNA pool  using Lipofectamine 2000 reagents  and following manufacturer’s instructions. Cells were incubated at 37°C 5% COIn both procedures, after the indicated treatment time, cells were rinsed once with ice-cold PBS and then lysed with Qiagen RNeasy Plus Kit . RNA quality was assessed using the Bioanalyzer  and spectrophotometer.ChIP was carried out by Active Motif (formerly Genpathway) as follows. Cells were fixed with 1% formaldehyde at room temperature for 15 minutes. Fixation was stopped by the addition of glycine to a final concentration of 0.125 M glycine. Chromatin was isolated from the sample by adding 10 ml lysis buffer containing PIPES, Igepal, PMSF and Protease Inhibitor Cocktail, followed by disruption with a Dounce homogenizer. Samples were pelleted by centrifugation and resuspended in buffer containing sodium deoxycholate, SDS, and Triton X-100. Lysates were sonicated using a Misonix Sonicator 3000 equipped with a microtip in order to shear the DNA to an average length of 300–500 bp. Lysates were cleared by centrifugation and the chromatin suspensions were transferred to new tubes and stored at −80°C. To prepare Input DNA (genomic DNA), two aliquots of 10 μl each (approximately 1/50 of each chromatin preparation) were removed and treated with RNase for 1 hr at 37°C, proteinase K for 3 hr at 37°C, and 65°C heat for at least 6 hr to overnight for de-cross-linking. DNAs were purified by phenol-chloroform extraction and ethanol precipitated. Pellets were resuspended in 1/5 TE buffer. Resulting DNAs were quantified on a Nanodrop spectrophotometer. Extrapolation to the original chromatin volume allowed determination of the yield for each chromatin preparation (as measured by the DNA content).Prior to use in ChIP, protein A agarose beads (Invitrogen) were pre-blocked using blocking proteins and nucleic acids for 3 hr. For each ChIP reaction, an aliquot of chromatin (30 μg) was pre-cleared with 30 μl pre-blocked protein A agarose beads for 2 hr. ChIP reactions were set up using pre-cleared chromatin and antibody AR  in a buffer containing sodium deoxycholate and incubated overnight at 4°C. Pre-blocked protein A agarose beads were added and incubation at 4°C was continued for another 3 hr. Agarose beads containing the immune complexes were washed two times each with a series of buffers consisting of the deoxycholate sonication buffer, high salt buffer, LiCl buffer, and TE buffer. An SDS-containing buffer was added to elute the immune complexes from the beads, and the eluates were subjected to RNase treatment at 37°C for 20 min and proteinase K treatment at 37°C for 3 hr. Cross-links were reversed by overnight incubation at 65°C, and ChIP DNAs were purified by phenol-chloroform extraction and ethanol precipitation. Quality of ChIP enrichment was assayed by qPCR using primers against known positive control site(s). Input DNA was queried at the same sites in parallel.ChIP DNA was amplified by following the Illumina ChIP-Seq DNA Sample Prep Kit protocol. In brief, DNA ends were polished and 5’-phosphorylated using T4 DNA polymerase, Klenow polymerase and T4 polynucleotide kinase. After addition of 3’-A to the ends using Klenow fragment (3’-5’ exo minus), Illumina genomic adapters were ligated and the sample was size-fractionated (200–250 bp) on a 2% agarose gel. After a final PCR amplification step , the resulting DNA libraries were quantified and tested by QPCR at the same specific genomic regions as the original ChIP DNA to assess quality of the amplification reactions. DNA libraries were sequenced on a Genome Analyzer II.Alignment of the 36-bp single-read sequences (“tags”) from ChIP-Seq to the human genome (hg19) was conducted by Active Motif with ELAND (Illumina CASAVA 1.5 pipeline) software. Tag density was calculated by dividing the genome into 32-nt bins and counting the number of 3’-end extended tags in each bin (Active Motif). Only sequence reads that pass quality filtering, with an alignment score of at least 10 and perfect genomic match were included for peak detection. AR-enriched genomic regions (binding sites) were identified by comparing the ChIPed samples with input sample using MACS algorithm  . Their primer sequences were included in Table AR binding sites were mapped to transcriptional start sites (TSS) of genes based on refFlat (hg19) table from UCSC Genome Browser. The classification of AR binding sites relative to genomic annotations (promoter/exomic/intronic/intergenic) and calculation of associated enrichment statistics were performed with RegionMiner tool and ElDorado database (Genomatix).Sequence conservation was assessed using phastCons conserved elements ,18 derivAR bound-sequences were searched for predefined motif matrices of transcription factors from MatBase library v8.3 vertebrate collection using RegionMiner (Genomatix). Over-representation statistics were reported as Z-score (the distance from the population mean in units of the population standard deviation) computed against genomic background (NCBI37/hg19). V$GREF-V$FKHD pair (module) is defined as two elements from 10 to 50 bp (middle to middle) of each other. Their occurrences were examined for distance distribution within the range.ab initio from repeat-masked AR-bound sequences. In cases where a binding site is longer than 500 bp, only 500 bp centered on its summit were used. In consideration of computational time, we preformed the search with 2500 top sequences in terms of binding score. MEME was run using “-dna -mod zoops -revcomp -evt 0.01” command line options. Specificity was assessed as Z-score from 100 randomly sampled groups of the same number of sequences of the same length from the same chromosomes as AR binding sites. To investigate the enrichment and score distribution of the above MEME-derived ARE consensus motif, we scanned the AR-bound sequences as well as randomly sampled genomic sequences with its position weight matrix using PATSER [MEME algorithm  was usedg PATSER  (v. 3e) Approximately 20 mg of tumor samples in RNALater were homogenized by means of Qiagen TissueLyser 2, for 2 min @ 20 Hz. Homogenates were then processed using Qiagen RNeasy Plus kit (Cat#74134). Samples were resuspended in 60μL water and 2 μg RNA from each sample were subsequently subjected to qRT-PCR using TaqMan RT-PCR ABI7900HT, in a two-step procedure, as following: 1) For reverse transcriptase step we utilized ABI High Capacity cDNA Reverse Transcription Kit . Cycle run was 10 min 25°C, 2 h 37°C, 5 min 85°C and cool down to 4°C. For the qPCR reaction we utilized ABI 2X universal Master mix  using ddCt (RQ) method for quantification. Cycle was 10 min 95°C, 15 s 95°C, 60s 60°C, 40 cycles. Primers used were ID# Hs00907244-ml for for AR and # 4352934E for GADPH (Applied Biosystems).3 in size the animals were castrated. Since PSA levels remain low in this in vivo model until the tumors are significantly large (400 mm3), the re-growth of tumors post-castration was interpreted as a sign that the animals entered into the castration refractory phase. Animals were then randomized based on tumor volume and treatment commenced. In general and unless otherwise indicated, compounds were given by oral gavage once daily. Vehicle formulation consisted of 0.9% benzyl alcohol, 1% Tween-80 and 98.1% methylcellulose (0.5%). Tumor volume was calculated by the formula: length x width x depth x 0.5236. To measure PSA, 15 μl serum was diluted 1:3 v/v in water and then 25 μl of the dilution samples were transferred to the ELISA plate for the assay . At the end of the study, animals were sacrificed and the tumors were extracted and treated with RNALater . Statistic analysis was performed using two-way ANOVA .VCaP (3.5million) cells  were implanted subcutaneously in CB17/lcr-Prkdc SCID mice, and when tumors reached about 200 mmin vitro reverse transcription, and target preparation following the GeneChip Expression Analysis Technical Manual (Affymetrix). Overnight hybridization of the fragmented cRNA on the GeneChip® Human Genome U133A 2.0 array and subsequent washing, staining and scanning steps were performed as suggested by the manufacturer (Affymetrix). Image analysis was done with the Expression Console (Affymetrix).RNA from VCaP cells treated with AR-siRNA, Compound 30 (10 μM) or corresponding controls underwent 1st and 2nd strand cDNA synthesis, Expression profiling data were RMA normalized with “affy” package of Bioconductor, followed by exclusion of spike-in controls (AFFX) and mixed cross-hybridization (_x) probe sets. Significance Analysis of Microarray (SAM) algorithm  implemenGene signature enrichment analysis was performed by comparing direct AR- activation/repression targets from small molecule antagonism with signatures collected from a variety of public databases and studies . Statistical significance of signature enrichment was determined using cumulative hypergeometric probability distribution as previously described  and corrAR: Androgen receptor; CRPC: Astration-resistant prostate cancer; ChIP-Seq: Chromatin immunoprecipitation coupled with massively parallel sequencing; TSS: Transcriptional start site; ARE: Androgen response element; SAR: Structure-activity relationship; TGI: Tumor growth inhibition.Pfizer Inc. at time of performing the studies described herein.All authors are or were employees of AF and GL conceived the experimental plan; ZZ and PR designed the analysis plan; MS and AF conducted cell treatment and ChIP-Seq profiling; ZZ conducted ChIP-Seq and integrative data analysis; WH conducted mRNA profiling; ZZ, HE, WH conducted microarray expression data analysis; MS conducted qRT-PCR experiment of AR expression; MS and AF conducted cell viability study; DB and AF conducted TGI and PSA inhibition study; JE conducted AR trans-activation study; NH conducted AR antagonist pharmacokinetic study; TN conducted AR nuclear translocation assay and KI67 IHC study; ZZ, AF, PR wrote and revised the manuscript. All authors read and approved the final manuscript.Figure S1. AR protein expression. VCaP cells were treated in the presence of 25nM control/non-targeted siRNA or AR-siRNA pool. The protein levels of AR were analyzed by western blot using anti-AR (Santa Cruz Cat# sc-815). Anti-Tubulin  was included for loading control. Figure S2. AR binding and cell type. Overlap of the AR binding sites between VCaP cells and other cell types from previous studies: 1; 2; 3. Binding sites based on earlier version of human genome were remapped to hg19 using UCSC liftOver tool. Overlap was defined by sharing of at least 1 bp. Figure S3. AR binding and sequence features. (A) Distance distribution between neighboring (10-50 bp) GREF and FKHD elements. (B) The motif identified de novo from AR-bound sequences appears to be a 15 bp perfect palindrome. (C) Motif and binding strength. AR binding sites were divided into two groups based on whether they had a significant occurrence of the palindromic motif in (B). Boxplots depict the distributions of their binding scores. Figure S4. AR and DAX1 form a tightly controlled feedback loop on steroid biosynthesis: AR and DAX1 counter-balance each other’s effect on steroidogenesis. AR also directly and positively regulates the expression of DAX1, whereas their physical interaction may serve to sense and prevent the over-production of DAX1 by AR. Dashed links refer to previously reported regulatory relationships, while solid links describe regulatory relationship identified in this study. Positive or stimulatory effects are represented by (+), and negative or inhibitory effects are represented by (−).Click here for filesFile1. AR binding sites list.Click here for fileTable S1. The distribution of AR binding sites relative to genomic annotations. Table S2. Gene signatures most enriched among AR-bound genes. Table S3. MatBase families most over-represented among AR-bound sequences, sorted by descending Z-score. Table S4. Transcription factor binding motifs associated with mode of AR regulation. Table S5. Selective drug-modulated direct downstream effectors of AR involved in steroid metabolism.Click here for filesFile2. Drug-modulated direct activation and repression targets of AR from small molecule antagonism.Click here for file


All recurrent tumors displayed new quantitative genetic alterations and AR mutations, whatever the treatment modalities. AR amplification was found after complete blockade. Increased expression of Her-2/neu with frequent ERK/AKT activation was detected in all variants. Combination of castration with a Her-2/neu inhibitor decreased recurrence risk (0.17) and combination with an mTOR inhibitor prevented it. Anti-hormone treatments influence risk of recurrence although tumor growth inhibition was initially similar. Recurrent tumors displayed genetic instability, AR mutations, and alterations of phosphorylation pathways. We postulated that Her-2/AKT pathways allowed salvage of tumor cells under castration and we demonstrated that their inhibition prevented tumor recurrence in our model.Almost all prostate cancers respond to androgen deprivation treatment but many recur. We postulated that risk of hormone escape -frequency and delay- are influenced by hormone therapy modalities. More, hormone therapies induce crucial biological changes involving androgen receptors; some might be targets for escape prevention. We investigated the relationship between the androgen deprivation treatment and the risk of recurrence using nude mice bearing the high grade, hormone-dependent human prostate cancer xenograft PAC120. Tumor-bearing mice were treated by Luteinizing-Hormone Releasing Hormone (LHRH) antagonist alone, continuous or intermittent regimen, or combined with androgen receptor (AR) antagonists . Tumor growth was monitored. Biological changes were studied as for genomic alterations, AR mutations and protein expression in a large series of recurrent tumors according to hormone therapy modalities. Therapies targeting Her-2 or AKT were tested in combination with castration. All statistical tests were two-sided. Tumor growth was inhibited by continuous administration of the LH-RH antagonist degarelix (castration), but 40% of tumors recurred. Intermittent castration or complete blockade induced by degarelix and antiandrogens combination, inhibited tumor growth but increased the risk of recurrence (RR) as compared to continuous castration (RR Androgen receptor (AR) controls cell proliferation and survival in the normal prostate and prostate carcinomas (PCa). Thus androgen deprivation is first-line treatment of PCa. Hormone therapy includes castration pharmacologically achieved with luteinizing-hormone releasing hormone (LH-RH) agonists or antagonists, AR antagonists as flutamide or bicalutamide or new treatment modalities such as inhibitor of 17–20 lyase  or MDV3100 i. Genomic changes occur during tumor progression but their role remains unclear, although clonal chromosome abnormalities have been found in PCa ii. Alteration of AR expression is frequent due to gene amplification iii. Activation of survival pathways is involved in hormone escape et al. showed that forced Her-2/neu expression modulates AR signaling and leads to androgen independence et al. showed that forced overexpression of AKT in LNCaP cell line accelerated tumor growth Whatever the hormone therapy, most tumors respond then acquire androgen independence and recur AR mutations, and growth factor expression/activation were studied. The involvement of phosphorylation pathways in hormone escape led us to test combination of tyrosine kinase inhibitors with pharmacological castration to reduce the risk of tumor recurrence.A key question in clinics is whether modalities of hormone treatment differently affect the risk of escape. To respond to this critical question, we used an experimental model of a hormone dependent of human prostate cancer (PAC120), derived directly from a patient and growing in immunodeficient mice. We evaluated the effect of different hormone treatment modalities on the immediate response and on the risk of recurrence; the biological changes associated with different treatments, as genome alterations, lnu) from Charles River  was used between passages 47 and 51. Tumor pieces of 20 mm3±5 (±20. 106 cells) where transplanted. All protocols followed institutional guidelines as put forth by the French Ethical Committee.PAC120, a hormone-dependent human-in-mouse PCa xenograft, 3). Intermittent castration consisted of 2 injections of degarelix at day 0 and 28 (one cycle), then stopped the treatment until the tumor reach back 2 times the initial volume. The complete androgen blockade is the association of degarelix monthly in combination with the anti-androgen receptor inhibitor . degarelix  Follow-up of tumor growth was done according to Marangoni et al. 3, mice were assigned to treatment  was used to screen tumor samples for AR mutations as previously described 5′-TGCGGCGGCGCAGTGCCGCTAT-3′) (nucleotides 2310–2339) and reverse (5′-GGTGCCATGGGAGGGTTAGATAGGGAG-3′) (nucleotides 4075–4101) primers flanking exons 1–8 . Thereafter, 100 ng AR cDNAs fragments were inserted into a yeast expression “gap repair” plasmid by homologous recombination, and after plating and incubation at 30°C for 72 h, yeast colonies were scored Briefly, total RNA was prepared from tumor samples . Reverse transcription was performed with the Omniscript reverse transcription kit (Qiagen) from 1 µg of total RNA. AR cDNA fragments were amplified with the forward , ERK1/2 , and PSA (Dako) were used at 1∶1000. Her-2/neu , AR , and p-ERK1/2  were used at 1∶500. Anti-beta-actin was used at 1∶5000 . Protocols are detailed in Procedure was previously published Light microscope histology studies used 4-µm-thick formalin fixed paraffin-embedded sections from PAC120, PAC120 residual tumors and AI variants stained with hematoxylin and eosin safran.We used the Mann-Whitney test, Kaplan-Meier survival plots, log-rank tests, and the Fisher’s exact test, according to respective experiments as detailed in p<0.001, log-rank test)  . Therefop = 0.03, and p<0.001, respectively)  .Flutamide or bicalutamide significantly delayed tumor growth (ctively) . Completctively)  with incnk test) . Completnk test) ; increasii & iii). Androgen independent (AI) variants displayed histological changes, such as mucinous, neuroendocrine-like, or adenoid differentiation features  of AI variants and occurred only in the AI derived after complete blockade (AIde-a) group . In these cases AR mRNA levels were increased of 10 to 20-fold, as were intratumoral PSA mRNA levels (a) group . FrequenA levels .AR mutations were detected using a functional yeast-based assay as described previously AR mutations were found in the PAC120 tumor but appeared in 28% (10/36) of AI variant tumors. An example of the profile that we obtained for AIde-a4 is shown in the AR mutations  was increased in all AI variants . These dn = 17) tested as compared with PAC120   , and pronk test)  versus c36 days was 59%  (Degarelix combined with everolimus completely prevented hormone escape of the 17 tumors treated. As single-agent everolimus delayed tumor growth and the TGIWhitney) .These results demonstrate a superior effect of castration when combined with HER-2/neu targeting antibodies or mTOR inhibitor.in vivo experimental study of the effect of different hormone treatments on the risk of hormone escape. We established a large series of AI variants to study the diversity of genetic and molecular events occurring after hormone escape. We used an hormone-dependent human PCa xenograft (PAC120) AR mutations, after multiple passages We conducted an Continuous pharmacological castration arrests tumor growth, escapes occur in 40% of cases. Antiandrogen monotherapy slowed down tumor growth. This reflects the clinical observations. In our model, complete androgen blockade increased the risk of hormone escape, as compared with castration alone. It seems that complete androgen blockade exerts a high pressure on the AR signaling pathway, leading to the activation of several survival pathways. Large randomized studies and meta-analyses showed that complete blockade did not prolong the time to progression in most patients AI variants displayed biological changes. Histology of tumors under castration showed numerous mitosis and necrotic areas, suggesting a balance between mitosis/cell survival/death and dismissing any dormant state of tumoral residue. AI variants displayed mixed patterns, mucinous or/and apocrine differentiation similar to the parental tumor and none features specific to hormone treatment modality.AR gene amplifications, were detected after complete androgen blockade as found in patients AR mutations which contribute to hormone escape by providing a larger receptivity of AR to other ligands et al. reported that hormone-naïve PCa tumor samples had very few AR mutations suggesting that these mutations offered little growth advantage and were in fact random “passenger” mutations AR mutations that affect receptor function AR mutations, although very few were common to all AI variants and a majority were not described We checked that the deeply altered karyotype of PAC120 remained quite stable through transplantation and time We examined Her-2/neu and p-AKT expression in PAC120 AI variants to determine whether kinase and phosphorylation pathways were involved in the hormone escape, as shown by others Other alterations can contribute to a ligand-independent activation of AR. Loss of PTEN, PI3K mutation, AKT1 and AKT2 amplification may activate AKT kinase. Increase of phospho-AKT levels was detected in bicatulamide-treated PCa Our data confirm that AR remains a central actor in PCa cell survival, and our data suggest that androgen receptor can be activated by alternative pathways resulting in androgen independence. Finally, we showed that combination treatment of castration with targeted molecular therapy significantly reduced the risk of hormone escape. This study might have great implication in the treatment of androgen dependent prostate cancer and dual inhibition is worth exploring in the clinical setting.Figure S1Schematic representation of the in vivo treatment regimens.(TIF)Click here for additional data file.Figure S2Relative mRNA expression of androgen receptor (AR) (black bars) and intratumoral prostate-specific antigen (PSA) (white bars) in androgen independent (AI) variants. *indicates variants with amplified AR gene copy number. Abbreviations used to identify variants are explained in the legend of (TIF)Click here for additional data file.Figure S3Hormone response profile in the AIde-a4 castration-resistant tumor. The responsiveness of the AR in the AIde-a4 castration-resistant tumor was assessed by using the yeast-based functional assay and compared with that of the wild-type AR as described in (TIF)Click here for additional data file.Figure S4Constitutive activity of Q693X androgen receptor variant. The nonsense mutation Q693X leads to a truncated androgen receptor variant, which is deleted of the entire ligand-binding domain and AF-2. When compared to the ligand-dependent activity of the wild-type AR, the Q693X AR variant demonstrated constitutive activity in the yeast-based AR assay. Histograms represent the relative number of colonies obtained in the presence of the indicated hormone or anti-androgen to that obtained in the presence of 100 nM DHT. DHT, dihydrotestosterone; And, androstenedione; Prog, progesterone; Bic, bicalutamide; Flut, flutamide.(TIF)Click here for additional data file.Table S1Number of mice assigned to each treatment group.(DOC)Click here for additional data file.Table S2In vivo PAC120 tumor response to different hormone treatments.(DOC)Click here for additional data file.Table S3Results of the comparative genomic hybridization array of DNA from androgen-independent (AI) tumors. Minimal regions were altered in more than 20% of AI variants. *Regions on 1 p, 19 p and 19 q were associated with a delayed time to hormone escape.(DOC)Click here for additional data file.Table S4Characteristics of the androgen-independent (AI) variants. Data for AIde01 to AIde04 were obtained from independent experiments (data not shown). Data for mRNA expression  and tumoral prostate-specific antigen (PSA), circulating PSA levels and Protein expression (see last column) are relative to their mean expression in PAC120 tumors (units are arbitrary).(DOC)Click here for additional data file.Methods S1(DOC)Click here for additional data file.


This randomised controlled and double-blinded pilot study evaluated if interactive virtual reality balance related games integrated within conventional rehabilitation sessions resulted in more superior retraining of dynamic balance compared to CR after stroke. 19 subjects diagnosed with a recent episode of stroke were recruited from a local rehabilitation hospital and randomly assigned to either a control or an experimental group. Subjects in the control groups underwent 60 minutes of conventional rehabilitation while those in the experimental groups underwent 40 minutes of convention rehabilitation and 20 minutes of self-directed virtual reality balanced rehabilitation. Functional Reach Test, Timed Up and Go, Modified Barthel Index, Berg Balance Scale, and Centre of Pressure of subjects in both groups were evaluated before and on completion of the rehabilitation sessions. Results indicate that the inclusion of interactive virtual reality balance related games within conventional rehabilitation can lead to improved functional mobility and balance after a recent episode of stroke without increasing treatment time that requires more health professional manpower. The world is aging and chronic conditions such as stroke are more prevalent . Global β = 0.37, P = 0.004). Poor balance moderately correlated  with poor ambulatory activity [Poor postural control after stroke has a significant impact on a patient's ability to achieve independence in activities of daily living and gait –10. Di Mactivity . A recenactivity . Despiteβ = 0.160, P < 0.001) and after stroke . However, due to the worldwide shortage of health care professionals especially in the area of rehabilitation [Dejong et al.  reportedlitation , patientlitation . Interaclitation . Virtuallitation , and 27 litation . HoweverThis double-blinded and randomised pilot study evaluated the effectiveness of introducing low cost interactive virtual reality balance related games within a conventional rehabilitation programme. The focus was to evaluate if including virtual reality balance related games during inpatient rehabilitation sessions without increasing therapy time would contribute to balance recovery among in-patients after a recent episode of stroke.The study was approved by Ang Mo Kio Hospital in Singapore and the Projects Committee of the School of Health Sciences  at Nanyang Polytechnic, Singapore (SHS/2009-2010/02/PT).A total of 19 in-patients  from the community rehabilitation hospital participated in this study which was conducted over two years. The inclusion criteria were all subjects who had recently experienced a first onset of stroke  and were graded to have moderate disability (mRS Grade 3) or moderate severe disability (mRS Grade 4) using the 6-point ordinal hierarchical Modified Rankin Scale. mRS is the most commonly used functional measure in stroke trials and takes around 5 minutes to complete. They also were evaluated to be medically stable and had a Mini-Mental State Examination score of greater than 23 and thus understood how to safely use virtual reality gaming consoles . ExclusiRandom Allocation Software assigned subjects to either 60 minutes of conventional rehabilitation group (control) or the experimental group which received 40 minutes of conventional rehabilitation with 20 minutes of interactive virtual reality balance related games . Subjects in the experimental group were introduced to either a Nintendo Wii-Fit or Microsoft Kinect game console system during rehabilitation. All subjects received 15 sessions of in-patient rehabilitation during their hospital stay. The Nintendo Wii-Fit programme required subjects to shift their weight during standing in response to the game . The Micr = 0.89) and interrater reliability (r = 0.98) and strongly correlated (r = 0.78) with BBS [One researcher who was not involved in the interventions and was blinded to the allocation of subjects to their groups' evaluated both groups Functional Reach Test (FRT), Timed Up and Go (TUG), Berg Balance Scale (BBS), Centre of Pressure (CoP), and Modified Barthel Index (MBI) before and after the intervention period. FRT is a quick and easy dynamic test of anterior-posterior stability evaluated when subjects perform one arm forward reach task. The test has an excellent test-retest (with BBS .r = −0.79) with Barthel Index [r ≥ 0.75), and construct validity (r ≥ 0.60) with Modified Barthel Index [TUG assesses functional mobility and claims to predict those at risk of falls if their timing is greater than 14.7 sec . Furtherel Index . BBS evael Index . Center of Pressure (CoP) sway was measured using the Nintendo Wii-Fit Board. This device evaluated balance and was reported to be comparable with force platforms with a high test-retest reliability (ICC = 0.77–0.89) . The ModU tests for between-group differences at baseline and after intervention. Wilcoxon sign ranked test determined within-subject changes between the baseline and post-intervention values in both groups. Data were analysed with intent-to-treat analyses. The level of significance was set at P < 0.05. The data were analysed using SPSS .Data collected from this pilot study were evaluated and found not to be normally distributed. Thus, data were evaluated with Mann-Whitney U tests found no significant differences in baseline measure of all outcomes measures in both groups . This implied both groups baselines were similar, and thus comparison after intervention would reveal intervention effect. There was a significant difference in FRT scores between the experimental and control groups after 15 sessions of rehabilitation . There were no statistically significant differences in all other outcome measures after intervention between the control and experiment groups .Mann-Whitney t groups . FRT wasThe introduction of low cost interactive virtual reality balanced related games within a conventional rehabilitation programme significantly improved TUG, FRT, and MBI scores . 15 sessions of convention therapy resulted in significant improvements in TUG and MBI scores .This randomised controlled and double-blinded pilot study found that introducing interactive virtual reality balance related games within the standard treatment time of conventional therapy sessions over 15 sessions was equally effective as conventional therapy alone to retrain balance after a recent episode of stroke. The inclusion of virtual reality games without providing any additional therapy time or professional intervention to the experimental group significantly improved their Functional Reach Test mean scores by more than the double compared with the control group . r = 0.807, P = 0.028) between these two outcome measures. Our findings also support the hypothesis that participation in interactive virtual reality balance related games was more effective than conventional exercises to maintain postural stability during walking as it replicates the load-unloading sway strategy at the hip [Smith et al.  reported the hip . Moreove the hip . Cumulat the hip . The results of this robust pilot study conducted on patients after a recent stroke extends finding found among “healthy” older adults that virtual reality games placed greater demands and challenges on one's neuromuscular system compared to conventional balance programmes . Our resConventional balance training during rehabilitation led to poor engagement and lack of interest by patients due to repetitive practice of the same exercises , 33. HowThe finding of this pilot study could encourage physical therapist to introduce interactive virtual reality games during rehabilitation to those who have physical and cognitive abilities to take responsibility for their recovery. Properly selected virtual reality games can be socially engaging, entertaining, and readily accepted and enjoyed by most patients , 39. PhyMoreover, the games can be performed safely and effectively at any time and at any location including within and outside rehabilitation sessions with older adults , 28, thoWe found that participation in 20 minutes of interactive virtual reality balance related games integrated within a rehabilitation session frees up 33% of physical therapist's time. Physical therapist could utilise the time gain with the introduction of virtual reality games with other patients that require more intensive therapy. Interactive virtual reality games should be welcome within rehabilitation session as this pilot study found it improved efficacy and efficiency of busy and scarce physical therapy professionals.Limitations. The main limitation of this pilot study was the small sample size. The results could be considered to be false positive. Post hoc evaluation indicated that future studies should involve a minimum of 50 subjects in each group to have a large effect size. Moreover, the selection of patients in this study reflects those with moderate disability based on the Modified Rankin Scale. Thus, the results cannot be generalised to rehabilitation of all patients after stroke. Furthermore, this study did not evaluate the long-term effects of interactive virtual reality balance related games within rehabilitation. “Correct” motor performance could be achieved by introducing motion sensors especially during practice sessions outside therapy time. This is important as early stage rehabilitation should maximise neuroplasticity by facilitating return of “normal” motor recovery rather than compensatory movement patterns. Interactive virtual reality balance related games are a viable adjunct to include within conventional rehabilitation sessions without increasing treatment time or requiring more professional attention. It was well received by in-patients and resulted in improvement in balance. Well-designed games based on therapeutic principles will empower patients to be involved in their rehabilitation while freeing up scarce therapist's time. A larger scale randomised control trial is required to confirm our findings and further evaluate the long-term effects of introducing virtual reality games as self-initiated exercises within the rehabilitation protocol of patients after stroke.


It has been previously demonstrated that autoantibodies against the β1/β2-AR exist in the sera of some patients with heart failure (HF) and these autoantibodies display agonist-like effects. Our aim in this study was to observe whether autoantibodies against the β3-AR (β3-AR Abs) exist in the sera of patients with HF and to assess the effects of β3-AR Abs on rat model of pressure overload cardiomyopthy. In the present study, the level of β3-AR Abs in the sera of HF patients was screened by ELISA. β3-AR Abs from HF patients were administrated to male adult rats with abdominal aortic banding (AAB), and the cardiac function was measured by echocardiographic examination and hemodynamic studies. The biological effects of this autoantibody on cardiomyocytes were evaluated using a motion-edge detection system, intracellular calcium transient assay, and patch clamp techniques. Compared to healthy subjects, the frequency of occurrence and titer of β3-AR Abs in the sera of HF patients were greatly increased, and β3-AR Abs could prevent LV dilation and improve the cardiac function of rats with AAB. β3-AR Abs exhibited negative chronotropic and inotropic effects and were accompanied by a decreased intracellular Ca2+ transient and membrane L-type Ca2+ current in cardiomyocytes. Our results demonstrated the existence of β3-AR Abs in the sera of patients with HF and found that this autoantibody could alleviate the cardiac dysfunction induced by pressure-overload in AAB rats. β Heart failure (HF) is a life-threatening clinical condition in which the heart cannot pump enough blood to the rest of the body. The clinical symptoms of HF include water-sodium retention, decreased perfusion of peripheral tissues and organs, which are the common final phase of many cardiovascular diseases . Despite1- and β2- adrenoceptor (AR) have been detected in the sera of patients with chronic HF iT) and L-type Ca2+ current (ICa-L) of cardiomyocytes induced by β3-AR Abs. A representative [Ca2+]iT profile was shown in 3-AR Abs upon cardiomyocytes [Ca2+]iT. 3-AR Abs on [Ca2+]iT in isolated rat cardiomyocytes. Purified IgGs (0.1 μmol/L) from β3-AR Abs positive HF sera decreased the peak systolic [Ca2+]iT from control value of 48.54 ± 12.41% to 30.26 ± 3.34%  in isolated rat cardiomyocytes. This effect was comparable with that induced by β3-AR agonist BRL37344 . 3-AR Abs (0.1 µmol/L) on ventricular membrane L-type Ca2+ current (ICa-L). As shown in 3-AR Abs markedly decreased the peak inflow ICa-L from control value of 1467 ± 223 to 626 ± 138 pA . This effect was comparable to that observed with β3-AR agonist BRL37344 (0.1 µmol/L), which decreased peak inward ICa-L from control value of 1467 ± 223 to 574 ± 129 .To clarify the mechanisms underlying the negative inotropic effects of β3-AR Abs in patients with HF were much higher than that of healthy subjects, and 2) β3-AR Abs could ameliorate cardiac dysfunction in AAB rats, and its negative inotropic and chronotropic effects and inhibition of L-type calcium channels may be responsible for the protective effects.The novel findings in this study were that 1) the occurrence frequency and the OD value of β3-AR was used as the antigen to screen for β3-AR Abs in the sera from patients with HF and healthy control subjects. The reason for choosing the second extracellular loop of the receptor is that this loop has been shown to be highly antigenic, immunogenic and important for receptor function in many G-protein-coupled receptors iT and ICa-L in isolated cardiomyocytes. And the inhibitory effects of β3-AR Abs on ICa-L and [Ca2+]iT may be the molecular mechanisms that are responsible for its negative inotropic effects. In addition, our current experimental results demonstrated that the agonist-like effects of β3-AR Abs were mediated by β3-AR because the chronotropic and inotropic effects of β3-AR Abs were completely blocked by bupranolol, a nonselective β-AR antagonist and by β3-AR antigen peptide, but not by nadolol, a selective β1- and β2-AR antagonist. These results suggested that the cardioprotective role of β3-AR Abs in pressure-overload hypertrophy may be attributable to its negative chronotropic and negative and inhibition of L-type calcium channels which could reduce myocardial oxygen consumption and intracellular calcium overlod, hence may ameliorate the pressure overload-induced pathological remodeling and cardiac dysfunction.In the present study, we observed that administering the β1-AR (β1-AR Abs) and β3-AR (β3-AR Abs) exist in the sera of patients with HF and it is of significance to compare the role of β1-AR Abs and β3-AR Abs in the development of HF. In recent years, a large number of investigations have demonstrated the involvement of β1-AR Abs in the pathogenesis of HF [1-AR Abs would permanently overstimulate the β1-ARs which result in a large amount of energy consumption and finally lead to HF. In contrast to β1-AR, stimulation of β3-AR could induce a negative inotropic effect [3-AR is markedly up-regulated in failing heart [1-AR. Thus the β3-AR Abs may serve as a “brake” to protect the heart from catecholamine overstimulation by means of its stimulatory agonist-like activities on β3-ARs. In the current study, we confirmed that β3-AR Abs from patients with HF exerted a cardioprotective role in the rat failing heart induced by pressure overload. We observed that administering the β3-AR Abs to rats with AAB could prevent LV dilation and improve the cardiac function of rats. Our findings are partly supported by several recent reports that β3-AR agonists had cardioprotective effects in pressure overload hypertrophy and HF [3-AR showed exacerbated pathological remodeling and impaired cardiac function [Previous reports and the present study show that both autoantibodies against the βis of HF -8. Theseis of HF ,12. Hencc effect . Moreoveng heart -26 whichy and HF ,28 and lfunction .3-AR Abs had substantial cardioprotective effects in pressure overload hypertrophy, which may alleviate the development of HF. These findings provide new insights concerning the significance of β3-AR Abs in the pathogenesis of HF. In conclusion, we have shown that β3-AR Abs could improve heart function, the mechanisms require further research. Our work leaves some unanswered questions for future study. Although we observed that β


Pulmonary congestion and edema are the major life-threatening complications associated with HF. The purpose of this study is to explore the relationship between the anti-β3-AR autoantibody and the expression of β3-AR in the lungs and heart for both aged patients and rats with HF.Evidences suggest that β3-AR peptides served as the target antigens in ELISA were used to screen the anti-β3-AR autoantibody in aged patients and rats. Two aged rat models were constructed based on aortic banding and sham-operation. The expression of β3-AR mRNA and protein in the lung and heart was measured in intervention and non-intervention groups by Western blot analysis at the baseline, 5th, 7th, 9th and 11th week, respectively.Synthetic β3-AR autoantibody in aged patients and rats with HF were higher than those in the control group (p<0.05). The expression of β3-AR mRNA and protein in pulmonary tissues decreased continually from the 7th week (p<0.05), followed by HF observed during the 9th week. The expression of β3-AR in myocardial tissues continued to increase after the 9th week (p<0.05), and the expression of both β3-AR mRNA and protein in the BRL group [HF group with BRL37344 (4-[-[2-hydroxy-(3-chlorophenyl)ethyl-amino] phenoxyacetic acid) (a β3-AR agonist) injection] was positively correlated with BRL37344 when compared with non-BRL group (HF group without BRL37344 injection) (p<0.05).The frequency and titer of anti-β3-AR autoantibody was detected in aged patients and rats with HF. The expression of β3-AR mRNA and protein in pulmonary tissues decreased continually, and began earlier than in the heart, but its expression in myocardial tissues increased continually and could be further promoted by β3-AR agonist.Anti-β Stimulation to β1 and β2-AR produces positive chronoscopic and inotropic effects. However, the activation of β3-AR induces a decrease in the contractility of myocardium 3-AR is obviously coupled to a Gi/nitric oxide (NO) pathway and can reveal negative inotropic effects in ventricular endomyocardial biopsy samples of heart transplant recipients 3-AR and cardiac function warrants further investigation, in aged patients or rats with heart failure.Growing evidences suggest that the autoimmune mechanism may play a major role in heart failure observed in both rats and human patients 2-AR 2-AR agonist can improve pulmonary function in patients with congestive heart failure, thus suggesting the involvement of β2-AR 2-agonist can increase alveolar fluid clearance in rats with heart failure 1-AR and β3-AR) in pulmonary tissues have gained less attention. Recent research has demonstrated very low expression of β3-AR mRNA in the lungs, and the authors have suggested that the expression of β3-AR mRNA could probably be accounted for by the presence of fat 3-ARs in human bronchi 3-adrenergic agonists may also increase alveolar fluid clearance during hypoxic lung injury in rats, and accelerate the amelioration of pulmonary edema Pulmonary congestion and edema are the major life-threatening complications associated with heart failure. The β-AR is widely distributed in lungs, where the dominant subtype is β3-AR autoantibodies can be detected in the plasma of aged patients and rats with heart failure. 2) The abnormal expressions of β3-AR mRNA and protein, and changes in the levels of anti-β3-AR autoantibodies in pulmonary congestion or edema emerge prior to the appearance of heart failure in aged rats. 3) These latter changes may involve continuing deterioration during heart failure, and may further be altered by the introduction of the β3-AR agonist.The major objectives of this study are summarized by three hypotheses: 1) The anti-βp<0.01, 50.0% vs. 8.6%) during the postoperative period (0–11 weeks). The major causes of early death were over anesthesia or heart failure, especially in the Banded group. From the 5th week, the rats from the Banded group (n = 90) were divided randomly into two groups as the BRL group [heart failure group with a β3-AR agonist called BRL37344 (4-[-[2-hydroxy-(3-chlorophenyl)ethyl-amino] phenoxyacetic acid) injection] (n = 50) and the non-BRL group (heart failure group without BRL37344 injection) (n = 40). Mortality rates were different at various stages in the non-Banded and BRL groups from the 5th to 11th week. The accumulative mortality rate in the BRL group was significantly higher than that in the non-BRL group , and these results suggested that β3-AR agonist (BRL37344) might be associated with an increased risk of death in aged rats with heart failure.Mortality rate with Banded group was significantly higher than that in the Sham group . However, at the 9th week, left ventricular function was significantly impaired in the BRL group compared to the non-BRL group (p<0.01). Thus, left ventricular function was progressively damaged.The hemodynamic parameters are summarized in th week , which was obviously higher than 12.7% (mean antibody titer: 64.3±3.1) in aged control group.The frequencies of anti-β3-AR autoantibody in the Banded group increased significantly from the 5th week, with a high peak (from 20.4% and 28.3±1.4 to peak 41.4% and 82.3±3.2) at the 10th week, followed by a decrease after the 10th week, although these results were not observed in the Sham group (p<0.05). It was worth noting that the frequencies and mean titers of anti-β3-autoantibodies in the BRL group increased markedly at the 5th week, and reached a maximum at the 10th week (from 37.5% and 68.4±2.3 to peak 66.5% and 142.6±1.1) when compared with the Sham group (p<0.05) (Similar results were observed in aged rats with heart failure. The frequencies and mean titer of anti-β(p<0.05) .th week after operation) prior to the appearance of heart failure in aged rats (the 9th week after operation). During the 2–7th week after operation, no significant difference was observed in the alveolar space, lung interstice or peri-alveolar blood vessels in the BRL group, non-BRL group or Sham group. However, from the 5th to the 7th week after operation, the peri-alveolar blood vessels in the BRL group reveal slight dilation and blood stasis. Subsequently, during the 7–11th weeks after operation, lung lesions in the non-BRL group were gradually characterized by interstitial edema, acinar cell vacuolization, and infiltration of inflammatory and congestion edema   .3-AR mRNA and protein in both BRL and non-BRL groups exhibited a positive correlation with the severity of heart failure (p<0.001). More importantly, compared with the non-BRL group, the BRL group revealed a more obviously positive correlation with the severity of heart failure (p<0.01). These results suggested that β3-AR agonist might further activate the expression of β3-AR mRNA and protein in heart of the aged rats with heart failure  pathway in ventricular endomyocardial biopsy samples of heart transplant recipients. The NO production leads to an activation of soluble guanylyl cyclase leading to an increase in intracellular cGMP i/α inhibition of adenylate cyclase is theoretically considered because part of the negative inotropic effect of β3-AR agonists is insensitive to NOS inhibition. Therefore, β3-AR might activate growth or metabolic signaling(e.g. through activation of MAP kinases). Thus, future works should be performed to demonstrate these mechanisms in more models.Three subtypes of β-receptors are expressed in normal hearts at various levels. The expression levels of β3-AR agonist (BRL-37344) may further increase mortality rate in aged rats with heart failure.The model of ascending aorta banding-induced chronic heart failure has been widely used to produce a time-dependent impairment of cardiac function 1-AR, and increased expression of β2-AR On the other hand, we have used male rats in the experiment because estrogen plays a cardioprotective role in female rat hearts. The effects of estrogen during ischaemia-reperfusion injury are associated with the decreased cardiomyocyte contraction and expression of β3-AR autoantibody in patients with heart failure are remarkably higher than those in the control group 3-AR autoantibody when compared with the normal control. Similarly, the positive rate in aged rats is positively correlated with the severity of heart failure. These results strongly suggest that the plasma level of anti-β3-AR autoantibody can act as a clinical reference index of the prognosis in patients with heart failure, especially aged patients.In recent years, several studies have reported that the positive rates of anti-β3-AR mRNA and protein reveal gradual decrease in aged rats with heart failure, exhibiting a negative correlation with the severity of heart failure. Additionally, β3-AR agonist (BRL 37344) can further inhibit the expression of β3-AR mRNA and protein in pulmonary tissues. Moreover, it has been reported that β3-AR agonist can increase alveolar fluid clearance during lung injury and accelerate the amelioration of pulmonary edema. We therefore speculate that, with the anti-β3-AR autoantibody, similar to a β3-AR agonist, these protective effects may greatly decrease or even disappear with gradually decreasing β3-AR expression in pulmonary tissues. Furthermore, β3-AR reveals a significant decrease in density and activity, thus resulting in a significant pulmonary congestion or edema.One of the most important findings from the present study is that the expressions of lung β3-AR may play a safety role during intense adrenergic stimulation such as stress and strong physical effort. Thus, over-expression of β3-AR may protect against cardiac damage induced by the high catecholamine level present in heart failure, particularly during the early stages. However, during the worst stages of heart failure, the down-regulation of cardiac β1-AR and β2-AR, and concomitant decrease in adenylyl cyclase activity result in a significant enhancement of cAMP-mediated negative inotropic effects, thereby enhancing myocardial depression 3-AR expression is up-regulated 2–3 times faster in the failing heart when compared with the non-failing heart 3-AR agonist may therefore exacerbate cardiac dysfunction and profoundly aggravate systolic and diastolic dysfunction.Multiple studies have established that in normal heart tissues, the negative inotropic effect induced by β3-AR expression in the heart. At the same time, the serum level of anti-β3-AR autoantibodies is associated with a 3- to 4-fold increase. Contrary to the results observed in the heart, a gradual decrease in the expression of β3-AR in the lung is observed. These data suggest that in the case of lung, the anti-β3-AR autoantibody, as β3-AR agonists, can increase continuous negative inotropic effects that can further inhibit the heart function, and at the same time, due to the significant increase in the expression of pulmonary β3-AR, the action of alveolar fluid clearance may be inhibited, resulting in increasingly impaired heart function. We therefore conclude that the serum level of anti-β3-AR autoantibody can act as a reference index of the prognosis in aged patients with heart failure, and indirectly reflect the expression of β3-AR mRNA and protein in the lungs and heart at different stages.In our aged rat model, increasing impairment of heart function is associated with a 3- to 4-fold increase in β3-AR mRNA and protein in pulmonary tissues reveals a gradual decrease, but the expression in myocardial tissues reveals an increase and can further be promoted by β3-AR agonist. The anti-β3-AR autoantibodies can increase gradually as the progression of heart failure. The serum levels of anti-β3-AR autoantibody may then act as a reference index, indirectly reflecting the expression of β3-AR mRNA and protein in lungs and heart at different stages.The ascending aorta banding-induced heart failure model in aged rats is an accurate and reliable model, but is associated with high mortality rate. During the development of heart failure, pathological changes of lung injury preceded myocardial injury. The expression of βThe present human study complied with the Declaration of Helsinki and was approved by the Ethics Committee and the Prescription and Therapeutic Committee (10-S-6) of Capital Medical University Beijing Chao-yang Hospital. All subjects provided written informed consented prior to admission into the study.All animal experiments were approved by the Institutional Animal Care and Use Committee of Capital Medical University (10-A-35). All surgeries were performed under isoflurane/oxygen anesthesia and all efforts were made to minimize suffering including the use of buprenorphine for pain management after surgery.3-AR autoantibody. Sixty-nine heart failure patients were selected for this study. Criteria for patient selection included: aged 65 years or older defined according to New York Heart Association (NYHA) functional classes II-IV; less than 45% left ventricular ejection fractions on echocardiograms; no apparent severe hepatic and renal dysfunction; no previous medication of ACEI, diuretic and/or digoxin. The normal control groups (NC) were comprised of 27 healthy individuals exhibiting normal clinical, ECG and echocardiography examinations, and matched ages and sexes with the heart failure patients.Outpatients from Beijing Chao-yang Hospital of China were the source of the samples collected for the determination the anti-β3-AR-selective agonist (BRL37344 (4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl) amino] propyl] phenoxyacetic acid) (Sigma-Aldrich Co. USA) at a dose of 0.4 nmol·kg−1·min−1 via a vena caudal injection sustained for 10 minutes, twice weekly for 6 weeks, while an equivalent dose of normal saline was administrated in the non-BRL group.Healthy 20 month-old male Wistar rats, weighing 500 to 550 g, were selected for this study. Animals were classified randomly into 2 experimental groups and a control group. Experimental animals (n = 140) were subjected to aorta banding, in which the ascending aorta was banded (steel wire area/ascending aorta area = 75%) according to the methods of Brown, et al max and dP/dtmin) by opened thorax. All pressures were measured with a pressure transducer connected to a polygraph recorder , stored, and displayed on a personal computer. The lung and heart tissues were rapidly excised and frozen in liquid nitrogen, and then stored at −80°C until use.Eight animals were anesthetized with urethane . A cannula connected to a pressure transducer was inserted along the carotid artery into the left ventricle to measure the following primary and derived variables: heart rate, left ventricle end-systolic pressure, left ventricle end-diastolic pressure and the maximal rate of rise and decline in left ventricle pressure  was calculated.The lung and heart were filled with 10% formalin for fixation, and coated in gauze for histopathological examination. All tissue specimens were cut into 5-mm-thick cross-sections, fixed in 10% formalin, dehydrated, and embedded in paraffin. Slices with 5 µm in thickness were obtained from the sections and stained with hematoxylin-eosin. The tissues were observed with a Brightfield microscope under 40× magnifications .th, 6th,7th, 8th,9th, 10th and 11th week after treatment. The sera were separated by centrifugation  for 10 min and stored at −80°C until needed for assay.Two milliliters of blood were withdrawn from the antecubital vein in patients with heart failure and in the control cases. Throughout the experiment of aged rats, 2 mL of blood samples were taken from the carotid arteries of subjects from all groups. Samples were collected before treatment, and at the 53-AR (residues176–202: Q-W-W-R-V-G-A-D-A-E-A-Q-R-C-H-S-N-P-R-C-C-A-F-A-S-N-M-C) were synthesized by the Biological Institute of CAMS &PUMC, Beijing, using the solid phase method of Merrifield. The purity of these peptides was evaluated by HPLC on a Vydac C−18 column and by amino acid analysis on an automated amino acid analyzer . The peptides in rats were nearly identical, except for residues shaded in the following sequence of β3-AR (residues173–199:Q-W-W-R-V-G-A-D-A-E-A-Q-E-C-H-S-N-P-R-C-C-S-F-A-S-N-M-C).The peptides with terminal cysteines corresponding to the sequence of the second extracellular loop of the human β3-AR autoantibody was determined by enzyme-linked immunosorbent assays (ELISA). Both the study population and rats were defined as being positive or negative based on the presence or absence of anti-β3-AR autoantibody. The ELISA protocol as described by Fu, et al. was used to screen for the presence of autoantibodies Anti-β3-AR by PCR was completed in a 50-µL reaction mixture containing 100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl2, 200 µM dNTPs, 20 pmol each of the primers, 1 unit of Taq polymerase, and 2 µL of DNA template. Amplification was performed with a thermal cycler  using the following parameters: initial denaturation at 95°C for 3 min; denaturation at 94°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 90 s for 40 cycles; and a final extension at 72°C for 10 min. For the β-actin reaction, a 50-µL reaction mixture containing 100 mM Tris-HCl (pH 8.3), 500 mM KCl, 200 µM dNTPs, 20 pmol each of the primers, 15 mM MgCl2 and 1 unit of Taq polymerase, was used. The conditions for the reaction were as follows: initial denaturation at 95°C for 3 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 51°C for 30 s, and extension at 72°C for 90 s; and a final extension at 72°C for 10 min. PCR products were separated using 1% agarose gel and visualized on a gel documentation system  after staining with ethidium bromide.Amplification of β3-AR . The primary antibodies were diluted in TBS-T . Either alkaline phosphatase conjugated goat anti-mouse (GAPDH) or goat anti-rabbit (β3-AR) antibody was diluted to 1:3,000 in TBS-T and used as a secondary antibody with 1 h incubation. The bands on the blots were visualized using western blue  and were semi-quantified using a computer image analysis system. The expression level of β3-AR protein was normalized to GAPDH.Lung and cardiac tissues were homogenized in ice-cold homogenization buffer. Total protein was isolated from tissues and the concentration was determined using the Bradford method. Crude protein extracts (80 µg) were separated on a 10% SDS-polyacrylamide gel and then transferred to nitrocellulose membranes. The membranes were blocked for 1 h at room temperature in TBS-T containing 5% non-fat milk, and probed with primary antibodies against GAPDH and βt-test. All other data were evaluated by analysis of variance. In all cases, a statistically significant difference was considered at p<0.05. An anti-β3-AR positive score were defined as a ratio [(sample OD - blank OD)/(negative contrast OD - blank OD)] of ≥2.1. Analyses were performed with the GraphPad Prism 4.0 software package .All data, with the exception of antibody titers, are expressed as mean value ± SEM. Antibody titers reported as geometric means and differences between data sets were evaluated by unpaired Student’s


GNMT is regulated by androgen and to map its AREs (androgen response elements). Real-time PCR analyses showed that R1881, a synthetic AR (androgen receptor) agonist induced GNMT expression in AR-positive LNCaP cells, but not in AR-negative DU145 cells. In silico prediction showed that there are four putative AREs in GNMT-ARE1, ARE2 and ARE3 are located in the intron 1 and ARE4 is in the intron 2. Consensus ARE motif deduced from published AREs was used to identify the fifth ARE-ARE5 in the coding region of exon 1. Luciferase reporter assay found that only ARE5 mediated the transcriptional activation of R1881. ARE3 overlaps with a YY1 [Yin and Yang 1 ] that was further confirmed by antibody supershift and ChIP (chromatin immunoprecipitation) assays. EMSA (electrophoretic mobility shift assay) and ChIP assay confirmed that AR interacts with ARE5 in vitro and in vivo. In summary, GNMT is an AR-targeted gene with its functional ARE located at +19/+33 of the first exon. These results are valuable for the study of the influence of androgen on the gene expression of GNMT especially in the pathogenesis of cancer.Androgen plays an important role in the pathogenesis of PCa (prostate cancer). Previously, we identified GNMT (glycine N-methyltransferase) as a tumour susceptibility gene and characterized its promoter region. Besides, its enzymatic product-sarcosine has been recognized as a marker for prognosis of PCa. The goals of this study were to determine whether  PCa (prostate cancer) is the third most common cancer for men throughout the planet, and the most common among men in North America, Europe and some parts of Africa . BesidesGNMT gene primarily expresses in the liver, pancreas and prostate , and COS-1 (a green monkey kidney cell line). For the hormone-treatment experiment, we replaced FBS with CS  FBS in steroid-depleted cell culture media.CT (threshold cycle) values were exported into EXCEL worksheets for analysis. Comparative CT methods were used to determine relative gene expression folds to β-actin, TBP or GAPDH, respectively.RT–PCR and real-time PCR were performed as described by Lee et al. previously . The folGNMT and a luciferase gene. Putative AREs were identified using MatInspector program (http://www.genomatix.de/). Based on the in silico screening, four putative AREs were identified: three AREs (ARE1-3) are located in the intron 1 of GNMT their locations were nucleotide numbers +266/+280, +379/+393 and +1114/+1128 of GNMT gene; another ARE (ARE4) is located in the intron 2 (+1788/+1802). The fifth putative ARE–ARE5 was identified by the investigator using a consensus sequence-nGnACnnnnnGTnCn deduced from those confirmed AREs published previously  for 30 min at room temperature (25°C). For our competition experiments, 50- and 100-folds of unlabelled double-stranded oligonucleotides were incubated for 15 min prior to adding the labelled probes. Antibodies to AR (sc-815) and YY1 transcription factor (sc-1703) were purchased from Santa Cruz Biotechnology. Complexes were separated on 4% (w/v) non-denaturing polyacrylamide gels, which were dried and analysed using autoradiography.Detailed procedures of EMSA have been described previously . In brier conARE  and 5′-Cr conARE . OligonuLNCaP cells were cross-linked with 1% (v/v) formaldehyde for 20 min at room temperature and the chromatins were obtained through sonication. Immunoprecipitation was performed with anti-AR antibody (sc-816), anti-YY1 antibody (sc-1703) or normal rabbit IgG at 4°C overnight. Precipitates were washed and the samples were extracted with elution buffer and heated to 65°C to reverse the crosslink reaction. Subsequently, the DNA fragments were purified with phenol/chloroform. For ChIP assay with anti-AR, a pair of PCR primers was designed to span the putative AREs of GNMT exon 1: forward primer (5′-GGCTGCCAGCAGTGCTTATG); reverse primer (5′-CGATATACAGCTGCCACACG). PCR products were run on 2% (w/v) agarose gel and stained with ethidium bromide for visualization. For ChIP assay with anti-YY1, the immunoprecipitated DNA was analysed by real-time PCR as described above with two primer sets: p1 forward (5′-GGCGCGTGTCACCATGTCCC), p1 reverse (5′-CCCAGCGAAGGAAGGCATCA); p2 forward (5′-CGCCGTGTTAGCCAGGATGGT), p2 reverse (5′-GAGAGACAGACAAGCTGAGGCT).To investigate the relationship between androgen stimulation and GNMT expression, LNCaP cells that express AR were treated with R1881 for 24 h. Then, the GNMT mRNA expression levels were analysed using real-time PCR. The results showed that compared with the ethanol solvent control, GNMT mRNA expression levels in LNCaP cells were induced by R1881 in a dose-dependent manner  A. Time-cin silico analyses with MatInspector program. The nucleotide sequences subject to the analysis spanning about 6.4 kb upstream and 3.7 kb downstream of the transcriptional start site of GNMT. The results showed that there are three putative AREs located in the intron 1 of GNMT: ARE1 (GTCTCAgccTGTACT) at +266/+280; ARE2 (AGAACAggcACTGCG) at +379/+393; ARE3 (AAACCAccaTGTTCT) at +1114/+1128. In addition, another ARE was found in the intron 2: ARE4 (GTGGCAtctTGTCCC) at +1788/+1802  deduced from those AREs published previously. A putative ARE  was located in the exon 1 coding region of GNMT . As shown in GNMT gene.Finally, we employed ChIP assay with PCR to confirm the interaction between AR and ARE5 GNMT through the AR signalling pathway. Our data showed that the mRNA level of GNMT in LNCaP PCa cells was induced by androgen agonist-R1881 in a time- and concentration-dependent manner  via separate YY1 and NF-Y binding sites [GNMT expression in a regular or AR-induced manner.It is important to note that in this study, we found a motif at +1118/+1126 in the intron 1 of  in vivo . Overexpng sites . Alternang sites . In adding sites . TherefoIt has been reported that activation of the PI3K (phosphoinositide 3-kinase)-PKB  pathway promotes AR ubiquitylation and leads to AR degradation via a proteasome-dependent pathway . PreviouGNMT is a direct AR target gene in PCa, we suspect that AR signalling may also play an important role in GNMT regulation in liver cancer. Vitamin A and its derivatives are known to induce hepatic GNMT and DNA hypomethylation in rats [GNMT is a susceptibility gene for both HCC and prostatic cancer ,11. It h in rats ,42. ReceIn conclusion, we found that GNMT is an AR-targeted gene with its functional ARE located at +19/+33 of the first exon in this study. These results are valuable for the study of the influence of androgen on the gene expression of GNMT especially in the pathogenesis of PCa and HCC.