Previous work has demonstrated that membrane lipid rafts associated with key signalling proteins mediate growth and survival signalling pathways in prostate cancer cells.During androgen ablation prostate cancer cells' growth and survival become independent of normal regulatory mechanisms. These androgen-independent cells acquire the remarkable ability to adapt to the surrounding microenvironment whose factors, such as neurotransmitters, influence their survival. Although findings are becoming evident about the expression of α1A-adrenoceptor we explored its presence by a biochemical approach in purified detergent resistant membrane fractions of the androgen-independent prostate cancer cell line DU145. Electron microscopy observations demonstrated the colocalisation of the α1A-adrenoceptor with caveolin-1, the major protein component of caveolae. In addition, we showed that agonist stimulation of the α1A-adrenoceptor induced resistance to thapsigargin-induced apoptosis and that caveolin-1 was necessary for this process. Further, immunohistofluorescence revealed the relation between high levels of α1A-adrenoceptor and caveolin-1 expression with advanced stage prostate cancer. We also show by immunoblotting that the TG-induced apoptosis resistance described in DU145 cells is mediated by extracellular signal-regulated kinases (ERK).In order to analyze the membrane topology of the α1A-adrenoceptor stimulation in androgen-independent prostate cancer cells via caveolae constitutes one of the mechanisms contributing to their protection from TG-induced apoptosis.In conclusion, we propose that α The α1A-AR subtype, in particular, is found in smooth muscle cells but its expression has also been described in epithelial cells 1A-AR is a member of the superfamily of G-protein coupled receptors (GPCR) mediating actions of the previously mentioned catecholamines in a variety of cells Prostate cancer is one of the most common forms of cancer in men and the second cause of cancer death in industrialized countries 1-AR antagonists are already used for the clinical treatment of benign prostate hyperplasia (BPH) 1-AR present in prostate smooth muscle cells 1-AR antagonists such as doxazosin on long-term BPH treatment. These agents have been demonstrated to inhibit prostate growth by inducing apoptosis in stromal and epithelial cells and are emerging as potential therapeutic regimens for the prevention and treatment of androgen-independent PCa 1A-AR agonist, stimulates their proliferation 1A-AR in androgen-independent PCa cells has yet to be established.α1-AR as well as the molecules involved in its signal transduction pathway are accumulated in caveolae, a subclass of membrane microdomains It has been described that the signalling and trafficking of several GPCR are regulated by specialized plasma membrane domains known as lipid rafts 1A-AR in androgen-independent epithelial cells and PCa progression. In this regard, it is tempting to link the presence of α1A-AR and cav-1 and to hypothesize that the α1A-AR could mediate via caveolae its functional effects on growth or survival of advanced stage PCa cells.To date, not much is known about the mechanisms underlying neurotransmitters involvement in the survival of PCa cells, let-alone the role of α1A-AR in androgen-independent PCa cells. Here, we investigate the presence of α1A-AR in caveolae of DU145 cells, an androgen-independent PCa cell line derived from brain metastasis. We analyze the consequence of α1A-AR stimulation by PHE on the receptor and cav-1 membrane distribution as well as its effect on the lipid composition of membrane raft fractions purified from DU145 cells. Furthermore, we describe the effect of PHE in the apoptosis resistance of these cells through activation of ERK and our results strongly imply the involvement of caveolae in this signalling pathway. Finally, by immunohistofluorescence and RT-PCR, we observe a positive correlation of α1A-AR and cav-1 expression and advanced stage PCa. The presence of α1A-AR-rich caveolae could therefore contribute to the generalized apoptosis resistance characterizing androgen-independent prostatic tissue.The objective of our work was therefore to explore the role of the α2-95% air atmosphere. These cells constitute a suitable model for brain metastatic cancer cells present in hormone-refractory prostate cancer.The androgen-independent human prostate cancer cell line DU145, obtained from the American Type Culture Collection, was maintained in culture in RPMI 1640 medium  supplemented with 10% (v/v) FCS , 5 mM L-glutamine and 2 mM kanamycin (Sigma). Cells were routinely grown in 50 ml flasks  at 37°C in a humidified 5% CO1A-AR  were transiently transfected (50 nM) with HiPerfect Transfection Reagent (Qiagen) in DU145 cells. Control siRNA (siCTL) experiments were performed by transfecting siRNA against Luciferase.Two ready-to-use siRNAs against α5′-CUUACGCUGAGUACUUCGA-3′. siADR1A-1 (mix of): 5′-CAGGAAAGAUGCAGAGGA-3′ and 5′-UUCCUCUGCAUCUUUCCU-3′. siADR1A-2 (mix of): 5′-GCGUCUACGUGGUGGCCA-3′ and 5′-UUGGCCACCACGUAGACG-3′.siLuciferase (siCTL) : CTGGAATAAGTTCAAATTCTT 2121 3′utr) (DUshcav-1)  using Nucleofector, as recommended by the manufacturer . We transfected 2×106 trypsin-treated DU145 cells with 3 µg of vector and then used the transfected cells to seed a 24 well plate (Nunc) at very low density. Clones underexpressing cav-1 were selected on the basis of their antibiotic resistance to puromycine and validated by Western Blot.Stable cell lines were obtained by transfecting DU145 cells with a Psuper vector encoding short hairpin RNA (shRNA) against cav-1 (sequence: 1A adrenoceptor (Santa Cruz Biotechnology), (1∶100) rabbit anti-caveolin-1 (Santa Cruz Biotechnology), (1∶50) mouse anti-caveolin-1 (BD transduction Laboratories), (1∶400) mouse anti-β-actin (Sigma), (1∶200) anti-calnexin , (1∶1000) mouse anti-cytokeratin 18 , (1∶1000) rabbit anti-PARP , (1∶100) mouse anti-bax (6A7) (Santa Cruz Biotechnology) recognizing an exposed epitope of bax in the activated conformation, (1∶1000) rabbit anti-procaspase 3 (UPSTATE), (1∶1000) anti-phospho-p44/42 MAP Kinase and (1∶1000) anti-p44/42 MAP Kinase . Secondary antibodies used for IB were: (1∶10000) horseradish peroxidase-linked anti-mouse or anti-rabbit (Chemicon). For IF, we used (1∶1000) Alexa Fluor® 488-labelled and (1∶2000) 546-labelled secondary antibodies (Molecular Probes).Primary antibodies used for immunofluorescence (IF) microscopy and immunoblotting (IB) were: (1∶100) rabbit anti-α2, 1.8; MgCl2, 1.2; NaHCO3, 5; NaH2PO4, 1; HEPES, 20; pH 7.3. All chemical products were provided by Sigma except TG (Alomone) and PD98059 .Cells were treated with 10 µM L-Phenylephrine Hydrochloride (PHE), 1 µM prazosin (PRA) and 10 µM PD98059 for three days (renewed every 24 h) and 10 µM Thapsigargin (TG) for 48 hours in RPMI medium. Short term PHE treatments were realized in a solution containing (mM): NaCl, 116; KCl, 5.6; CaClCells were grown in three 60-mm dishes per condition and drugs were applied as described above. After treatments, cells were trypsinized, harvested and resuspended in 0.2 ml sterile PBS. 1 ml of cold 70% ethanol was added onto cell suspensions while vortexing. Samples were centrifuged, washed in sterile PBS and then incubated with ribonuclease (2 µg/ml) for 15 min. Propidium iodide  was then added and allowed to incubate for an additional 30 min. DNA content was measured by exciting propidium iodide at 488 nm and measuring the emission at 520 nm (FL3) using a flow cytometer (Beckman coulter Epics XL4-MCL with Expo32 acquisition). Experiments were repeated four times.g for 10 min at 4 °C and the amount of protein was assessed by the BCA method . Equal amounts of proteins were subjected to SDS/PAGE (16% gels). Finally, the proteins were transferred on to nitrocellulose membranes using a semi-dry electro-blotter (Bio-Rad). After 1 h saturation in 5% non-fat milk  only for anti-phospho-p44/42 MAPK), membranes were incubated overnight with diluted primary antibodies (see “Antibodies and Reagents”). The membranes were then washed (3×10 min) with a TNT buffer  and treated with the corresponding horseradish peroxidase-linked secondary antibodies , (1∶10000) diluted in TNT/milk (or TNT/BSA) for 1 h at room temperature. After several washes in TNT buffer, the membranes were processed for chemiluminescent detection using the Super Signal West Dura chemiluminescent substrate (Pierce) according to the manufacturer's instructions. The membranes were finally exposed to X-Omat AR films . The intensity of the signals was evaluated by densitometry and semi-quantified using the ratio for each sample between the intensity of protein of interest divided by the actin or calnexin intensity. Each experiment was repeated at least twice.The cell culture medium was discarded and flasks were washed with iced PBS. Cellular proteins were then extracted using RIPA buffer  with 5 mM EDTA and anti-proteases cocktail  for 30 min on ice. After scraping, any insoluble material was removed by centrifugation at 30000×g. The supernatant corresponding to crude lipid raft extract was isolated and 0.2% Triton X-100 was added. A discontinuous 5-step Optiprep® (Axis-Shield PoC AS) gradient was formed with buffer D (buffer B+1% Triton X-100) (w/v) in order to obtain 1.66 ml of 35%; 2.5 ml of 20%; 2.5 ml of 15%; 2.5 ml 10%; 0.83 ml of 5%. Crude lipid raft extract was made dense with sucrose (230 mg for 0.5 ml of lysate) and laid at the interface between gradients 35% and 20%. Ultracentrifugation at 165400×g for 4 h at 4°C in 50 Ti rotor (Beckman Coulter) was followed by collection from the bottom up of 1.1 ml fractions. 1.1 ml of 20% (v/v) TCA (Trichloroacetic Acid) was added to each fraction in order to precipitate proteins. Tubes kept on ice for 20 min were centrifuged at 10000×g at 4°C for 10 min. The precipitate was washed with methanol, centrifuged then re-suspended in 4% (v/v) SDS. Since samples were not highly concentrated in proteins and detection by classical western blot was difficult to obtain, samples were loaded on nitrocellulose membrane in a 96-well Dot-Blot unit according to the manufacturer's instructions (Bio-Rad). Briefly, the ten fractions were spotted onto nitrocellulose membrane in series. When dry, the membrane was incubated in blocking solution for 1 h (TNT/milk) then hybridized with the desired antibodies following classical IB procedure as described above (see “Western Blot”). Precautions were accordingly taken in keeping some important factors constant for the treated and non-treated conditions. The cell density used for the experiments was constant and when cold detergent extraction was carried out both the concentration of the detergent and the ratio of cell number/detergent concentration were the same. Each well was loaded with 25 µl from each fraction. This standardized method was repeated at least three times and was used to evaluate changes in the partitioning of a target molecule in DRM, avoiding false estimations when based on the relative amounts with regard to all other molecules present.Cells were rinsed, scrapped, centrifuged and the supernatant was removed. The pellet was resuspended with isolation buffer B (according to Axis-Shield S33 Application Sheet) and homogenized with a Dounce homogenizer (Kontes) of 15 µm pestle clearance then centrifuged for 10 min at 1000×Lipids were extracted according to the method of Folch et al. Extraction and saponification were performed according to the previously described procedure The above quantification protocols for lipids and cholesterol were repeated three times for three individual experiments in non-treated (control) and treated DU145 cells with PHE for 10 min. As the amounts of lipids and cholesterol quantified in each fraction (ng/ml or µg/ml) varied slightly among experiments, results were normalised by determining mean values for the three experiments represented by the percentage of lipid and cholesterol in each fraction in control conditions compared to PHE-treated conditions.This method was based on a published protocol For transmission electron microscopy, Cells were fixed in 2.5% glutaraldehyde prepared in 0.1 M cacodylate buffer and post-fixed in 1% osmium tetroxide in the same buffer. After acetonitril dehydration, the pellet was embedded in Epon. Serial thin sections (90 nm) were cut using a Reichert Ultracut E ultramicrotome and collected on 150 mesh hexagonal barred copper grids. After staining with 2% uranyl acetate prepared in 50% ethanol and incubation with a lead citrate solution, sections were observed on a Hitachi H-600 transmission electron microscope.Resection BPH specimens from human prostate frozen in liquid nitrogen-cooled isopentane and kept in “Tissue-Tek®” at −80°C before 10 µm sections were prepared at −20°C with a cryostat, mounted on glass slides and proceeded to IF study. Normal and cancerous prostate tissues were supplied by tissue-array slides (SuperBioChips Laboratories), deparaffinized according to manufacturer's indications (Cliniscience) before preparation for IF according to 1A-AR isoform 1 amplicon was amplified with 5′-AGACCAATCCTCCTGTACCAC-3′ (forward) and 5′-CTCTGCATCTTTCATGTCCTAG-3′ (reverse). The 220 pb cav-1 amplicon was amplified with 5′-AGTGCTCCTGTTCTCCCTTC-3′ (forward) and 5′-CTTGTCGATGGCTTCCTTCAC-3′ (reverse) (Eurogentec). The 236 pb GAPDH amplicon internal control was amplified with 5′-TTCACCACCATGGAGAAGGC-3′ (forward) and 5′-GGCATGGACTGTGGTCATGA-3′ (reverse). The 241 pb cytokeratin 18 amplicon was amplified with 5′-TGAGTCAGAGCTGGCACAGA-3′ (forward) and 5′-TGGTGTCATTGGTCTCAGACA-3′ (reverse). Finally, the 210 pb cytokeratin 14 amplicon was amplified with 5′-TGCGAGATGGAGCAGCAGAA-3′ (forward) and 5′-TGCCATCGTGCACATCCATGA-3′ (reverse). α1A-AR, cav-1, cytokeratin 14 and 18 mRNA expressions were validated by gel density analysis by using GAPDH mRNA as internal control.Reverse transcription-PCR was carried out as previously described Human BPH biopsies were obtained from consenting patients following the local ethical considerations. All experiments involving patient tissues were carried out under approval number ‘CP 01/33’, issued by the ‘Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale de Lille'.t tests (for comparing two groups) or analysis of variance tests followed by Dunnett (for multiple control versus test comparisons). Differences were considered significant when p<0.05 (*), p <0.01 (**) and p <0.001 (***).1A-AR with cav-1 1A-AR signalling is known to localize in caveolae 1A-AR, we used the “rip-off” technique developed in adherent cells allowing the isolation and observation by electron microscopy of large areas of the plasma membrane 1A-AR immunogold staining  colocalizes with cav-1  in plasma membrane microdomains of about 50–100 nm . Dot blot analysis of DRM fractions revealed distribution of cav-1 in low density gradient (5%–20%) fractions 1–6 and α1A-AR in fractions 1 and 3–6 in control (CTL) conditions. After 10 minutes treatment by 10 µM PHE cav-1 distribution was observed in low density gradient (5%–10%) fractions 1–5 and α1A-AR was now detected in all fractions 1–6 (including low density fraction 2) (q11 (an α1A-AR effector) contents showed a shift from fractions 4–5 to fractions 1 to 5 after PHE treatment and remain present in higher density fractions, suggesting that receptor activation does not involve all Gαq11 proteins present at the membrane level. We also assessed the purinergic receptor P2Y2 used as a negative control for the specificity of modification in protein distribution induced by PHE. α1A-AR and P2Y2 are both GPCR sharing similar signalling pathways. P2Y2 is located in the same fractions whether DU145 cells were treated or not , sphingomyelin (SM), phosphatidylcholine (PC) and cholesterol 1A-AR signalling, we explored the effect of PHE stimulation on the DU145 cell surface morphology  simultaneously with PHE . Our results propose that α1A-AR may associate with cav-1 in resting cells and upon receptor stimulation clustering of cav-1-rich units form α1A-AR-containing structural surface caveolae.Caveolae are formed by the polymerisation of caveolins leading to the clustering and invagination of existing cholesterol-sphingolipid rich domains in the cell plasma membrane mulation . The sammulation , d. Heremulation , b as comulation , a. Furtwith PHE , c. Cav-1A-AR signalling as it is further explained.We first quantified cav-1 in DU145 non-transfected cells (DU145 WT) and in stably transfected DU145 cells with either an empty plasmid (DUshCTL) or a plasmid expressing shRNA against cav-1 , a. Thes1A-AR signalling in the survival of DU145 cells, we induced cell apoptosis by a 48 h treatment with 10 µM thapsigargin (TG), a known inhibitor of sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). As a consequence of inhibiting SERCA, TG induces a substantial depletion of calcium stores, creating perturbations in cellular calcium known as reticular stress coupled with the ability to engage mitochondrial-dependent apoptotic pathways In order to study the role of α1-AR specificity of the apoptosis resistance induced by PHE, PRA was simultaneously used with PHE. Indeed, the resistance to TG-induced apoptosis was abolished when 1 µM PRA was added to the pretreatment solution (A three-day pre-treatment by 10 µM PHE prior to TG treatment induced a significant 25% decrease in the number of DU145 WT cells (white columns) in SubG1 phase as well as a 20% decrease in DUshCTL (black columns) , b. In osolution , D. We tsolution . The preWe then explored the expression of target proteins known to regulate mechanisms of apoptosis induction or resistance such as the caspases and the poly (ADP-ribose) polymerase (PARP) by western blot analysis , a. Scanin vivo1-AR.PARP, a 116 kD nuclear poly (ADP-ribose) polymerase is one of the main cleavage targets of caspase-3 1-AR. DAPI-stained nuclei are indicative of the number of cells present in the observation field. The absence of apoptotic figures (chromatin condensation and nuclear fragmentation) in cells expressing activated Bax is expectable as Bax activation is an early-stage apoptotic process as opposed to nuclear fragmentation.Bax is a 23 kD pro-apoptotic protein, member of the Bcl-2 family. The critical events in the activation process of Bax are: its translocation to mitochondria and its N-terminal conformational change closely coupled to mitochondrial membrane insertion and oligomerisation 1A-AR is abundant in the fibromuscular tissue in normal and hyperplastic prostates 1A-AR and cav-1 expression with established features of PCa as well as BPH in comparison to normal prostate, we performed α1A-AR and cav-1 immunostaining on tissue microarrays containing specimen cores from 40 patients. Cytokeratin 18 immunostaining was used as a label of apical epithelial cells present in acini. Normal tissue regions from 9 of these patients were present on these slides. The figures shown here are representative of all samples observed. We first confirmed the specificity of the α1A-AR antibody by the use of two siRNA against α1A-AR (siADR1A-1 and siADR1A-2)  , a. The 1A-AR and cav-1 in a series of cancerous and hyperplastic human prostate samples. We analyzed six PCa samples obtained from patients in hormone-refractory phase and characterized as androgen-independent and six BPH samples  we underlined significant elevated mRNA levels of α1A-AR and cav-1 in androgen-independent cancer compared to BPH. We propose that their simultaneous expression could account in part for the α1A-AR signalling via caveolae in androgen-independent PCa epithelial cells.Finally, we were interested in assessing the mRNA levels of α samples , a. Relaromising , b. We f1B-AR activates ERK in submandibular gland cells via a mechanism dependent of membrane microdomain integrity It is now well established that ERKs are overexpressed in advanced stage prostate cancer and play a significant role in prostate tumorigenesis Various locally produced and circulating factors maintain prostate cancer growth by acting through cellular receptors. Increasing evidence supports the involvement of GPCR in neoplastic transformation of the prostate 1A-AR in human prostate pathology 1A-AR and its effector proteins are differentially distributed in surface caveolae of cardiac cells A growing number of recent data has shown the involvement of α1A-AR in androgen-independent PCa epithelial cells and its functional role in the proliferation and survival of these cells remain unknown. The present study is the first to demonstrate the localization of α1A-AR in caveolae of the androgen-independent PCa epithelial cells DU145. Our results from the exploration of the lipid-protein contents of purified DRM from these cells were particularly revealing. Noticeably, we provide evidence of significant increase in DRM lipids upon agonist stimulation of the α1A-AR. We propose that this elevated content of raft-specific lipids consequently leads to alterations of DRM density as it is known that high lipid composition causes floating of membranes to lower density fractions after centrifugation 1A-AR and cav-1 content is accordingly distributed toward lower density fractions.Despite the above findings, the precise surface localization of the α1A-AR stimulation. It is well established that caveolae are formed by the polymerisation of caveolins, leading to the clustering and invagination of a subset of sphingolipid/cholesterol-rich membrane domains Furthermore, lipid composition alterations may be explained by coalescence of cav-1-containing membranes. In agreement with this suggestion, we observed a remarkable surface membrane reorganisation and formation of a great number of cav-1-rich invaginations following α1A-AR signalling 1A-AR may not necessarily be associated to caveolae. In order to study the importance of the caveolar localisation of α1A-AR, we examined its functional role in the presence and absence of cav-1. We demonstrated that agonist stimulation of the α1A-AR enhanced resistance to reticular stress-induced apoptosis in DU145 cells through the Bax and the caspase-3 pathway. Importantly, we found that cells underexpressing cav-1 showed no resistance to TG-induced apoptosis, thus implying the necessity of caveolae integrity in this process.A large body of work indicates that cav-1 expression is essential for caveolae formation and function 1A-AR signalling in DU145 cells contributes to their resistance to TG-induced apoptosis via caveolae. Additionally, our data add to the growing evidence that cav-1 promotes survival and growth of PCa cells 1A-AR and cav-1 in DU145 cells may contribute to favouring α1A-AR signalling pathway via caveolae amplifying the effects on the survival of these cells.Our results strongly suggest that α1A-AR expression variations according to prostate pathological state. Our study was innovative in the investigation of both α1A-AR and cav-1 expression in normal, cancerous and BPH samples. We show that the elevated expression of α1A-AR in prostate epithelial cells is correlated with prostate pathological progression. During prostate cancer, the organ's architecture is greatly compromised, characterized among others, by proliferation of epithelial cells and invasion of prostate ducts and acini 1A-AR as compared to normal or hyperplastic prostate. Accordingly, we demonstrated increased mRNA levels of cytokeratin-18, α1A-AR and cav-1 in androgen-independent PCa samples as compared to BPH samples. In agreement with previously published data our results propose that the role of α1A-AR in BPH is related to its presence in the fibromuscular stroma 1A-AR in advanced stage PCa epithelial cells provide a strong rationale for the understanding of the significance of α1A-AR signalling in androgen-independent PCa.To date, no relevant data describes α2+ signalling 2+ concentration (data not shown). This observation strongly suggests that resistance to TG-induced apoptosis in DU145 by PHE does not involve the Ca2+ signalling pathway. α1A-AR signalling is complex and various lines of evidence demonstrate that α1A-AR stimulation can activate a variety of signalling pathways depending on cell type, receptor expression and agonist concentration 1A-AR activate ERK-related survival pathways 1A-AR in DU145 androgen-independent prostate cancer cells. Interestingly, it has been demonstrated that human prostate biopsies have increased levels of activated ERKs in malignant tissues as compared to those in benign specimens Previous work from our laboratory has demonstrated that TG-induced apoptosis as well as apoptosis resistance induced by EGF in DU145 cells are mechanisms dependent of intracellular Ca1A-AR. Our data show that caveolae integrity is necessary for α1A-AR function in these cells. This study reveals that α1A-AR and cav-1 expressions are positively correlated with the pathological state of human prostate and are highly expressed in advance PCa tissue samples. Our data strongly suggest that the protective function of the α1A-AR via caveolae described in DU145 cells, may contribute in synergy with other factors, to the apoptosis resistance mechanisms of PCa cells and enhancement of their survival.In conclusion, we demonstrate that caveolae of androgen-independent prostate cancer cells DU145 are involved in the apoptosis resistance induced by the αFigure S1Representative cell cycle profiles of propidium iodide (PI)-stained (A) DU145, (B) DUshCTL and (C) DUshcav-1 cells obtained by flux cytometry as described in the “(0.56 MB PDF)Click here for additional data file.Figure S2Representative examples of Hoechst-stained (A) DU145 wild-type, (B) DUshCTL and (C) DUshcav-1 cells. Cells were (a) non-treated, (b) treated by 10ÂµM TG for 48 h, (c) pre-treated by 10ÂµM PHE for 3 days followed by 48 h 10ÂµM TG or (d) pre-treated simultaneously by 10ÂµM PHE and 1ÂµM PRA for 3 days followed by 48 h 10ÂµM TG (as described in the “(2.59 MB TIF)Click here for additional data file.


Neurospora crassa is an excellent system to study evolution and biological function of SSRs.Simple sequence repeats (SSRs) have been successfully used for various genetic and evolutionary studies in eukaryotic systems. The eukaryotic model organism N. crassa. The distribution of tri-nucleotide (nt) SSRs, the most common SSRs in N. crassa, was significantly biased in exons. We further characterized the distribution of 19 abundant SSR types (AST), which account for 71% of total SSRs in the N. crassa genome, using a Poisson log-linear model. We also characterized the size variation of SSRs among natural accessions using Polymorphic Index Content (PIC) and ANOVA analyses and found that there are genome-wide, chromosome-dependent and local-specific variations. Using polymorphic SSRs, we have built linkage maps from three line-cross populations.We identified and characterized 2749 SSRs of 963 SSR types in the genome of N. crassa, 3) there are different levels of evolutionary forces in variation of amino acid repeats, and 4) SSRs are stable molecular markers for genetic studies in N. crassa.Taking our computational, statistical and experimental data together, we conclude that 1) the distributions of the SSRs in the sequenced N. crassa genome differ systematically between chromosomes as well as between SSR types, 2) the size variation of tri-nt SSRs in exons might be an important mechanism in generating functional variation of proteins in  Simple sequence repeats (SSRs) refer to the sequences that are one to six-nucleotides (nt) repeated in tandem in a genome. SSRs have many advantageous features for various biological studies: SSRs are ubiquitous and abundant in a genome, highly variable and suitable for high-throughput applications -8. In adde novo genesis) or that they are brought about in a primal form into a receptive genomic location by mobile elements (adoptive genesis). These two hypotheses are both adequate for explaining the ubiquitous distribution of SSRs. However, there remains much to be understood to elucidate which one is right and how the non-random distribution of SSRs has emerged in the eukaryotic genome , a well-known pathogen causing a shoot or tip blight of numerous pine species and some other conifers, is 0.43 [N. crassa seems to be relatively high. To estimate a mean PIC value objectively, the tested SSR loci should be randomly sampled. However, unbiased sampling, as done in our study, is not easy to achieve even in sequenced organisms. Since the PIC calculations in many studies, including those mentioned above, are based on the available SSR marker set rather than a random sample, they could produce biased estimates of the PIC for the genome.The estimated PIC value was comparable and relatively high for SSR markers compared to other organisms -60 where is 0.49 , and the is 0.43 . CompareN. crassa implies that the SSR marker system has sufficient resolution/polymorphism to be used for genetic studies. Even though our current study of polymorphism of the selected SSR types uses a rather small sample of 7 strains, our PIC estimation of the SSR type AC/CA was consistent with the estimation using a larger sample of 32 strains  and allele length homoplasy [Recently, molecular marker techniques for assisting efficient mapping/gene-cloning have been developed in a system -68. All N. crassa natural accessions  are correlated with various factors including chromosomes, genomic categories, SSR types, repeat numbers, and total SSR lengths. This suggests that the factors affecting the mutation rate could be random across the genome. Thus, the non-random distribution pattern of SSRs presumably reveals the functional significance of SSRs. The size variations of tri-nt SSR in exons might be an important mechanism in generating functional variation of proteins in the N. crassa. Using statistical analyses, we concluded that there are both genome-wide and local effects in size variation of SSRs. Since genetic mobile elements are inactive in the most of N. crassa genome, the detected size variation of SSRs cannot be explained by transposable elements as demonstrated in other systems. Considering their high PIC values, SSRs are good genetic markers for intra-species populations of N. crassa. However, since the polymorphism level is locus specific, more thorough empirical characterizations of size variability of SSRs across the genome are necessary to increase their efficiency as molecular markers.We conclude that the distributions and size variations of the SSRs in Neurospora genome sequence, release 7, was downloaded from the Broad Institute [n is equivalent to (CA)n, (TG)n, and (GT)n, while (AGC)n is equivalent to (GCA)n, (CAG)n, (CTG)n, and (TGC)n. Lastly, we determined the abundance of each SSR motif and unit size in the different genomic regions by normalizing the size of the corresponding genomic region. To describe the abundance of SSRs in different genomic region, we chose to use the "relative abundance", which is calculated by dividing the number of SSRs by mega base-pair (MB) of sequences in our analyses.The 39.2 Mb nstitute , and wasnstitute . We usedNeurospora genome, we strived to select SSRs randomly from the Neurospora genome. We divided the genome into 250 kb windows and selected SSRs randomly within each window. A total of 164 SSR loci consisting of di- to hexa-SSRs with various sequence motifs were chosen for further analysis. The scatter plot of the selected markers showed that they were evenly distributed in the genome (Data not shown). With the SSRs selected, we designed oligos using Primer3 software  in flanking sequence to amplify the targeted SSR loci. The range of the annealing temperatures in each primer set was between 50°C and 60°C and the primer pairs yielded amplification products between 100 and 350 bp.To characterize the overall pattern of polymorphism of the SSRs in the ® (Applied Biosystems).For semi-automated genotyping analysis, the 5' M13 sequence was attached to a forward primer in order to incorporate a florescent dye into the PCR product. Fluorescent dye labelled M13 forward primer and a marker specific reverse primer were used to generate fluorescent-labelled PCR product as previously described . The comWe generated the predicted amino acid sequences with an assumption that exonic tri-nt SSR sequences had an equal chance to be translated in all the possible reading frames of the tri-nt repeats. For example, SSR sequences GCTGCTGCTGCTGCTGCT can be translated in three different frames: 1) GCT GCT GCT GCT GCT GCT, which will be translated into Ala-Ala-Ala-Ala-Ala-Ala, 2) CTG CTG CTG CTG CTG CTG, which will be translated into Leu-Leu-Leu-Leu-Leu-Leu, and 3) TGC TGC TGC TGC TGC TGC, which will be translated into Cys-Cys-Cys-Cys-Cys-Cys. Only one of the three possible reading frames would be used to generate the "observed" amino acid repeats.The measurement of the allelic diversity or polymorphism information content (PIC) value was first described by Botstein et al.  and modiN. crassa strains are divergent and not related among each other .where Pij represents the frequency of the jth allele for marker i, and summation extends over n alleles. The allelic polymorphism of the 162 SSR markers in the seven natural accessions, FGSC#2223, FGSC#4825, FGSC#4720, FGSC#4715, FGSC#3223, FGSC#4724, and FGSC#2478, were calculated following the formula. The genome structure of seven P = 0.001. Subsequently, Monte Carlo simulation with 500 iterations was used to test the marker locus order generated by GMENDEL.The 564 F1 progenies  abundance is given by A = Y/L. Our modelling strategy incorporates the frequency and length information by assuming that the counts are Poisson random variables with expected values proportional to their associated lengths. If E denotes the expected value of a count, then the expected rate is R = E/L. A log-linear model is used to describe how these rates vary as a function of SST type, genomic region category, and chromosome. The fit of a particular model can be assessed by comparing the empirical abundance values, A, to maximum likelihood estimates of expected rates which satisfy the model assumptions, using the model deviance statistic,where the summation is over all 266 cells in the 19 × 7 × 2 contingency table. Our most general model has the form,or equivalently,where T denotes the main effect of SSR type, C denotes the effect of chromosome, G is the main effect of genomic location category, and T × G allows for type by region interaction, that is, differential type effects by genomic category..All data presented in the current report are freely available via our webpage, SSRs, Simple sequence repeats; nt, nucleotide; AST, abundant SSR types; PIC, Polymorphic Index Content; AAR, amino acid repeats; GO, gene ontology.QS, JP and YL generated the bioinformatic data. TK analyzed bioinformatical data and experimental data. JB, HGG, and TK performed statistical analyses. KL conceived of the project and participated in its design. TK and KL wrote the manuscript. All authors read and approved the final manuscript.List of 2749 SSR loci in the Neurospora crassa genomeClick here for fileGO analysis for proteins containing amino-acid repeatsClick here for fileNeurospora crassa genomeAbundant SSR types (AST) in the Click here for fileThe distribution of the randomly selected SSRs by the unit numberClick here for fileNeurospora crassa genomeThe physical location and PIC values of 131 SSR loci in the Click here for fileComparison of PIC values of the AC/CA SSR type in two different population sizesClick here for fileThree hypotheses for the size variation of SSRsClick here for fileThe list of 33 SSR loci for statistical analysesClick here for fileThe marker quality of the mapped SSR lociClick here for file


In the majority of castration-resistant prostate cancer (CRPC), prostate-specific antigen (PSA), product of a gene that is almost exclusively regulated by the androgen receptor (AR), still acts as a serum marker reflecting disease burden, indicating that AR signaling is activated even under castrate level of serum androgen. Accumulated evidence shows that transcriptional ability of AR is activated both in ligand-dependent and -independent manners in CRPC cells. Some androgen-independent sublines derived from originally androgen-dependent LNCaP prostate cancer cells overexpress the AR and PSA, for which silencing the AR gene suppresses cellular proliferation. The overexpression of the AR confers androgen-independent growth ability on androgen-dependent prostate cancer cells. Some patient-derived prostate cancer xenograft lines also acquire castration-resistant growth ability secreting PSA. More recent publications have shown that the AR activated in CRPC cells regulates distinct gene sets from that in androgen-dependent status. This concept provides very important insights in the development of novel anti-prostate cancer drugs such as new generation anti-androgens and CYP17 inhibitors. Although almost all prostate cancers initially respond to androgen-deprivation therapy (ADT) comprising medical or surgical castration, ADT is usually not permanently effective or curative, and most tumors eventually regrow after a certain period. Before docetaxel was recently proved to improve survival of prostate cancer patients after ADT failure ,2, thereRecent works have accumulated direct and indirect evidence indicating that, in CRPC, AR signaling is activated even under the castrate level of serum androgen. This concept is very important since it means that AR itself and its downstream signaling can be still a therapeutic target in CRPC. However, molecular mechanisms for re-activation of AR in CRPC are complex and heterogeneous. Thus, it is indispensable to demystify detailed pathways for castration-resistant progression of prostate cancer. This review outlines recent advances in prostate cancer research and experimental evidence for the essential role of AR in CRPC.In hormone-naive status, androgens circulate in blood in either a bound  or free form. The majority (~95%) of circulating androgens is testosterone, produced by Leydig cells in testis under control of the pituitary hormone luteinizing hormone (LH). Free serum testosterone can transfer across plasma membrane into cytoplasm of prostatic epithelial cells, where unbound AR is abundant. Testosterone is then reduced by 5α-reductase into dihydrotestosterone (DHT), a more potent form of AR ligand. Upon binding of androgen, AR translocates to nucleus in a dimerized form and regulates gene expression by binding to specific hormone response elements.Normal prostate epithelium and most of untreated prostate cancer cells are highly dependent on the androgen/AR axis and ablation or blockade of the signaling pathway results in severe regression of the tissue. Prostatic epithelial cells in culture stop proliferating at G1 cell-cycle phase (G1 arrest) upon androgen deprivation and then go to apoptosis. With the exception of rare small cell carcinomas of the prostate, most untreated human prostate cancers express AR at both the mRNA and protein level and respond to ADT. Physiologically, 95% of serum androgens are produced by testicular Leydig cells, while the rest are secreted by the adrenal gland. In ADT, surgical castration by bilateral orchiectomy, or medical castration by the administration of luteinizing hormone-releasing hormone analogue (either agonist or antagonist), suppress serum testosterone levels. Combined androgen blockade (CAB) comprises administration of an anti-androgen and surgical or medical castration. Castration suppresses serum testicular androgen levels and residual testicular and adrenal androgens are blocked by anti-androgens.Molecular mechanisms for CRPC that progresses, by definition, despite castrate levels of serum testosterone can be categorized into roughly two concepts, namely AR-independent and -dependent pathways. AR-independent pathway, which is beyond the scope of this review, includes AR-independent activation of survival or anti-apoptotic pathway and mechanisms involving subpopulation of intrinsically AR-independent cancer cells. It has been known that androgen ablation induce activation of anti-apoptotic molecules such as BCL-2 , ClusterThe presence of a primarily AR-independent subpopulation in prostate cancer cells has long been suggested as a potential mechanism for castration-resistant progression of prostate cancer . Althougde novo ectopic androgen synthesis and AR hypersensitivity or promiscuous activation caused by overexpression, mutation or truncation, interaction with co-factors, and posttranslational modifications. These discoveries have led to the development and introduction of novel therapeutic agents targeting the AR signaling axis toward clinical use.Thus, there is certainly an AR-independent subset of CRPC, however they are considered as a small minority, whereas the vast majority of CRPC maintain AR-mediated signaling activity and dependence on it. Reactivation of AR signaling in CRPC is clinically evident by re-elevation of PSA in most cases, and there is a response to secondary or tertiary hormonal therapy in the majority of them. Additionally, constant nuclear expression of AR in CRPC cells indicates that the AR signaling remains active despite the androgen withdrawal . It is fExpression profiles of castration-resistant tumors by Holzbeierlein and colleagues demonstrated that the gene expression changes detected in response to androgen ablation therapy were no longer present and that many of AR-driven genes were expressed, suggesting a reactivation of the androgen response pathway in the absence of exogenous androgen . It was de novo synthesis of DHT in the LNCaP xenograft model for CRPC using liquid chromatography-radiometric/mass spectrometry (LC/MS) technique [Locke and colleagues demonstrated intratumoral echnique . LNCaP technique . CRPC alechnique  and apprechnique .One of the hallmarks of CRPC is overexpression of AR. A number of reports have shown increased expression of AR in human CRPC tissues based on gene expression –35 and iet al. [AR gene amplification partly accounts for the high expression of AR in some CRPC cases. Although AR plays an essential role in the carcinogenesis of prostate cancer, only a small minority of untreated prostate cancers harbor copy number alteration of AR gene ,35,45,46et al. ) as wellAs for posttranscriptional modification, Yeap and colleagues demonstrated that AR mRNA stability is a major determinant of AR gene expression in prostate and breast cancer cells  and idenMicro RNAs (miRNAs) modulate translation of mRNA by base-pairing interactions with 3′-UTR through destabilizing the mRNA or by repression of protein synthesis in actively translating ribosomes. However, association between miRNA-mediated regulation of AR and development of CRPC had not been evident until very recently . A systeAR is regulated also in posttranslational manners. It has been known that half-life of AR protein is prolonged in the presence of agonistic steroids including testosterone, dihydrotestosterone, R1881 (synthetic androgen), and other nonandrogenic steroids in prostate cancer cells ,55 and oin vivo for castration-resistant progression and identify potential targets in CRPC patients.More recently, some molecules that are responsible for AR stabilization have been identified. Thomas and colleagues demonstrated that Stat5  stabilizes AR and potentiate castration-resistant progression of prostate cancer cells . They alAR sometimes recognizes steroidal or non-steroidal ligands other than testosterone or dihydrotestosterone like an agonist, which leads to aberrant activation of AR in the absence of androgen. Clinicians often observe apparently a paradoxical PSA decrease upon discontinuation of first generation non-steroidal anti-androgens including flutamide and bicalutamide, the so called “anti-androgen withdrawal syndrome (AWS)” first described by Kelly and Scher . This phin vivo [Hara and colleagues established a bicalutamide-resistant subline from LNCaP cells and found the cells harbor Trp741Cys/Leu mutation in the AR gene, which recognized bicalutamide as an agonist . Our groin vivo ,68. Indein vivo –72. Thusin vivo . Howeverin vivo , indicatSome clinical and experimental evidence indicate that there are several mechanisms that confer AR activation to prostate cancer cells relatively or completely independent (hereafter referred to as simply “ligand-independent”) of activating ligands. There are a number of co-factors for AR (extensively reviewed in ref. –76), andAdditionally, FOXA1 was shown to regulate a large number of genes considered to be AR-pathway-specific through binding and activating the glucocorticoid receptor . In thatPosttranslational modifications including phosphorylation, acetylation, sumoylation, ubiquitination and methylation that contribute to ligand-independent activation of AR transcriptional activity have been widely studied ,90. AmonIn addition to serine/threonine kinases, involvement of some tyrosine kinases has been suggested for castration-resistant activation of AR. Mahajan and colleagues found that activated Cdc42-associated tyrosine kinase (Ack1) phosphorylated AR protein at Tyr-267 and Tyr-363 and promoted castration-resistant growth of LNCaP and LAPC-4 xenograft tumors . Guo andin vivo [The PI3K/AKT pathway also plays complicated roles both in castration-naive and -resistant prostate cancers. Reports have shown that androgen withdrawal results in an increase of PI3K/AKT pathway activity both in cultured cells ,101 and in vivo ,103, whiin vivo , there hin vivo ,103,105.in vivo –108. Cinin vivo . Collectin vivo .N-terminal domain (NTD) and DNA-binding domain (DBD) but lacks ligand-binding domain (LBD) and C-terminal domain (CTD) have been recently discovered as novel molecular mechanisms for the castration-resistant progression of prostate cancer. It was already known that 22Rv1 prostate cancer cells expressed smaller 75–80 kDa AR species that are distinct from wild-type ~112 kDa AR protein [Truncated AR species that composes only  protein . Antibod protein . After s protein , more th protein .Indeed, the AR truncated variants are frequently found in CRPC tissues ,119 and Thus, AR drives CRPC progression in various fashions. Indeed, recent advances in transcriptome and genome revealed that AR is one of the most frequently altered genes in prostate cancer . MoreoveWith a castration-resistant status, prostate tumor models showed that AR is still required for growth. Chen and colleagues knocked down AR using short-hairpin RNA (shRNA)-expressing lentivirus in castration-resistant subline of LAPC-4 and LNCaP cells and showed the growth of xenograft tumors from both cells were retarded compared to control infected with control shRNA-expressing virus . AnotherAnother effort is to target NTD that can theoretically and indeed cover AR truncate variants as a target . NTD conAR promoter is suggested as an associated molecular mechanism [Some castration-resistant cell lines express little AR compared to their androgen-dependent parental lines ,61 as weechanism . It was echanism . In thesechanism . TherefoAR drives CRPC even under castrated serum androgen level. There are a variety of mechanisms for the castration-resistant activation of AR and the AR in CRPC regulates distinct gene sets from that in androgen-dependent prostate cancer. These indicate that AR persistently stays at the center of therapeutic targets even after castration-resistant progression of prostate cancer.


Clematichinenoside AR is a promising lead compound for the treatment of rheumatoid arthritis. A systematic research for the related impurities in AR bulk samples is still lacking. For the safe use of this natural product in future clinical practice, the structure and content of each constituent, including the main ingredient as well as the impurities in AR bulk sample must be characterized in detail.Clematis manshurica Rupr. (Ranunculaceae) with the potential of treating rheumatoid arthritis. Five impurities were characterized, and impurity 2 (Clematomandshurica saponin F) is a new triterpenoid saponin isolated from this product. Optimum separation for clematichinenoside AR and five related impurities was carried out on an Agilent octadecylsilane bonded silica gel column  using a gradient HPLC method. The validation results showed good sensitivity, specificity, linearity(r2>0.9992) precision(RSD<1.63%), accuracy(recoveries in the range of 95.60%-104.76%) and robustness. Three AR bulk samples containing all the impurities were examined by two methods, and the stability of correction factors for the determination of related impurities was discussed. The proposed stability-indicating method was suitable for the quality control of this natural product.A simple and stability indicating RP-HPLC method was developed and validated for determining the purity of clematichinenoside AR (AR), a natural product from the roots of Five related impurities of clematichinenoside AR were characterized, including a new triterpenoid saponins firstly found in clematichinenoside AR bulk samples. In the simple chromatographic method for determining clematichinenoside AR and its related impurities in bulk samples, the correction factor was better for the quality control in the relative stable concentrations. Clematis chinensis Osbeck and Clematis manshurica Rupr. (Ranunculaceae). According to previous pharmacological studies, clematichinenoside AR has a potential anti-inflammatory effect in arthritic rats, and its mechanism may involve the inhibition of the expression of NF-κB p65 subunits, TNF-a and COX-2 [1H NMR, 13C NMR, COSY, DEPT, HMBC, HSQC and ROESY).Radix et Rhizoma Clematidis (Wei-Ling-Xian) has been used as an anti-inflammatory, antitumor, and analgesic agent in traditional Chinese medicine (TCM) with a long history ,2. Tritend COX-2 -11. In pnd COX-2  and a LCnd COX-2 . Our prend COX-2 . BesidesAll the chemical reference substances, including clematichinenoside AR and related substances were prepared in our laboratory, and the purity of each compound was confirmed to be higher than 98% by HPLC. Three batches of AR bulk samples were supplied by Chia Tai Tianqing Pharmaceutical Company. HPLC grade acetonitrile were purchased from Merck , other analytical grade reagents were purchased from Nanjing Chemical Reagent Co., Ltd. .Analyses were primarily performed on an Agilent 1200 HPLC system , equipped with a variable wavelength detector (VWD). The HPLC separation method was developed on an Agilent reversed phase octyldecyl silica column . The mobile phase consisted of solvent A(water) and solvent B(acetonitrile) with the following gradient program: 30% B in 0~5 min; 30%~35% B in 5~12 min; 35%~60% B in 12~17 min; 60% B in 17~20 min. .For the isolation, a glass column with ODS material and semi-preparative HPLC instrument using an Agilent 1200 with Agilent Eclipse XDB-C18  were used to enrich and purify impurities. AR bulk sample (Lot: 20110610) dissolved in water was subjected to ODS column chromatography and eluted with gradient water–methanol. Each collected fraction was analyzed by TLC and HPLC. The fractions of the same compound were pooled together and purified by HPLC instrument. These impurities were used as reference substances in the following studies after structural elucidation by TOF-MS and NMR.AR reference standard solution (2.0 mg/mL) and five impurity solutions (1.0 mg/mL) were prepared respectively in a mixture of acetonitrile and water  as standard stock solutions. The mixed standard solution was obtained by transferring the appropriate volumes of every stock solution into a 5 mL volumetric flask and diluting the mixture with the mobile phase in order to get standard solutions containing 1.0 mg/mL AR and 0.05mg/mL of each impurity (at 5.0% concentration relative to AR).20D) was −38.1 . The IR spectrum showed the presence of hydroxyl groups at 3425 cm-1 and carbonyl groups at 1640 cm-1.Impurity 2 (Clematomandshurica saponin F) was a white, amorphous powder, and was freely dissolved in water and methanol, mp 231-232°C. The optical rotation instrument using pyridine-d5.HR-ESI MS gave a strong molecular ion with the m/z value of 1821.8175, suggesting the molecular formula as C13C NMR spectral data reported in Table 1H and 13C NMR spectra. The 13C NMR spectrum showed 82 carbon signals. There were 28 signals at upfield (δ 0–60), in which 25 signals were due to aglycone and three signals from C-6 of the three rhamnose . 39 signals were shown from δ 60 to δ 90, in which δ 81.9 and δ 63.9 were from the aglycone. 9 signals exhibited from δ 90 to δ 100  were signals of the sugar anomeric carbons. There were two signals showed from δ 110 to δ 150, which were from the aglycone. The signal at δ 176.5 was due to carbonyl group. The 13C NMR spectral data reported in Table 1H NMR spectrum exhibited nine signals of the sugar anomeric protons at δ 5.04 , 6.25 , 5.80, 4.91, 5.07, 5.40, 6.20, 4.97, 5.81 and methyl signals of the three rhamnose at δ 1.67, 1.57, 1.51. Also there are six methyl signals of aglycone shown in the 1H NMR spectrum at δ 1.15, 1.10, 1.06, 0.94, 0.87, 0.85. The downfield shift of C-3 and the upfield shift of C-28 suggested that the hydroxyl group of C-3 and the carbonyl group of C-28 were both glycosydated. The chemical shift of C-23 moved to the downfield at δ 63.96 because of the hydroxylation. The acid hydrolysis showed the presence of arabinose, glucose, rhamnose and ribose. Compared with the data in the literature [The 82H134O44, C76H124O39, C69H124O34, and C64H104O29, respectively. Their chemical structures were suggested as clematichinenoside AR6[2[Impurities 1, 3, 4, 5 were obtained as white, amorphous powders. The TOF-MS revealed impurities 1, 3, 4 have double charged molecular ion at m/z 933.4001, 852.3874, 794.3646, and gave impurity 5 has molecular ion at m/z 1335.6913, which corresponds to a molecular formula of Coside AR6, and cleA simple, efficient and reliable method was developed to examine AR and five impurities in bulk samples. AR and its related impurities all showed maximum absorption at 203 nm. Therefore, the detection wavelength was set at 203 nm, flow rate and column temperature were optimized in order to achieve the desired resolution and tailing factor of closely eluting impurities using simple chromatographic conditions. Under this condition, all the peaks were clearly separated. The proposed method was validated with respect to system suitability, specificity, robustness, linearity, accuracy, precision, LOD and LOQ. Figure To investigate the effect of the HPLC column on the separation and tailing factor under the optimized conditions, octyldecyl silica gel columns from three different vendors were evaluated for the analysis of AR and its impurities. These HPLC columns included: (1) Thermo ODS Hypersil ; (2)Diamonsil C18 3)Agilent TC-C18. Based on the results from the laboratory HPLC system, all the resolutions were more than 1.50, but the Agilent TC-C18 provided the best resolutions. The tailing factors of AR and impurities obtained on the Agilent TC-18 column were between 0.8 and 1.2, which indicated good peak shapes for all compounds. Tailing factors of Imp-1, 5 on the Diamonsil C18 column were less than 0.8 and the numbers of theoretical plates for Imp-2, 3, 4 and AR were less than those obtained on the Agilent TC-C18. Besides, the tailing factors of Imp-1, 5 were bigger than 1.5 and was not suitable for tailing factor requirement on the Thermo ODS Hypersil. Thus, the Agilent TC-C18 column demonstrated the desirable performance and was selected for further studies. The results were listed in Table Agilent TThe system suitability tests were conducted using the working solution containing AR (1.0 mg/mL) and five impurities (each at 0.05 mg/mL) by five repeated injections with the optimized method. It was observed that the tailing factors for all compounds were between 0.8 and 1.2, and the resolutions were greater than 1.50. These results met the HPLC method requirements for separation and quantification of AR and related impurities in bulk samples.2O2 at 60°C for 1 h, illumination of 1.2 million lux hours for 10 days and heat at 60°C for 8 h, respectively. Photolytic and thermal studies were conducted by exposing the samples both in solution (1.0 mg/mL) and in solid state. Samples were withdrawn at the appropriate times, the pH was adjusted to neutral and the samples were subjected to HPLC analysis after suitable dilution (1.0 mg/mL) to evaluate the ability of the proposed method to separate AR from its potential impurities. The bulk drug and degradation samples were examined by peak purity testing utilizing DAD detector. The purity factor obtained from AR peak was higher than threshold, which demonstrated the spectral homogeneity. Degradation of the sample at 60°C and illumination of 1.2 million lux hours did not show any difference in terms of chromatographic behavior compared to a fresh one, but in the acid and basic conditions, the content of Imp-5 increased significantly. Besides, clematichinenoside AR was transformed into Imp-4 in acidic condition. All the results demonstrated that degradation products formed during the stress studies were well separated from AR which proved that the adopted method was specific  with each impurity at 5.0% concentration (0.05 mg/mL). Each RSD (%) of peak areas for all components was below 2.0% as shown in Table The standard solutions containing both AR and impurities were injected and the peak height of each component was recorded. The limit of detection (LOD) and limit of quantitation (LOQ) for AR and five impurities were calculated by normalization of the signal/noise ratio (S/N) to 3 for LOD and S/N to 10 for LOQ, see Table -1 for impurity 2 and 1, from 10 to 100 μg·ml-1 for impurities 3, 4 and 5. The adopted method demonstrated good linearity for clematichinenoside AR (r2>0.9994) and five impurity components (r2>0.9992).The linearity of test solution was prepared at six concentration levels, 0.01 to 2.0 mg/mL for AR, from 6 to 60 μg·mlThe accuracy of the method was checked for three known concentration levels  added to a sample containing all compounds and peak area was recorded. The mean analyte recoveries and their recovery rates obtained for each level are described in detail in Table -1) were purposely subjected to small modifications. The temperature, the flow rate and the combined effects as the three factors were studied on the characteristics of the chromatogram of AR and its impurities. The tailing factors for all compounds were between 0.8 and 1.2 and the resolutions were greater than 1.50.For an analytical method to be robust, it must be able to produce quantitative results despite small changes in the experimental parameters, which may occur in a typical testing laboratory. The robustness of the method was studied by employing an experimental design, where the temperature of the column (25°C −35°C) and the flow rate of the mobile phase (0.8-1.2mL·minR/CR).Due to the difficulties and expenses to prepare all the related reference standards, the application of the external standard method was limited, so correction factor was used in the quantification of related substances. According to ICH guiding principles, if the response factors of drug substance and relevant impurity are not close, we can use the drug substance as a standard to estimate the levels of impurities with providing a correction factor. The method of calculating correction factor is the same as conversion factor . The corR: peak area of standard substance; Cs: concentration of standard substanceAs: peak area of internal standard substance; Cs: concentration of internal standard substance; AIn this study, AR was selected as internal standard, and each related impurity was as standard. The correction factor of each substance was obtained as the mean values calculated from five different concentrations. The results from five gradient concentrations showed that the correction factors of Imp-1 (F1) was 1.00 ± 0.04, the Imp-2 (F2) was 1.13 ± 0.02, the Imp-3 (F3) was 1.00 ± 0.02, the Imp-4 (F4) was 0.90 ± 0.04,and the Imp-5 (F5) was 0.61 ± 0.02. The RSDs for corrections for all compounds were below 5%. Additionally, there were other two approaches used to calculate the correction factors. The first one was calculated by the ratio of equation slope of AR and its impurities. F1 was 1.00; F2 was 1.01; F3 was 0.95; F4 was 0.82 and F5 was 0.59. The above results showed that F1 and F5 were similar while the others had significant difference. The linearity data showed that the interception of impurities 2 and 4 were not less than 1. This indicated that we couldn't neglect the influence of interception. The second one was only by one single concentration solution. But Table -1) and the UV detection wavelength (200 nm-206 nm). The results were shown in Table In the quality control of this natural product, the value of correction factor may change greatly if it was performed in different laboratories. Therefore, the stability of correction factor was investigated by ruggedness and robustness tests. In ruggedness test, five columns  were investigated. All these columns meet with the requirements of system suitability parameters which was shown in Table The validated method was applied to quantitate impurities in three batches of AR bulk samples by using two methods: external standard method as method I, and single reference standard by five correction factors were used to determine the impurities of the same samples as method II. The sample solutions were prepared at 1 mg/mL. The contents of impurities relative to AR were summarized in Table The quality of bulk drug not only depends on the adopted procedure, but also on the side-reaction products, unreacted raw materials and intermediates, because they may render unwanted toxicological effects. Hence, thorough examination of related impurities plays a very important role in controlling the quality of bulk drug in the final product ,19. The HPLC: High-performance liquid chromatography; ICH: International Conference on Harmonization; UV: Ultraviolet; LOD: Limit of detection; LOQ: Limit of quantification; SD: Standard deviation; RSD: Relative standard deviation; ELSD: Evaporative Light Scattering Detector; RP-HPLC: Reverse phase high-performance liquid chromatography; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; TOF-MS: Time-of-flight mass spectrometry nuclear magnetic resonance; HMBC: Heteronuclear Multiple Bond Correlation; HR-ESI MS: High resolution electrospray ionization mass spectrometry; Rs: Resolution; As: Tailing factor; α: Selectivity; k’: Capacity factor; NTP: Number of theoretical plates; AR: Clematichinenoside AR.The authors declare that they have no competing interests.LFL planed and supervised the whole work, YZ carried out the experiments and analyzed the data statistically, YG, JS, XLZ and LY participated in writing the manuscript. All authors read and approved the final manuscript.


Allergic Rhinitis is a common Ear, Nose and Throat disorder. Asthma and Allergic Rhinitis are diseases with similar underlying mechanism and pathogenesis. The aim of this survey was to highlight current treatment trends for Allergic Rhinitis and Asthma.A questionnaire was emailed to all registered consultant members of the British Association of Otorhinolaryngologists - Head and Neck Surgeons regarding the management of patients with Allergic Rhinitis and related disorders.Survey response rate was 56%. The results indicate a various approach in the investigation and management of Allergic Rhinitis compatible with recommendations from the Allergic Rhinitis and Its Impact on Asthma guidelines in collaboration with the World Health Organisation.A combined management approach for patients with Allergic Rhinitis and concomitant Asthma may reduce medical treatment costs for these conditions and improve symptom control and quality of life. The definition of Allergic Rhinitis (AR) was formulated by Hansel in 1929 [The nasal passage and paranasal sinuses are an integral part of the respiratory tract and patients may have rhinitis without sinusitis, but not sinusitis without rhinitis, hence the term rhinitis has been replaced in modern ENT literature by the more accurate term rhinosinusitis . AR and The aetiology of AR is multi-factorial and the diagnostic and therapeutic choices remain diverse. The aim of this questionnaire survey was to highlight the treatment trends in the management of AR amongst UK-based otolaryngologists.A questionnaire (Appendix 1) was emailed via ENT UK to the email addresses of all registered consultant members of the British Association of Otorhinolaryngologists - Head and Neck Surgeons (BAO-HNS). Survey recipients were asked 1) about their familiarity with the Allergic Rhinitis and Its Impact on Asthma (ARIA) guidelines in collaboration with the World Health Organisation (WHO), 2) about the type of investigations, treatment regimes and follow-up arrangements for AR patients and 3) about whether they give to patients advice leaflets on life style changes including education and allergen avoidance. All participants had the opportunity to answer anonymously.A total of 551 questionnaires were emailed. There were 309 replies (56% response rate). All consultants were familiar with the association between AR and Asthma but only 63% of the respondents were familiar with the ARIA guidelines. Seventy seven (25%) of otolaryngologists managed AR patients of which a 20 - 30% proportion had associated symptoms or a diagnosis of Asthma . Only nine consultants (3%) arranged pulmonary spirometry Figure .Fifty six per cent of otolaryngologists preferred the management pathway of starting treatment and then discharging AR patients to General Practitioners (GPs) for further follow-up. However three (1%) consultants managed and subsequently referred AR patients to a respiratory physician Figure .In total two hundred and forty (78%) of otolaryngologists provided patient leaflets on life-style changes including education and practical tips on allergens avoidance .Figure AR is a multi-factorial disease with a worldwide disabling effect on all individuals irrespective of age and ethnic background. In 2001 the ARIA in collaboration with the WHO workshop published guidelines for healthcare professionals in order to highlight the latest updates on the aetiology of AR emphasizing the association between AR and Asthma and proposing a management algorithm .Risk factors for AR include a combination of environmental and genetic interactions . The diaAn overwhelming 99% of UK ENT consultants use intra-nasal steroids to treat AR. This trend is supported by several studies demonstrating that intra-nasal steroids are the most efficacious and cost effective first line treatment for AR ,13.et al in a cochrane review showed sublingual immunotherapy to be a safe and effective option in managing AR [et al in a recently published meta-analysis advocate the use of allergen injection immunotherapy as an effective treatment with a low risk of adverse effects in the management of AR [Allergen specific immunotherapy for AR was first described in 1911 by Noon and involves the administration of a gradually increasing quantity of an allergen extract . Patientaging AR . Furthernt of AR . Howevernt of AR .The importance of educating patients or carers with advice leaflets with information regarding AR including allergen avoidance cannot be over emphasised and was actively supported by 78% of responders of this survey. This enables individuals to be actively involved in the management of their disease resulting in an overall improvement in patient satisfaction, treatment compliance and outcomes . Such prInterestingly, only 3% of otolaryngologists arranged pulmonary spirometry and 1% of all respondents referred AR patients to a respiratory physician for management of any associated lower respiratory tract pathology. Several studies though have demonstrated that AR patients with no underlying Asthma do have impaired pulmonary function and reversible airway obstruction ,22. In aThis survey demonstrates as expected a diverse approach to the management of Allergic Rhinitis. An interesting finding of the survey is that despite good evidence on the relationship between Asthma and AR, most otolaryngologists often do no think "outside the specialty", for example they would rarely use spirometry as a diagnostic aid.A combined treatment strategy of allergen avoidance, pharmacotherapy, immunotherapy and education applied to patients with AR and concomitant Asthma as recommended by the ARIA guidelines may reduce medical treatment costs and improve symptom control and quality of life.The authors declare that they have no competing interests.RSN: Primary Author of manuscript. PDK: Assistant Author of manuscript and Editing. EGT: Contribution to Literature Search and Proof Reading. AK: Senior Clinician and contribution to Proof Reading and Editing. DKN: Assisted writing manuscript and designing questionnaire. All authors read and approved the final manuscript.Dear colleague,Pease could you complete this survey with your responses and return it by clicking the SUBMIT button below. We thank you for your valuable time in completing this survey.1. Are you familiar with the association between Allergic Rhinitis and Asthma?• Yes• No2. Are you familiar with the Allergic Rhinitis and its impact on Asthma (ARIA) guidelines in collaboration with the World Health Organisation?• Yes• No3. What proportion of your patients with Allergic Rhinitis have got symptoms or diagnosis of Asthma?• <10%• 10 - 20%• 20 - 30%• 30 - 40%• 40 - 50%• >50%4. If you suspect Allergic Rhinitis what investigations would you consider ?• None• Skin prick test to known allergens• Serology tests e.g. RAST, IgE• Peak flow meter (air flow litres/minute)• Pulmonary spirometry5. If you suspect Allergic Rhinitis in a patient which of the following management plans would you consider?• Start treatment and review• Start treatment and discharge patient to GP for follow-up• Start treatment and refer to a Respiratory Physician6. If you suspect Allergic Rhinitis which of the following would you use for treatment ?• Antihistamine - oral• Antihistamine - intra-nasal• Steroid - intra-nasal• Steroid - intra-bronchial• Steroid - oral• Decongestant - oral• Decongestant - intra-nasal• Anti-cholinergic (Ipatropium Bromide)• Anti-leukotriene• Specific Immunotherapy - sublingual• Specific Immunotherapy - subcutaneous7. How long do you prescribe nasal sprays for• 3 months• 6 months• >6 months8. What is the frequency of nasal spray administration do you prescribe ?• Two puffs once a day• Two puffs twice a day• Other (please specify)9. Do you provide leaflets for patients about life-style changes including education and avoidance of allergen(s)?• Yes• NoThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6815/11/3/prepub


Epigenetic mechanisms such as chromatin accessibility impact transcription factor binding to DNA and transcriptional specificity. The androgen receptor (AR), a master regulator of the male phenotype and prostate cancer pathogenesis, acts primarily through ligand-activated transcription of target genes. Although several determinants of AR transcriptional specificity have been elucidated, our understanding of the interplay between chromatin accessibility and AR function remains incomplete.We used deep sequencing to assess chromatin structure via DNase I hypersensitivity and mRNA abundance, and paired these datasets with three independent AR ChIP-seq datasets. Our analysis revealed qualitative and quantitative differences in chromatin accessibility that corresponded to both AR binding and an enrichment of motifs for potential collaborating factors, one of which was identified as SP1. These quantitative differences were significantly associated with AR-regulated mRNA transcription across the genome. Base-pair resolution of the DNase I cleavage profile revealed three distinct footprinting patterns associated with the AR-DNA interaction, suggesting multiple modes of AR interaction with the genome.in vivo evidence that a significant fraction of AR binds only to half of the full AR DNA motif. These findings indicate a dynamic quantitative relationship between chromatin structure and AR-DNA binding that impacts AR transcriptional specificity.In contrast with other DNA-binding factors, AR binding to the genome does not only target regions that are accessible to DNase I cleavage prior to hormone induction. AR binding is invariably associated with an increase in chromatin accessibility and, consequently, changes in gene expression. Furthermore, we present the first  The androgen receptor (AR), a ligand-activated member of the nuclear receptor superfamily, plays a critical role in the male phenotype and prostate cancer biology. AR expression results in context-specific transformation of prostate epithelial cells -5, and pAR-mediated transcriptional specificity is highly regulated, and the AR associates with proteins that possess co-activator or co-repressor function . AR bindRecent reports examining nucleosome positioning in relation to AR binding have found that local nucleosome depletion and increased chromatin accessibility accompanies the AR binding to DNA ,17. HoweThe mapping of DNase I hypersensitive (DHS) sites is an accurate method to identify different types of active gene regulatory elements within accessible chromatin -20. Morein vivo evidence of AR binding to both half and full AR DNA recognition motifs. Together, our work reveals that active chromatin remodeling occurs during androgen nuclear receptor activation.To determine the relationship between AR-dependent chromatin accessibility changes and AR-mediated transcription, we performed DNase-seq and mRNA-seq on the well-established androgen-sensitive prostate cancer cell line LNCaP, before and after hormone induction. AR binding sites were obtained from three published studies describing AR ChIP-seq experiments on LNCaP cell lines. Another member of the nuclear receptor superfamily, the glucocorticoid receptor (GR), was recently found to bind predominantly in DHS sites that exist prior to GR ligand hormone treatment . In agreTo assess the relationship between accessible chromatin and AR activation, we performed DNase-seq on independent growths of LNCaP cells that were cultured with (LNCaP-induced) or without (LNCaP) the synthetic androgen R1881 (12 hours). Using previously published methodologies and a standard analysis pipeline , we idenP-value cutoff of 0.05. The DHS sites in each cell condition cover approximately 3% of the human genome  to identify the degree of accessibility changes, which we refer to as ΔDNase regions. At the strict threshold, we identified 2,586 regions with strict ΔDNase increase after androgen induction and no regions of signal decrease. The loose threshold identified 18,692 regions with loose ΔDNase increase and 1,467 regions with loose ΔDNase decrease . This differential count also indicated that more regions display an increase in DNase-seq signal with androgen treatment, supporting the edgeR results  promoter . In addition, several other significantly enriched motifs were detected in both ΔDNase increase and decrease regions Figure  that corBased on our motif analysis of ΔDNase regions and recent reports of AR binding to nucleosome-depleted regions marked by acetylated H3  and H3K4Each of the three individual AR ChIP studies displayed consistent overlap patterns with DHS sites. In each individual experiment approximately 20% of all AR binding sites occurred within DHS sites that are present both before and after hormone treatment (poised DHS sites). An additional 20% to 30% of AR binding sites overlapped DHS sites following androgen induction. Thus, results from each dataset suggest that slightly less than half of all AR binding sites in DHS regions are poised Figure  and the Given the observation that a substantial number of AR binding sites occur within LNCaP-induced only DHS sites, we examined the association between AR binding events and quantitative chromatin remodeling. To test this, we evaluated AR sites that overlapped regions with increased DNase-seq signal (strict and loose ΔDNase increases). As expected, AR ChIP-seq peaks identified only within LNCaP-induced DHS sites  did not overlap DHS sites. To determine if this is due to a peak-calling threshold, we decreased the stringency threshold for identifying DHS sites to either the top 200,000 or top 400,000 DNase I sensitive regions. Overlap with AR ChIP-seq indicates that the proportion of AR binding sites binding in a poised versus qualitatively remodeled region was consistent regardless of the threshold, and that a substantial proportion of AR binding occurs in non-DNase I sensitive regions of the genome even after relaxing the DHS peak thresholds Figure . Even afde novo motif analysis. First, we searched for motifs enriched in AR binding sites defined by ChIP-seq peaks that did not map within DHS sites. Only one motif was enriched within these regions by our de novo analysis, which resembled both the canonical AR motif and a motif derived de novo from AR ChIP-seq sequences that fall within poised DHS sites  and CTCF . Loose ΔDNase increases were significantly enriched near both up- and downregulated genes (P < 0.001). We noticed that both strict ΔDNase increases and loose ΔDNase decreases were enriched near downregulated genes with borderline significance. The reverse comparison, in which we associated differentially regulated genes to ΔDNase regions within 20 kb of the transcriptional start site  for LNCaP-induced cells . Increasing the value of k consistently identified the same three general patterns in LNCaP-induced DNase-seq data within the AR motif, with multiple clusters aggregating to each general pattern . DNase-seq signal was represented by a vector of DNase I cuts spanning 15 bp around the center of the AR motif. We identified three reproducible clusters, each of which represented part of the observed composite footprint Figure . These cs Figure , with thTMPRSS2 requires an AR dimer. Consistently, we observed a DNase-seq digestion pattern similar to that shown in cluster 3 within this enhancer element  and time point of 12 hours as compared with 4 hours by He et al. and 1 hour by John et al. Similar to He et al., who also utilized a quantitative measure of change in DNase-seq signal, we observed that less than half of AR binding targets poised chromatin and we were also able to associate AR-induced chromatin remodeling with AR-induced transcriptional changes, suggesting that the mechanism of chromatin remodeling and its phenotypically relevant association with differential transcription requires longer periods of receptor activation. Importantly, we used a different statistical measurement of quantitative change in DNase-seq signal to reach the same result and conclusion. In our study and those by He et al. and John et al., we note that the degree of nuclear receptor binding within regions of poised chromatin decreases with increased hormone treatment time . Although this observation is confounded by differences in receptor, receptor ligand, sequencing depth and DNase-seq protocol among the mentioned studies, these data suggest that more extensive comparative analyses over a full time course of ligand stimulation of both AR and GR are needed to fully understand the similarities and differences of different hormone receptors with respect to their interaction with chromatin.Several prior reports also support AR-induced chromatin remodeling ,17, inclet al. . Our datr the GR , where aWhile the majority of high confidence AR binding occurred in regions sensitive to DNase I cleavage, a substantial proportion of AR binding events occurred in regions of low DNase-seq signal. It is possible that inconsistent and/or intermittent nucleosome depletion at these genomic regions decreases DNA accessibility and limits detection by our assay; this attribute of nucleosome depletion appears to be associated with a slightly different AR motif. Consistently, we also found that AR binding  is significantly lower in non-DHS regions than in DHS regions. Thus, it is plausible that regions that are identified with weaker AR binding and lower DNase-seq signal may experience a dynamic equilibrium of nucleosome and nuclear receptor binding, as has been previously proposed . Loci wiin vivo and endogenous evidence of functional AR monomers that have been suggested to exist as a stable subpopulation of AR molecules [in vivo, and these modes are associated with different co-factors. These observations are consistent with a recently proposed model of a transient interaction between nuclear receptors such as the AR and DNA rather than a stronger and more stable AR-DNA interaction [AR footprinting analysis further revealed the complexity of the AR-DNA interaction. The aggregate DNase-seq signal around AR motifs demonstrated a relatively weak but consistent pattern of protection that corresponds to the expected binding pattern, consistent with other DNase I footprinting studies . In addiolecules . Only theraction . Our anaThe dynamics of AR-DNA binding are likely impacted by additional co-factors that may facilitate AR binding directly or indirectly. Distal regulatory elements identified by DNase-seq displayed an enrichment of SP1 and E2A/TCF3 motifs within DHS specifically accessible in LNCaP cells compared with 113 independent cell lines. TCF3, a basic helix loop helix factor involved in Wnt/β-catenin signaling ,48, reprOverall, these lines of evidence combined with our results warrant further investigation of SP1 in the context of AR binding and AR-induced chromatin remodeling. Our analyses show that qualitative and quantitative assessment of chromatin accessibility by DNase-seq is an important and useful tool for elucidating AR biology in prostate cancer cell line models.LNCaP cells were obtained from ATCC and maintained according to manufacturer instructions. Prior to cell treatment with either 1 nM R1881 (methyltrienolone) or vehicle (ethanol), cells were grown in Roswell Park Memorial Institute-1640 media containing 10% charcoal-dextran stripped serum for 60 hours.6 cells were harvested for each condition (± androgen). Nuclei were extracted and digested with optimal concentrations of DNase I enzyme. After confirmation of adequate digestion, DNase I-digested ends were blunt ended, and a biotinylated linker was ligated to these ends. Fragments with linker attached were isolated, digested with MmeI, and captured using streptavidin-conjugated magnetic beads. A second linker was ligated to the MmeI-digested end, and then the fragments were amplified and subsequently purified via gel electrophoresis. These sequencing libraries were sequenced on the Illumina GAIIx sequencing platform . Three biological replicates were processed for each cell growth condition. Sequencing results were aligned to the human reference genome (NCBI Build 37) using the Burrows-Wheeler aligner (BWA)[P < 0.05. Gene-relative categories were defined as previously described [DNase-seq was performed as previously described ,53. Briener (BWA). Alignmener (BWA). Discretescribed .To determine regions of significant change in DNase-seq signal with androgen induction, we used the edgeR bioconductor package ,55. The P < 0.05). Finally, neighboring windows that were identified as having a significantly higher DNase-seq signal in a condition were merged. To generate a normalized differential tag count for regions, the number of DNase-seq tags within each LNCaP and LNCaP-induced DHS region was determined and normalized to the average number of tags in either all LNCaP or LNCaP-induced DHS site. For each of the 175,796 union DHS regions, the normalized number of tags in LNCaP in the region was subtracted from the normalized number of tags in LNCaP-induced to give the differential tag score for each region.This approach allows for inclusion of regions that contain an increase or decrease in DNase-seq signal such that they cross the threshold defining a DHS site. The defined union set was then divided into overlapping windows of 300 bp. DHS regions smaller than the window size were expanded to the window size. Regions larger than the window size were tiled with overlapping windows, where the overlap varies depending on the size of the hypersensitive region to tile. We start by finding the number of windows that would fit completely inside the defined DHS site using the default overlap. If these windows discard fewer than 10% of the bases on each edge of the DHS site, we tile the site using these windows. If using the default overlap would cause us to lose more than this edge threshold, we add another window and adjust the overlap so that the windows exactly cover the entire DHS region. We find that these windows cover almost all of the DHS bases in the original, while minimizing the number of non-DHS bases considered for the downstream analysis. Our approach created approximately 550,000 windows for differential analysis among five replicates . The number of tags mapping to each window in each replicate were extracted, and regions with a sum total of less than five reads were eliminated. We then used edgeR to call windows with significantly different counts in each pairwise comparison at two thresholds: strict (FDR < 0.05) and loose  from the same cell growth as used for DNase-seq and hybridized to Affymetrix Exon 1.0 ST arrays  using a standard protocol. Resulting .CEL files were summarized into expression measures at the gene-core level using Affymetrix Power Tools and Robust Multi-array Average (RMA) normalization . DiffereRNA for use in mRNA-seq was isolated from three independent growths of LNCaP with or without androgen using the Ambion miRVANA miRNA isolation kit . Induction of canonical AR target gene expression was confirmed by qPCR, and RNA quality was verified using an Agilent Bioanalyzer . All RNA used for subsequent library preparation had an RNA integrity number greater than 9.0. mRNA-seq libraries were created using the Illumina mRNA-seq protocol and kit then sequenced on the Illumina GAIIx platform. Resulting sequence data was aligned to the human reference genome (NCBI Build 37) first using BWA . Reads uet al. [To establish the relationship between differential chromatin and differential expression, we tested for significance in overlap in both directions, that is, we tested if ΔDNase regions tend to be located near differentially expressed genes, and then tested if differentially expressed genes tend to have a ΔDNase region nearby. We first assigned each DHS site to its nearest gene and intersected these nearest genes with each AR-regulated gene set  from the JASPAR database. The MA0007.1 matrix was trimmed by discarding low information base pair positions surrounding positions 4 to 18, resulting in a 15 bp PWM. PWM motif scores that scored lower than the 90i to all other runs  were compared. Each cluster center from a single run was matched to another cluster center in another run in a pairwise manner that identifies maximum correlation; this procedure was performed across all pairs of runs to assess the similarity and reproducibility of results over multiple runs of the algorithm.For cluster correlation analysis, the k-means algorithm  was run de novo motifs 25 bp up- and downstream from PWMs classified into each cluster. TomTom was used to match significant motif matches to publicly available motifs (E < 0.05).For aggregate visualization of clusters, we tabulated DNase-seq tag counts 100 bp around AR PWM matches classified into each cluster within 'R1881 intersect' ChIP peaks that were DHS in both LNCaP-induced and LNCaP. MEME was used to search for DNase-seq data from this study can be visualized using the UCSC Genome Browser . SpecifiAR: androgen receptor; bp: base pairs; BWA: Burrows-Wheeler Aligner; ChIP-seq: chromatin immunoprecipitation coupled with high-throughput sequencing; CTCF: CCCTC-binding factor; DHS: DNase I hypersensitive; DNase-seq: DNase I hypersensitivity analysis coupled with high-throughput sequencing; FDR: false discovery rate; FOX: Forkhead box; FAIRE: formaldehyde-assisted isolation of regulatory elements; GR: glucocorticoid receptor; kb: kilobase pairs; mRNA-seq: messenger RNA abundance measured by high-throughput sequencing; NF1C: nuclear factor 1 C-type; PCR: polymerase chain reaction; PWM: position weight matrix; qPCR: quantitative polymerase chain reaction; RMA: Robust Multi-array Average; SOM: self-organizing map.The authors declare that they have no competing interests.AKT, TSF, GEC and PGF designed the experiments. AKT and LS performed the experiments. GAC contributed AR ChIP-seq data and revised the manuscript. AKT, GGY, YS, NCS, BST, SGG and UO analyzed the data. AKT, TSF, GEC and PGF wrote the manuscript. All authors revised the manuscript and approved the final version.Supplemental information containing four supplemental figures and three supplemental tables.Click here for fileTable S4 detailing RefSeq genes identified as differentially expressed in response to androgen receptor activation by mRNA-seq. mRNA from three biological replicates of LNCaP and three biological replicates of LNCaP-induced was extracted and sequenced. Genes differentially regulated by AR activation were determined using edgeR at a threshold of FDR < 0.05.Click here for fileTable S5 showing a comparison of androgen receptor-regulated genes as determined by mRNA-seq to previously published androgen receptor-regulated gene lists. Genes identified as differentially regulated by AR activation are shown along with all other RefSeq genes, ranked in order by the number of published studies that identify the gene as AR-regulated.Click here for file


Recent evidence implicates dysregulation of UCH-L1 in the pathogenesis and progression of human cancers. Although typically only expressed in neurons, high levels of UCH-L1 have been found in many nonneuronal tumors, including breast, colorectal, and pancreatic carcinomas. UCH-L1 has also been implicated in the regulation of metastasis and cell growth during the progression of nonsmall cell lung carcinoma, colorectal cancer, and lymphoma. Together these studies suggest UCH-L1 has a potent oncogenic role and drives tumor development. Conversely, others have observed promoter methylation-mediated silencing of UCH-L1 in certain tumor subtypes, suggesting a potential tumor suppressor role for UCH-L1. In this paper, we provide an overview of the evidence supporting the involvement of UCH-L1 in tumor development and discuss the potential mechanisms of action of UCH-L1 in oncogenesis.Ubiquitin carboxyl-terminal hydrolase L1  is an abundant, neuronal deubiquitinating enzyme that has also been suggested to possess E3 ubiquitin-protein ligase activity and/or stabilize ubiquitin monomers  In . In 5]. ynculein , 18. It Although UCH-L1 is almost exclusively expressed in neurons , proteomThe potential oncogenic function of UCH-L1 is supported by a number of clinical studies demonstrating that UCH-L1 expression level in tumors is inversely correlated with patient survivability , 36, 37.In vitro tumorigenesis studies show that UCH-L1 stimulates oncogenic transformation and invasion in nonsmall cell lung carcinoma [To determine whether upregulation of UCH-L1 is a result of oncogenic transformation or itself a driving force of tumorigenesis, the direct involvement of UCH-L1 in cancer pathogenesis has been investigated. arcinoma  and coloarcinoma  cells, sarcinoma . Of thesarcinoma . Furtherarcinoma . Collectin vitro [in vivo in a murine xenograft model [Recently, it has been suggested that UCH-L1 promotes cancer cell motility and invasion, which may contribute to its oncogenic role. Overexpression of UCH-L1 in HCT8 colorectal cancer cells has been reported to enhance cell migration . Additioft model . UCH-L1 ft model . These sDespite growing evidence implicating UCH-L1 as a positive regulator of tumor growth and development, the mechanism by which UCH-L1 conveys oncogenesis is not fully understood. Many of the investigations into the role of UCH-L1 in cancer have focused on upregulation of UCH-L1 in tumor tissues and cancer cells. However, little is known about changes in UCH-L1 enzymatic activity during tumorigenesis. Although one group has observed a decrease UCH-L1 hydrolase activity in cervical carcinoma tissues and an increase in hydrolase activity in transformed keratinocytes , the rolIn contrast to the body of literature identifying UCH-L1 as an oncogene, several reports have been put forth suggesting UCH-L1 acts as a tumor suppressor during the pathogenesis of certain cancers , 46, 50.There are a number of possible reasons for the discrepancies in the observed oncogenic and tumor suppressor functions of UCH-L1 in tumorigenesis. First, studies suggesting UCH-L1 attenuates prostate cancer progression , 50 focuCurrently, the precise mechanism(s) of UCH-L1-mediated tumorigenesis are not fully understood. Previous studies have identified several cancer-related signaling processes that are regulated by UCH-L1, which may contribute to its role in oncogenesis. In particular, UCH-L1 has been implicated in the regulation of cell cycle progression, cell survival, cell motility, and invasion .Disruption of UPS function has been implicated in cancer pathogenesis and progression  and manyα-synuclein through its putative, dimerization-dependent E3 ubiquitin ligase activity [in vivo and substrates other than α-synuclein have not been identified, alterations in UCH-L1 function may disrupt K63-linked polyubiquitination, possibly altering nonproteasomal cellular processes to promote tumorigenesis  [β-catenin-induced EMT  . Recentluced EMT . TherefoIn summary, emerging evidence suggests that UCH-L1 is a potent oncogene that promotes tumor growth and development during the progression of many forms of cancer. However, the exact role of UCH-L1 in oncogenesis remains controversial, as UCH-L1 has been suggested to function as a tumor suppressor in certain tumor types. The observed involvement of UCH-L1 in the regulation of cell cycle progression, cell survival, and metastasis may explain its oncogenic role. However, further studies are needed to clarify the exact mechanisms of action of UCH-L1 in tumorigenesis. Continued investigation into the function of UCH-L1 in cancer may tell us whether or not UCH-L1 can be used as a diagnostic marker. UCH-L1 is upregulated in many cancer tissues and, therefore, high levels of UCH-L1, particularly in nonneuronal tissues, may serve as an early detection biomarker for tumors. Furthermore, UCH-L1 itself could be a potential therapeutic target, which may have benefits for the treatment of cancer. Elucidation of the role of UCH-L1 in cancer may lead to a better understanding of the molecular pathogenesis of tumors as well as potentially facilitate the development of novel cancer therapeutics and diagnostics tools.