Inhibition of androgen receptor (AR) signalling represents the conventional medical management of prostate cancer. Ultimately this treatment fails because tumors develop an incurable, castrate resistant phenotype, resulting in an unmet need for new treatments in prostate cancer. The AR remains a viable therapeutic target in castrate resistant disease, such that novel ways of downregulating AR activities are attractive as potential treatments. Here we describe a mechanism by which the AR can be downregulated by the MDM2 antagonist Nutlin-3, resulting in loss of pro-survival c-FLIP gene expression and apoptosis. We additionally show that loss of c-FLIP sensitises prostate cancer cells to Nutlin-3. Finally, we demonstrate that the unrelated MDM2 antagonist Mi-63 also impinges upon AR signalling, supporting the concept of future treatment of prostate cancer with MDM2 antagonists. Numerous lines of evidence implicate the androgen receptor (AR) in all stages of prostate cancer, the most common male malignancy. Pharmacological inhibition of AR activity by androgen deprivation therapy (ADT) is the mainstay of treatment in prostate cancer, where curative surgery is contraindicated . The majEvasion of apoptosis is widely accepted to be important in tumorogenesis and chemotherapeutic resistance. The pro-survival FLICE-inhibitory protein (c-FLIP) is a master regulator of apoptosis that blocks activation of the extrinsic (death receptor-mediated) apoptotic pathway by inhibiting the formation of the death-inducing signalling complex (DISC) and subsequent inactivation of PROCASPASE 8 –11; the in vivo, including inhibition of LNCaP prostate cancer xenograft growth [p53, the most commonly mutated tumor suppressor gene in human cancers, encodes a transcription factor responsible for cell cycle arrest and apoptosis in response to DNA damage and chemotherapeutic drugs . Interest growth , 18. Intt growth , 20. Altt growth , raisingin vivo is unclear, as is the question of whether newer anti-androgens such as enzalutamide (MDV3100) [Previous work has indeed demonstrated that Nutlin-3 treatment reduces AR protein levels and produces cell cycle arrest and apoptosis in prostate cancer cells expressing active AR and wild-type p53 , 23. AddMDV3100) , which aMDV3100) , might aHere we address some of these questions by providing new insight into Nutlin-3 activity in prostate cancer cells. We show that sensitivity to Nutlin-3 treatment correlates with AR dependency in different cells models, that otherwise have the same p53 response. This suggests that AR signalling is an important determinant of Nutlin-3 efficacy, beyond the p53 response, and offers an explanation for the marked sensitivity of LNCaP cells to Nutlin-3. We go on to show that Nutlin-3 treatment increases AR-MDM2 interactions resulting in reduced AR levels, loss of AR from the pro-survival c-Flip gene promoter, downregulation of c-FLIP expression and subsequent downstream cleavage of pro-apoptotic CASPASE-8. Consequently, Nutlin-3 combined with anti-androgen treatments, or AR depletion, results in widespread apoptosis. Conversely, Nutlin-3 combined with anti-androgen treatment did not enhance cell cycle arrest beyond that observed with Nutlin-3 alone, implying that apoptosis is the key mechanism at play. We propose that prostate cancers retaining AR and p53 signalling might have special significance in the clinical application of MDM2 inhibitors in order to prevent or delay the development of CRPC, which inevitability emergences with the conventional use of anti-androgens.50) was approximately 3μM for both LNCaP and LNCaP(CR) cells, the less AR-dependent LNCaP(hi) cells exhibited an IC50 of 6μM Nutlin-3. Finally, we treated LNCaP cells with the direct AR antagonists enzalutamide or casodex in combination with Nutlin-3 for 72 hr  cells. Application of 4-10μM Nutlin-3 to either cell line, for 24 hr, resulted in a reduction in the number of cells in S-phase to similar levels between the cell lines Figure . AdditioUpon studying apoptosis in LNCaP cells versus LNCaP(hi) cells, we noted a striking difference between the cell lines in their response to Nutlin-3 Figure . WhereasIn order to understand the mechanism through which Nutlin-3 impacts upon AR signalling, we examined the AR-MDM2 interaction. LNCaP cells were treated with Nutlin-3 for 15-30min prior to AR immunoprecipitation Figure . This shThe subcellular localisation of AR and MDM2 was also examined in response to Nutlin-3 in LNCaP cells Figure . In untrc-FLIP gene is important in prostate cancer [c-FLIP expression, we studied both transcript and protein levels in LNCaP cells exposed to pro-apoptotic concentrations of Nutlin-3. After 48hr, exposure of LNCaP cells to Nutlin-3 reduced c-FLIP transcript levels by more than 80%  in LNCaP cells. Having shown a decrease in AR levels in response to Nutlin-3, we postulated that recruitment of AR to cis-acting androgen response elements (AREs) might also be impaired in this context. Specific oligonucleotides were used to amplify a region of the human c-FLIP promoter containing 4 characterised AREs upstream from the transcription start site. As shown in Figure c-FLIP promoter to levels detected with a non-specific control HA antibody  cells. Casodex resistant LNCaP(CR) cells have been previously described . ProlifeCell cycle profiles were obtained at 24hr exposure to Nutlin-3, whilst cells remained viable, by permeablising in 1% Triton-X-100 in PBS followed by treatment with 100 μg/ml RNase and 500 μg/ml propidium iodide for 10 min. Cells were then washed in PBS prior to analysis.Apoptosis was measured using either active caspase-3  or Annexin V / propidium iodide staining  as described by manufacturer. No cells were gated out of analysis. In the case of caspase-3 a discrete population was observed and quantitated respective to the unstained population see . For AnnAntibodies included MDM2 (Santa Cruz), p53 and p21 , AR (BD Biosciences), Caspase-3, 8 and PARP as described . Nutlin-Ni-NTA agarose (Qiagen) was used to purify His-tagged ubiquitin modified proteins under reducing conditions as previously described . Immunop


Diminished ovarian reserve(DOR) is associated with female infertility and poor response to ovarian stimulation. Our objective was to assess the effect of dehydroepiandrosterone(DHEA) on DOR women and to explore whether the improvement of ovarian response after DHEA supplementation was dependent on the expression levels of androgen receptor(AR).n = 53), which received DHEA supplementation (25 mg three times a day) for 8 weeks, and the control group (n = 50), which did not receive DHEA, before the IVF cycles. Serum hormone levels, antral follicle count(AFC) and the expression of AR and FSH receptor(FSHR) in granulosa cells(GCs) were measured, meanwhile ovarian response parameters and IVF outcomes were compared. The GCs from another 36 DOR women were cultured with different concentrations of DHEA in vitro. Then, we compared the expression of AR and FSHR in GCs according to the different numbers of oocytes retrieved both in DHEA and control group.A prospective cohort study was performed in the Department of Human Reproductive Medicine, Beijing Obstetrics and Gynecology Hospital during August 2014 to August 2016. 103 DOR women who completed the study were divided into the DHEA group (P = 0.047), DHEAs(P = 0.019) and AR mRNA expression in GCs(P = 0.049). In vitro experiment, the protein and mRNA expression of AR and FSHR in the preovulatory GCs were significantly increased in response to DHEA supplementation(P <0.05). No significant differences were found in ovarian reserve, ovarian response, or IVF outcomes between the two groups. Subgroup analyses showed the levels of AR and FSHR mRNA in GCs were significantly increased in DHEA group with ≥5 oocytes retrieved(P <0.05).In the present study, DHEA supplementation resulted in significantly higher levels of serum T. Retrospectively Registered 19 March 2015.The Chinese Clinical Trial Registry ( Womehave DOR –6. Thesehave DOR . Althouget al [2, AMH and inhibin B (INHB) significantly improved after DHEA supplementation [It is believed that androgens could promote folliculogenesis and potentiate the effects of gonadotropin(Gn) , 9. In ret al  reportedet al . A prospentation . A randoentation . Howeverentation . Similarentation  found thet al found that AR promoted follicle progression from pre-antral stage to the antral stage by a GCs-specific ARKO mouse model [The mechanism of actions following DHEA administration remains speculative. It has been reported that DHEA affects early follicle maturation by regulating androgen receptor (AR) transcription, increasing FSH receptor (FSHR) expression, modulating FSH activity in granulosa cells (GCs) and increasing the number of growing preantral and small antral follicles –22. It wse model . DespiteIn the present study, we aimed to assess the effects of DHEA on ovarian reserve, ovarian response, and pregnancy outcomes of DOR women undergoing IVF treatment and to compare the expression of AR and FSHR in preovulatory granulosa cells both in vivo and in vitro and to investigate the relationship between AR, FSHR expression in granulosa cells and the numbers of oocytes retrieved in order to explore the potential benefits of DHEA supplementation.2 > 80 pg/ml, AMH <0.5-1.1 ng/ml and AFC ≤5 on day 2-3 of a spontaneous cycle. Patients were excluded if they had a diagnosis of polycystic ovary syndrome (PCOS), leiomyoma, endometriosis, metabolic disorders, or a history of ovarian or pelvic surgery, pelvic irradiation, hormonal treatment or DHEA supplementation.In our study, participants gave their written informed consent after a detailed explanation regarding the meaning of ovarian deserve and information about the possible beneficial effects and potential side effects of DHEA. All procedures and sample collection methods were approved by the Human Ethics Committees of Beijing Obstetrics and Gynecology Hospital, Capital Medical University. The subfertile women undergoing IVF or intracytoplasmic sperm injection-embryo transfer (ICSI-ET) therapy were due to tubal and/or male factors or failure of intrauterine insemination (IUI) for 3 times. The inclusion criteria included  age <40 n = 54), patients were administered a 25-mg DHEA capsule three times daily for 8 weeks prior to their first IVF cycle. Another 52 patients comprised the control group. Of the 106 patients, 1 withdrew from the DHEA group, 1 was lost to follow-up and 1 went to another hospital in the control group. Accordingly, 103 patients completed the entire study (53 in the DHEA group and 50 in the control group). The preovulatory GCs from another 36 DOR patients were collected and cultured for in vitro experiment. This study was registered with the Chinese Clinical Trial Registry (ChiCTR-IPR-15006126).A prospective, cohort study was conducted from August 2014 to August 2016 in Department of Human Reproductive Medicine, Beijing Obstetrics and Gynecology Hospital. A total of 106 patients who understanded both the advantages and disadvantages of each method were divided into a treatment group and a control group according to whether they received DHEA  treatment before their first IVF cycles. In the DHEA treatment group (DOR women received the Gonadotropin-releasing hormone (GnRH)-antagonist protocol. Exogenous gonadotropins  or Letrozole followed by 150 IU/d human menopausal gonadotropin (hMG)  were administered until the follicles reached maturity. GnRH-antagonists  were initiated when the largest follicle reached a diameter of 13-14 mm. Human chorionic gonadotropin (hCG)  was administered when the leading follicle reached a diameter of 18 mm or at least two follicles reached 17 mm in diameter. Oocyte retrieval was performed 34-36 h after hCG administration. A maximum of two embryos were transferred 2-3 days after retrieval. Luteal support was started on the day following oocyte retrieval and continued for an additional 9-10 weeks. The achievement of a biochemical pregnancy was assessed by the serum hCG assay 14-16 days after the embryo transfer. A clinical pregnancy was defined as visualization of a gestational sac under ultrasonography 30-35 days after the embryo transfer.2, T, and luteinizing hormone (LH). Serum basal FSH, LH, E2 and T were measured using the Beckman Coulter Access II Immunoassay system, and basal DHEAs was measured using the DHEAs Enzyme-linked Immunosorbent Assay Kit(ELISA) kit . Serum AMH and INHB levels were measured using an AMH and INHB ELISA Kits . All experiments were performed according to the manufacturer’s instruction. The intra-assay coefficients of variation (CVs) were less than 10% for AMH and INHB, less than 9% for DHEAs. The inter-assay CVs were less than 15% for AMH, INHB and DHEAs. The detection range was 0.24-11.78 ng/ml for AMH, 19.67-147.62 pg/ml for INHB and 8-400 μg/dl for DHEAs. The AFC in both ovaries were measured by trans-vaginal ultrasonography scans on day 2-3 of menstrual cycle in all patients.Serum was collected on Day 2-3 in the menstrual period before and after DHEA treatment prior to ovulation induction. All samples were stored at -80 °C until they were assayed. Serum samples were assayed for AMH, INHB, DHEAs, FSH, Eg for 10 min at room temperature and follicular fluid was removed. Granulosa cells were separated from red blood cells and others by centrifugation through 50% Percoll  for 15 min at 1000 g. The GCs in the intermediary layer were harvested. Following centrifugation and washing with phosphate-buffered saline (PBS), the cells were resuspended in RPMI-1640 containing 10% foetal bovine serum (FBS) and 1% antibiotic-antimycotic , then centrifuged at 400 g for 2 min at room temperature. The supernatant was discharged and the GCs were placed at -80 °C for RNA extraction.Human granulosa cells were collected from the follicular fluid of dominant follicles (follicle diameter ≥ 14 mm) undergoing oocyte retrieval and purified by density centrifugation from follicle aspirations, as described by Nie et al. and Shi et al , 27. Eac2 at 37 °C. The cultured GCs were then removed from the dishes after treatment with 0.25% trypsin at 37 °C and collected by centrifugation.Preovulatory GCs from another 36 DOR patients without DHEA supplementation were purified, pooled and seeded in 6-well or 12-well plates to ensure that they attained 40–50% confluence by the next day. Then, GCs were treated with a DMSO control or different concentrations of DHEA  (Sigma) for 24 or 48 h in a humidified atmosphere of 5% COTotal RNA was extracted from human ovarian GCs using the TRIzol reagent  according to the manufacturer’s instructions. cDNA was synthesized using 2 μg of RNA as previously reported . Quantit3VO4, 125 mM NaF, 2.5 mg/ml aprotinin, 25 mg/ml trypsin inhibitor, and 25 mM PMSF). The protein concentrations were determined using a BCA Protein Assay Kit . A total of 20 μg of the total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electrotransfer onto nitrocellulose membranes. The membranes were blocked with 5% skim milk for 1 h and incubated overnight at 4 °C with specific primary antibodies . After washing the membranes three times with 0.1% Tween in Tris-buffered saline (TBST), the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies  at room temperature for 1 h. The reactive bands were visualized using an enhanced chemiluminescence kit (Thermo Fisher Inc. Shanghai. China). The final results were analysed using Scion Image software (http://scion-image.software.informer.com/).Western blot analysis was performed as described by Yang et al . BrieflyU test. The chi-square test was used to compare fertilization, embryo quality, embryo implantation, and clinical pregnancy rates between the two groups. Bonferroni adjustment was for multiple comparisons. The Spearman correlation coefficient was used to assess the relationship between T, DHEAs and other variables. A P value <0.05 was considered to be of statistical significance.All data were analysed using SPSS 21.0 software . The results are expressed as the means ± standard deviations (SDs), and normally distributed data were compared by means of independent samples t-tests. Non-normally distributed data were compared using the Mann-Whitney 2, T, P, AMH, INHB, and DHEAs) and AFC, were not significantly different (P >0.05) between the DHEA and control groups . The serum FSH level (9.51 ± 1.78 IU/L) decreased, while AFC (5.34 ± 1.68) along with the levels of AMH (0.96 ± 0.16 ng/ml) and INHB (49.01 ± 15.56 pg/ml) increased in the DHEA group; however, there were no significant differences between the DHEA and control groups (P >0.05)  Table .Table 2C2 (1903.28 ± 1178.43 pg/ml vs. 1578.93 ± 956.63 pg/ml) and endometrial thickness on the hCG day (9.73 ± 1.66 mm vs. 9.29 ± 2.11 mm) were increased, while the total Gn dose (2292.45 ± 842.27 IU vs. 2555.00 ± 963.94 IU) and Gn stimulation duration (9.79 ± 1.71 d vs. 10.18 ± 1.90 d) were decreased. There were increased numbers of retrieved oocytes (4.51 ± 2.40 vs. 3.70 ± 2.00), higher rates of embryo implantation (23.08%) and clinical pregnancy (33.33%) in the DHEA group, although there were no significant differences between the DHEA and control groups  and the control group (n = 29). The results were summarized in Fig. p = 0.049). FSHR mRNA expression increased in DHEA group but there was no significant difference between the two groups (p = 0.064).We next detected the expression levels of AR mRNA and FSHR mRNA in preovulatory GCs collected from corresponding follicular fluid of DOR patients so as to find whether their expression was influenced by DHEA pre-treatment during COS. The mRNA levels of the AR and FSHR were quantified by real-time PCR and the relative levels were compared between the DHEA group  for 24-48 h, DMSO as control. We observed that the AR mRNA and protein expression levels increased with increasing DHEA concentration. The expression of AR reached the highest level at 30 ng/ml DHEA, and subsequently decreased Fig. . Similarr = 0.457), high-quality embryos (r = 0.462), AFC(r = 0.310), E2 on HCG day (r = 0.215), AR mRNA expression (r = 0.769) and FSHR expression (r = 0.616), and were inversely related to the age (r = -0.253). The serum DHEAs levels were positively correlated with the number of retrieved oocytes (r = 0.579), AR mRNA expression (r = 0.362) and FSHR expression (r = 0.320), and were negative correlated with bFSH (r = -0.245)  and that DHEA supplementation augments ovarian stimulation in poor responders , 31. As n = 16) and <5 in group B (n = 14). As shown in Fig. P <0.05). The control group was also divided into two subgroups (C and D) based on the number of oocytes retrieved. The number of retrieved oocytes was ≥5 in group C (n = 12) and <5 in group D (n = 17). The expression of AR and FSHR mRNA increased, but they didn’t reach significant difference (p = 0.268 and p = 0.057)  based on the number of oocytes retrieved. The number of retrieved oocytes was ≥5 in group A (57) Fig. .Fig. 3ThDiminished ovarian reserve is associated with a poor ovarian response (POR) to gonadotropin stimulation and IVF outcomes. Whether pretreatment with DHEA improves the ovarian response in DOR patients remains debated. In the present study, no statistically significant improvement in ovarian reserve markers, ovarian response, or IVF outcomes was found. However, subgroup analysis showed that the expression of AR and FSHR mRNA in GCs were significantly increased in women with ≥5 oocytes retrieved after DHEA pretreatment, suggesting that DHEA may improve the ovarian response in DOR women through promoting the expression of AR and FSHR in GCs.2 levels on hCG day, the numbers of oocyte retrieval and fertilization slightly increased after DHEA pretreatment. It appeared that DHEA had a positive effect on ovarian response in DOR women and we need a large sample research to verify the efficiency. Gleicher N et al [Although many studies have focused on the effects of androgens and AR on follicular development, the conclusions are controversial. A recent meta-analysis indicated the clinical pregnancy rate was statistically increased in DOR patients who were pre-treated with DHEA, there was no significant improvement on the number of oocytes retrieved and the cancellation rate, and there was a non-significant difference in the clinical pregnancy rate when data were restricted to RCTs . DHEA is N et al  reportedThe action of androgen is mediated by AR, as a member of nuclear receptor superfamily encoded by an X chromosomal gene, interacts directly with target genes to regulate follicle development and function , 39. MeaTo further investigate the regulation action of DHEA on AR and FSHR, we cultured preovulatory GCs from another 36 DOR women with different concentrations of DHEA in vitro. AR and FSHR mRNA and protein expression was increased in a concentration-dependent manner Fig. . The isoIn the present study, we found positive correlations between the serum T, DHEAs and oocytes retrieved, the expression of AR and FSHR mRNA Table , indicatIn conclusion, DOR women who received DHEA supplementation display increased expression of AR mRNA in GCs, suggesting that DHEA pretreatment upregulate the AR expression. The selective beneficial effect of DHEA on ovarian response in DOR women may be associated with the upregulation of AR and FSHR.


Neoceratium has particularly remarkable features that include its easily recognizable outline and large size. This ubiquitous genus shows consistent presence in all plankton samples and has been a model for numerous studies since the end of the 19th century. It has already been described as a good candidate to monitor water masses and describe ocean circulation. We argue that the sensitivity displayed by Neoceratium to water temperature also makes it relevant as an indicator of ocean warming. The advantages and interests of using Neoceratium species to monitor climate change on a large scale are reassessed in view of recent advances in understanding their biology and ecology.Among the planktonic dinoflagellates, the species-rich genus  Ceratium as curious anchor-shaped or three-horned organisms. Of the dinoflagellates, the most famous genera are probably Ceratium Schrank and Protoperidinium Bergh, emend Balech, 1974, the history of which is very similar. As for Protoperidinium, the first descriptions of Ceratium taxa are very ancient and for both genera, the marine species have only recently been separated from freshwater ones and grouped under a different genus name, after studies provided morphological evidence favoring such a division [Neoceratium, proposed by Gómez et al. [Ceratium.Most scientists who have had the opportunity to observe marine phytoplankton under a microscope describe z et al. , identifNeoceratium. An observed increase in the number of records during the last 25 years of the 20th century likely corresponded more to an increasing trend in the number of publications in all fields. Indeed, numerous studies that both do or do not focus on phytoplankton, mention, cite or list Neoceratium species  whereas others show a robust outline  tending towards compactness. The slender forms may thus reflect an adaptation of a species to improve their floatability in warmer waters characterized by lower viscosity. On the other hand, the higher viscosity of colder waters would offer better floatability to cells thus allowing the development of compact and robust forms (Neoceratium genus [The first illustrations of y Müller  date froy Müller . Since tfic taxa . From hist forms . Accordium genus . To clarum genus ). AlthouNeoceratium one of the preferred models in studies focusing on phytoplankton, in particular experimental studies investigating different aspects of dinoflagellate ecophysiology, including their bioluminescence [Neoceratium constitute a great advantage in terms of enabling easy discrimination and isolation from a sample. Abundance, frequency and easy recognition are all important factors to consider when choosing a biological model as together they ensure a constant supply of cells and permit the reproduction of experiments and measurements. Accordingly, Neoceratium represents an ideal candidate as a biological model.Its frequent occurrence in water samples has made nescence ,10, flagnescence ,12, cellnescence ,14,15, dnescence , trophicnescence ,18,19. TNeoceratium is also an interesting model for studying water masses and current regimes. For instance, analysis of the valuable time-series provided by the Continuous Plankton Recorder in the North Atlantic Sea has revealed a strong relationship between the occurrence of dinoflagellates, including numerous Neoceratium species, and the description of the water circulation [Neoceratium breve which is considered as an indicator of Equatorial Surface Waters [culation . In the culation  as some e Waters ,22.Neoceratium species are restricted to marine regions characterized by specific thermal conditions [Neoceratium could be defined by the analysis of sampling stations [Neoceratium species appears to be one of the rare dinoflagellates that can be considered as Arctic-boreal taxa [In addition, several biogeographical investigations have confirmed that the majority of nditions ,23. A landitions : an arctstations . In the eal taxa .Neoceratium, i.e., ubiquity, frequency, taxonomic richness and sensitivity to temperature, suggest this genus may provide interesting ecological indicators to monitor global warming. However, some prerequisites are needed to justify the relevancy of an ecological indicator. The selection of ecological indicators should ensure their meeting several criteria. They should be easy to measure, have high sensitivity and known response to stress, have predictable responses to stress, be anticipatory , display disturbance and changes over time , and show low variability in terms of responses [Neoceratium meeting several of these conditions  are sufficient to perform taxonomic identification of Neoceratium taxa [Neoceratium is exclusively based on morphological criteria which mainly consider the global size and shape as well as the curvation of the horns [Neoceratium taxa at the species and infraspecies levels [Neoceratium occurrence. Although phytoplankton have a short generation time, and should theoretically respond very quickly to environmental changes, their dynamics are driven by different ecological factors which results in large annual and inter-annual variability in terms of species assemblages [Neoceratium species show consistent all year round and year-to-year presence [Neoceratium species to water temperature and how they respond to temperature increase may therefore permit the anticipation of the likely response of the genus to future ocean warming. Numerous studies have thus investigated the response of Neoceratium to warming. In terms of abundance, warming seems to have a positive effect on the Neoceratium species, as with other dinoflagellates [Neoceratium species [Neoceratium species increased in the North Sea during the post-90s while the central point of the spatial distribution pattern of Neoceratium furca moved northward as a likely consequence of climate change [Neoceratium [Neoceratium composition during the last century, as shown by a study conducted in the northwestern Mediterranean Sea, based on comparing old data gathered in the literature to new data obtained from analysis of recent samples [Neoceratium species in the Ligurian Sea, which tended to become closer to the assemblage characterizing the Tyrrhenian waters. In addition, a decrease in species richness was observed during the warm season in surface samples while it increased in vertical ones, suggesting that deeper localization may represent another possible phenological response of stenothermic taxa to warming. Clear patterns have thus been identified in the response of Neoceratium to increases in water temperatures, in terms of biomass, composition and phenology.Monitoring one or several ium taxa . The idehe horns . Recentls levels . The useemblages . This mapresence ,27,30. Tgellates . In the  species . This tre change . Phenoloceratium . WarmingNeoceratium as ecological indicators is also relevant with regards the need to anticipate changes in the community structure and/or the environment due to climate change. Indeed, phytoplankton presents short generation times, which ensure their fast response to and thus our early detection of any modification or perturbation of the environment.The choice of phytoplanktonic species like Neoceratium has been studied for decades and as such the literature provides a lot of information describing its response to natural perturbations and anthropic stresses. This is an important prerequisite to evaluate a target response to a specific change (here warming) from the established pattern of known responses [Neoceratium species and clustering them according to their biogeographical affinities [Neoceratium species based on the review of phytoplankton studies [Neoceratium species in the different marine basins, or to identify patterns of biogeographical extension.esponses . Thus, dfinities ,4,23,24  studies ,38,39,40Neoceratium does not really meet this criterion as variability of morphology is one of its features known to be influenced by several parameters, including water temperature. Some of these morphological changes reflect physiological processes that have, until recently, been completely ignored. For example, the successful cultures of Neoceratium ranipes revealed that the variations in the number and length of digitations at the extremities of the antapical horns  that extended their distribution range from the eastern basin and the Atlantic with warming [Monitoring total on range . In facton range  and cilion range . In Euroon range . As an e warming .Monitoring the long-term trends in the richness of specific species can also constitute a good indicator of warming. In particular, some stenotherm species have been shown to disappear from surface waters in warmer conditions . This tyNeoceratium assemblage previously described. The thermal range of a species may differ from one basin to another. For instance, one recent study focusing on the annual cycle of Neoceratium in the Ligurian Sea underlined some discrepancies with the thermal ranges of occurrence previously described in biogeographical analyses conducted in other oceanic regions than the Mediterranean Sea, especially with regards to some warm-water species occurring in colder waters than expected [Surveying one or a small number of specific also is able to reflect a trend in the response to warming with the advantage of considerably limiting the monitoring effort. For such a survey, the selection of the target species should depend on geographical location and consider the species’ biogeographical characteristics as well as any local expected . The numNeoceratium and reinforce the interest in using this genus as a biological model in marine research studies. Finally, the monitoring of Neoceratium species as an indicator of ocean warming offers undisputable advantages including moderate counting effort as the only genus which is both cosmopolitan and perennial with easily recognizable cells for easy isolation from samples. In addition, current literature provides a rich database made up of regional species checklists and biogeographical resources. This biological model may also meet the requirements of new ecological indicators by reflecting the phytoplankton composition rather than being solely based on chlorophyll-derived biomass.These recent data provide new insights into the diversity and taxonomic richness of Neoceratium species, in the context of modern oceanographic campaigns aiming to assess marine biodiversity on a global scale. One example would be the 2.5 year Tara Oceans expedition undertaken to explore the complex properties of the planktonic ecosystems [The development of digitization tools over the last few decades will also provide new perspectives for plankton surveys on ocean or even worldwide scales . For insosystems .


AR) and location of the Janus boundary (α) separating the polar and apolar regions of the particle. The chemical factor indicated the supplementary wettability (β) of the two sides of the particle with identical deviations of apolarity and polarity from neutral wetting. These two factors competed with each other to determine particle configurations at the interface. In general, the critical value of β (cβ) required to preserve the pinned configuration was inversely proportional to the values of α and AR. From the numerical calculations, the empirical relationship of the parameter values of Janus ellipsoids was found; that is, AR > 1, the cβ value is consistent with the boundary between the tilted only and the tilted equilibrium/upright metastable region in their configuration phase diagram. We believe that this work performed at the single particle level offers a fundamental understanding of the manipulation of interparticle interactions and control of the rheological properties of particle-laden interfaces when particles are used as solid surfactants.We investigated the geometric and chemical factors of nonspherical Janus particles  with regard to the pinning and unpinning behaviors of the Janus boundary at the oil–water interface using attachment energy numerical calculations. The geometric factors were characterized by aspect ratio ( Typical colloidal particles with appropriate wettability can be irreversibly adsorbed at fluid–fluid interfaces  ,2. StronAR) is stronger than the chemical effect , whereas particles under the opposite conditions adopted upright configurations. Janus particles with tilted configurations likely deformed their surrounding fluid interface due to unpreferred wetting , and the resulting interface deformation led to lateral capillary interactions between particles to minimize the surface areas of the deformed interfaces [AR values adopted tilted configurations and exhibited strong capillary attractions. In contrast, low AR particles adopted upright configurations and attractive interactions occurred negligibly.Janus particles that possess chemical and/or geometric anisotropy can improve the stabilization efficiency of Pickering emulsion systems when used as stabilizers ,13,14,15terfaces ,37,38,39terfaces ,34,40. AAR and wettability values, two energy minima appeared in the attachment energy profile; one corresponding to an upright configuration, and the other to a tilted configuration—one of the two configurations should be kinetically stable. With regard to Janus ellipsoids, it is unknown whether the Janus boundary of the particles can be detached or unpinned from the fluid–fluid interface, regardless of configuration. These pinning and unpinning behaviors might depend on the relative effect of chemical and geometric anisotropy. Indeed, it was previously reported that the Janus boundary of Janus spheres could be unpinned from the interface when the boundary significantly deviated from the central region of the particles [AR value is larger than 1.The configurational behaviors of two types of non-spherical Janus particles  have been theoretically investigated by numerically calculating the attachment energy at the fluid–fluid interface as a function of orientation angle ,17. Witharticles ,32. In tAR), the location of the Janus boundary (α), and the supplementary wettability (β) as shown in AR = c/a. Depending on the value of AR, the ellipsoids are prolate or oblate at AR > 1 and AR < 1, respectively, with a sphere formed at AR = 1. We designate all studied particles as Janus ellipsoids. The Janus boundary that indicates the wettability separation line (WSL) dividing polar and apolar regions is characterized by an elevation angle, α. The supplementary wettability is defined as α = 180°) and apolar (α = 0°) spheres when trapped at an oil–water interface, respectively  and a particle immersed in the aqueous phase (wE) areijS is the surface area (i = P (polar) or A (apolar)) exposed to a fluid phase (j = w (water) or o (oil)), ijγ is the corresponding surface energy of surface i and fluid j, IS) corresponds to the cross-sectional area of the particle at vα, which is the elevation angle measured from the major axis to the fluid interface , as shown in The respective energies of a particle at the oil–water interface ; therefore, the shapes of the attachment energy profiles obtained from the equations should be identical. Since the use of either Equation (6) or (7) predicts the same configurational behavior, Equation (6) is used in this work. To calculate the surface area ijS, a numerical method using the hit-and-miss Monte Carlo method is employed [owγ = 50 mN/m, corresponding to the interfacial tension of a decane–water interface.The difference between Equations (6) and (7) is the reference energy state  ,41. In tAR > 1, there are four possible cases in their configuration behaviors, depending on AR and wettability values, such as the upright only, the upright equilibrium/tilted metastable, the tilted equilibrium/upright metastable, and the tilted only regions [vα) as well as the orientation angle (0° < rθ < 90°), in which rθ = 0° and 90° indicate the geometries of the Janus boundary parallel and perpendicular to the interface, respectively. In this case, the attachment energy is scanned by varying the vα values at a constant rθ. The minimum attachment energy is then calculated at the given rθ value, and the same procedure is repeated for different values of rθ to find a global energy minimum, Notably, for Janus prolates with  regions ,17. For For spherical Janus particles with supplementary wettability  should satisfy the condition of α = v,minα) and unpinning  behaviors of the Janus particles with respect to the interfaces can be determined through two competing factors: geometric and chemical anisotropy. To minimize the attachment energy in Equation (6), a Janus particle tends to be aligned toward increasing the displaced area (IS) at the oil–water interface, simultaneously increasing the surface area of the preferred wetting state  [Pinning (oderate) . In thisAR and α) and wettability (β) on the pinning and unpinning behaviors of Janus ellipsoids, the attachment energy , 1 (sphere), or 2 (prolate). Since the supplementary wettability (β) is used and Janus particles with α ≤ 90° are considered, α > 90°.To investigate the effects of geometry  and preferred wetting (AoS) in Equation (6) when α = v,minα.For the Janus ellipsoids with AR and β values can also adopt the tilted configuration. As shown in the configuration phase diagram in β and low AR values only possess the upright or the pinned configuration (light blue region), whereas two coexisting regions (pink and yellow regions) appear when particles carry the opposite properties . For instance, for particles with AR = 2 and β = 10° or 22° , configuration behavior depends on the relative geometry and wettability effects. As shown in AR = 0.5, the value of v,minα at β, and is not consistent with α if the value of β is not sufficiently high. The condition of v,minα = α indicates that a pinned geometry is found at a critical value of β, in which cβ = 67°. Similar results are found for spheres with AR = 1  around the central regions of the particles; thus, the geometric effects become stronger than the wettability effects. Note that the value of cβ = 30° for the Janus sphere obtained from the numerical calculation is consistent with the theoretical prediction of When the particles are geometrically asymmetric , strong wettability effects are required to preserve the pinned configuration. As shown in AR = 0.5, the value of v,minα consistently decreases as β increases, and does not match the value of α = 30° up to β = 50°. Due to large values of IS around the central regions of the oblate, geometric effects are likely to be dominant to wettability effects, resulting in particles with central regions located at the interface, adopting an unpinned configuration. For Janus ellipsoids with lower AR values, wettability effects gradually become significant. As shown in AR = 1 and 2 are pinned to the interface when the critical wettability value is cβ = 60° or 22°. Note that the value of cβ increases as the particles become more geometrically asymmetric (cβ = 30° and 8° for particles (α = 60°) with AR = 1 and 2, respectively).For Janus ellipsoids with a high degree of geometric asymmetry  is inversely proportional to the values of α and AR, which are due to geometric effects. As the values of AR decrease, the particles demonstrate reduced attachment energy when the interface is located at the central regions of the particles. When the value of α decreases , a higher value of β is required to adopt a pinned configuration. More quantitatively, to find a relationship of cβ, α, and AR, the cβ values are plotted as a function of α, as shown in AR values are shown in the inset plot in AR = 1, an empirical relationship for Janus ellipsoids is obtained, Additionally, the critical values of AR > 1) with β < cβ is unpinned, they rotate and adopt the tilted configuration to further decrease their attachment energy by increasing the displaced area (IS) [cβ value corresponds to the boundary between two configuration regions—the tilted only and the tilted equilibrium/upright metastable regions.Finally, it is notable that when the Janus boundary of Janus prolates (rea (SI) ,17 and, α and AR) and chemical (β) anisotropy values. For geometrically and chemically symmetric Janus ellipsoids (α = 90°), the Janus boundary was always pinned to the interface, regardless of the values of AR and β. Contrarily, for geometrically asymmetric Janus ellipsoids with α ≠ 90°, the Janus boundary was unpinned when chemical effects (β) were not sufficiently high. In general, particles with large values of α and AR required stronger wettability to preserve the Janus boundary pinned to the interface. It was also found that determination of the critical values of β as functions of α and AR yielded the empirical relationship cβ corresponded to the boundary between the tilted only and the tilted equilibrium/upright metastable regions in their configuration phase diagram. In the case of non-supplementary wettability where AR ≤ 1). For Janus prolates that can adopt tilted configurations, it was reported that the relative strength of Pθ and Aθ significantly affected the tilted angle and the configuration behaviors [Pinning and unpinning behaviors of the Janus boundaries of Janus ellipsoids at the oil–water interface were quantitatively investigated using numerical calculations of the attachment energy. Particle configurations were determined via two competing factors: geometric (ehaviors . We beli


Variation in the 10 toll-like receptor (TLR) genes has been significantly associated with allergic rhinitis (AR) in several candidate gene studies and three large genome-wide association studies. These have all investigated common variants, but no investigations for rare variants (MAF ≤ 1%) have been made in AR. The present study aims to describe the genetic variation of the promoter and coding sequences of the 10 TLR genes in 288 AR patients.Sanger sequencing and Ion Torrent next-generation sequencing was used to identify polymorphisms in a Swedish AR population and these were subsequently compared and evaluated using 1000Genomes and Exome Aggregation Consortium (ExAC) data.TLR10-TLR1-TLR6 locus was the most variable, while the TLR7-TLR8 locus was consistently showing a much lower level of variation. The AR patients had a total of 37 promoter polymorphisms with 14 rare (MAF ≤ 1%) and 14 AR-specific polymorphisms. These numbers were highly similar when comparing the AR and the European part of the 1000Genomes populations, with the exception of TLR10 where a significant (P = 0.00009) accumulation of polymorphisms were identified. The coding sequences had a total of 119 polymorphisms, 68 were rare and 43 were not present in the European part of the 1000Genomes population. Comparing the numbers of rare and AR-specific SNPs in the patients with the European part of the 1000Genomes population it was seen that the numbers were quite similar both for individual genes and for the sum of all 10 genes. However, TLR1, TLR5, TLR7 and TLR9 showed a significant excess of rare variants in the AR population when compared to the non-Finnish European part of ExAC. In particular the TLR1 S324* nonsense mutation was clearly overrepresented in the AR population.The overall level of genetic variation was clearly different among the 10 TLR genes. The TLR1, TLR5, TLR7, TLR9 and TLR10. This further emphasizes the frequently reproduced TLR10-TLR1-TLR6 locus as being involved in the pathogenesis of allergic rhinitis.Most TLR genes showed a similar level of variation between AR patients and public databases, but a significant excess of rare variants in AR patients were detected in The online version of this article (doi:10.1186/s12881-017-0379-6) contains supplementary material, which is available to authorized users. Allergic rhinitis (AR) is a global illness with a well-recognized impact on quality of life and work performance. It is characterized by nasal obstruction, secretion and itching and is often associated with symptoms of the eyes, fatigue and asthma . The herTLR1, TLR6 and TLR10 are located in a 90 kbp region on chromosome 4, TLR7 and TLR8 are located in a 57 kbp region on the X chromosome, whereas the remaining genes are located in separate loci on chromosome 1 (TLR5), chromosome 3 (TLR9), chromosome 4 (TLR2 and TLR3) and chromosome 9 (TLR4). Earlier studies of genetic variation have reported a large number of polymorphisms for all 10 TLR genes . The 1000Genomes data set was then subdivided into four separate populations; individuals of European , African , Asian  and South American origin . Allele frequencies were calculated for each variation using the same allele as referent for all populations. Missense mutations identified in the study population and in the 1000Genomes population were investigated using SIFT [Publically available information on the polymorphisms in the 10 TLR genes were extracted from dbSNP  [date  accessed]) were also used to test for accumulation of rare variants in the coding regions of the ten TLR genes. The non-Finnish European population of ExAC consists of > 30.000 individuals and were used for the extraction of polymorphisms (excluding indels) present in the coding region of the ten TLRs. A one-sided simulation test was used to investigate for the probability of an excess of rare variants in the AR patients compared to ExAC data.Three different statistics describing the spectrum of variation were calculated to investigate for the accumulation of rare TLR variants in AR patients. The first statistic calculated the number of sites where the minor allele frequencies (MAF) were ≤ 1% in patients (AR population) and controls (EUR population). The second statistic calculated the number of variants that were unique to either AR patients or EUR controls and the third statistic compared SNPs detected in patients and controls using information obtained in SIFT and PolyPhen-2 analysis. A one-sided permutation test was used to test all three statistics for equality of the populations and the three statistics were summarized for each individual gene and for the sum of all genes. One-sided tests were used since it was the possible accumulation of rare variants in the AR-population that was under investigation. The permutation test randomized the alleles among patients (AR population) and controls (EUR population) for each variable site and each of the three statistics were calculated 100 000 times. The Polymorphisms where at least one of the AR and EUR populations showed a MAF ≥ 0.05 were also tested for association with AR using the normal approximation test. For detailed descriptions of bioinformatics and genetic analysis see Additional file TLR2 and TLR5 had slightly lower quality scores with > 90% of bases having a Phred score of 20 or higher in > 75% of individuals. A total of 37 polymorphisms were detected, 25 of these were present in dbSNP and 12 were not (Table TLR10 harbouring the highest number of variants (13), equalling 35% of all detected polymorphisms. TLR1 and TLR6 which both reside in the same locus as TLR10 also show higher than average (3.7) numbers of polymorphisms (5 polymorphisms each). TLR7 and TLR8 which are located in a small region on the X chromosome, both show lower numbers of polymorphisms .The 288 AR patients were screened for polymorphisms in the putative promoter regions of the 10 TLR genes using Sanger sequencing in both directions. The promoter regions were defined as the region 50 bp downstream to 500 bp upstream of the start of exon 1 of all the genes. The sequence data was generally of high quality with > 95% of bases having a Phred score of 30 or higher in > 95% of individuals. TLR8 with an allele frequency difference of 0.09 yielded an uncorrected P-value < 0.05. The number of rare (MAF ≤ 1%) and population-specific variants do not show a positive correlation between the populations but they are on the other hand so few that the correlation coefficient is uninformative. Two things are directly apparent from Table TLR10 there is both a higher number of SNPs with MAFs ≤ 1% and a higher number of population-specific SNPs in AR patients. This yields a P-value of 0.00009 for AR-specific SNPs that is sufficiently low to pass a Bonferroni correction  were extracted from the 1000Genomes project and compared with data from the AR patients Table . The toton Table . It shouTLR10 shows the highest number (18) with TLR1 (14) and TLR6 (11) again showing high numbers of polymorphisms. Also in this case TLR7 (8) and TLR8 (9) showed a lower than average (11.9) number of variable sites.The Ion Torrent sequencing of the 288 AR patients identified a total of 119 polymorphisms in the coding sequences of the 10 TLR genes, 68 with an allele frequency ≤ 1%, 43 not present in the EUR population and 21 not previously described in dbSNP  out of a total of 22 tests performed.A comparison of the results of the AR and EUR populations shows that both the total number of polymorphisms and the number of rare polymorphisms are slightly higher in the EUR population, 119 versus 142 and 68 versus 90 Table . The disTLR7 again shows a significant result and yields an uncorrected P-value of 0.0034. In addition, TLR1, TLR5 and TLR9 also gave rise to significant results with TLR9 showing the lowest P-value, indicating an excess of rare variants in the AR population compared to ExAC data.The non- Finnish European population of ExAC were used as an additional control population. Due to the different structure of the data it was used in a simulation test to investigate the probability for an accumulation of rare variants (MAF ≤ 1%) in the AR population. As in the test for AR-specific variants vs the EUR population, TLR1, TLR5 and TLR10 had the highest numbers of non-synonymous variants with 10, 11 and 13 variants, respectively. The 3 nonsense mutations were S324* in TLR1, R447* in TLR2 and R392* in TLR5 , whereas it was not present at all in the EUR population or the non-Finnish population of ExAC (investigating 66 616 chromosomes). The corresponding frequencies for the R447* mutation in TLR2 was estimated to a single copy in the AR population and none in the EUR population. In TLR5 the estimated allele frequencies of the R392* mutation were similar at 0.059 and 0.060 in the AR and EUR populations, respectively. In order to evaluate a potential accumulation of damaging variants, a permutation test based on the SIFT and PolyPhen-2 scores were used. The one-sided test showed no significant differences between the AR and EUR populations.The analysis above covered all variants. An obvious alternative is to focus on variants that alter the gene products i.e. 3 nonsense mutations and 69 missense mutations. Forty-four out of the 69 missense mutations had MAFs ≤ 0.01. TLR5 (rs2072493) and in TLR8 (rs3764880), respectively. Both were missense mutations classified as “benign” by PolyPhen-2. The allele frequency differences were 0.056 and 0.086 corresponding to P-values of 0.04 and 0.008, respectively. In both cases, the AR population had a lower frequency of the rare allele.SNPs with MAF ≥ 5 were also tested individually for allele frequency differences between the AR and EUR populations. Two SNPs showed significant allele frequency differences and were located in We have adopted a broad perspective in the present study where we investigate all 10 TLR genes for both rare (MAF ≤ 1%) and common (MAF > 1%) polymorphisms in both promoters and coding sequences. The polymorphisms are also subdivided into different classes e.g. nonsense and missense where missense mutations are further evaluated based on functional predictions. The primary objective of the present study was to describe the genetic variation of the TLR genes in a Swedish AR population in comparison to the general variation seen in background populations similar to our own. A second objective was to identify rare candidate mutations that are increasing risk for AR. The main results of this study is summarized in Fig. TLR2 and TLR5 show slightly lower quality, but since these two genes do not give rise to any significant signals these genes were not evaluated further. The low coverage (2–6X) NGS data from the 1000Genomes project are more likely to create both false positive and false negative data. However, since both the total number and the number of rare polymorphism are highly similar for the two populations, this seems not to be a large problem. Another concern is the use of publicly available background populations as control groups since AR is a common disease with a prevalence of 10–20% [In the present study different sequencing methods were used to create the different datasets. Since different sequencing platforms have different error profiles, direct comparisons can be problematic. The TLR exome data analyzed in this study have high coverage (>50X) in 1000Genomes and ExAC data, and in our Ion Torrent sequencing data (~200X), for more information see [Additional file TLR10 gene is the most variable in both populations, in the promoter as well as in coding regions. The other two genes in the same region, TLR1 and TLR6, are also well above average with regard to the number of variable sites. The other extreme is represented by TLR7 and TLR8 which are consistently showing a lower level of variation. These differences may be due to several different factors. It should be remembered that even under strict neutrality the level of variation is expected to vary between regions, i.e. the level of variation observed may be due to chance events. X-linked loci have slightly lower effective population size which may have contributed to the lower level of variation in TLR7 and TLR8. In addition, since active genes are involved it can safely be assumed that purifying selection is operating on nonsense and some of the missense mutations. If the effects of these changes are recessive, the effect of selection is stronger on X-linked loci, thus further reducing the level of variation. This effect will be present for all sites in linkage disequilibrium with the selected loci.A general observation that is not directly related to the occurrence of AR is the fact that the overall level of genetic variation is clearly different among the 10 TLR genes and that this to a large extent is related to their chromosomal locations. The TLR8 showed significant frequency differences between the AR and the EUR population. These SNPs are all located in the same haploblock and reflects the association observed earlier for the TLR7-TLR8 region [TLR8 which was also demonstrated in a previous study [Only a handful of the more frequent polymorphisms (MAF ≥ 5%) that could be tested individually yielded significant differences between the two investigated populations. SNPs in the promoter and the coding sequence of 8 region . Howeverus study .TLR1 S324*, TLR2 R447* and TLR5 R392*) are located in the leucine-rich repeats and truncates the receptors so that neither the transmembrane nor the toll-IL1 receptor domain will be translated and are therefore damaging to the function of the proteins. The TLR1 S324* nonsense mutation was clearly overrepresented in the AR population compared to the EUR population and the non-Finish European ExAC population. The frequency observed in AR patients for S324* is 45 times higher than the most common nonsense mutation in TLR1 in the complete ExAC database. Thus, the TLR1 S324* mutation is a strong candidate for being associated with AR.Many of the missense variants in the coding sequence may well be damaging and result in lowered fitness. However, it is likely that a majority of these are not involved in the development of AR since approximately equal numbers were found in cases and controls. The 3 nonsense polymorphisms  competitive binding of ligands of stimulatory TLRs 2) TLR10 competing for hetero-dimerization of other TLR2 family members (TLR1 and TLR6) 3) direct inhibitory effects of TLR10 via P13k signaling pathway [Investigation of rare variants in the promoter regions showed no general accumulation in AR patients relative to EUR controls. The major exception was the promoter region of R10 gene , suggest pathway .TLR1, TLR5, TLR7 and TLR9 showed significant accumulations of rare variants in AR patients. SNPs in the TLR1 locus have previously been associated with allergic disease in a number of large meta-GWAS [TLR7 have been associated with the development of asthma, rhinitis, and AD in a Danish population [TLR9 were reported to play a role in the predisposition to asthma [The analysis of the coding sequence showed no strong general accumulation of rare or AR-specific polymorphisms in AR patients relative to EUR controls. However, in the simulation tests for rare variants using ExAC data, eta-GWAS –19, 21 aeta-GWAS . Geneticpulation  and alsopulation . In a sto asthma  and wereo asthma . Thus, tTLR10-TLR1-TLR6 locus which was identified by a large number of SNPs. Following these studies, a replication attempt of the SNPs identified in the three meta-GWAS was performed using both the original phenotype definitions and a more strict AR definition [TLR10-TLR1-TLR6 locus. This locus produced significant P-values for SNPs located in a region that were larger than 100 kbp and encompassed all three genes. Another meta-GWAS investigating a combined phenotype of asthma and hay fever also identified this locus as associated with the disease [TLR10, TLR1 and TLR6 expression are shown in Additional file TLR10-TLR1-TLR6 region is correlated with AR. The overrepresentation in the AR population of a set of variants in the promoter of TLR10 and in the coding region of TLR1 including the S324* mutation, identify these polymorphisms as candidate mutations and add further details to the associations observed for common SNP from this region in the meta-GWAS and the replication study. The lack of significant associations of common variants in the TLR10-TLR1-TLR6 locus is probably a matter of power in the present study. There are in fact allele frequency differences for a number of SNPs, but at a non-significant level. Nonetheless, the rare variants found in this study coupled with previous strong association to common variants highly implicate the TLR10-TLR1-TLR6 region as a risk-locus for AR.Three large meta-GWAS identified a total of 37 loci associated with AR where four of these loci were reported by all three studies –19. One finition . A total disease . In addi disease . The patTLR1, TLR5, TLR7, TLR9 and TLR10 genes in AR patients compared to public background populations, indicating that rare variants may also contribute to the disease. Further studies investigating the contribution of rare variants in AR using larger populations consisting of matched cases and controls are much needed, but the results of the present study may serve as a starting point for future studies.This study was motivated by the lack of re-sequencing studies in allergic diseases. Previous association studies have identified common variants in the TLR genes to be associated with AR. The present study describes the genetic variation of these genes in a population selected for AR, which is an important step to understand the genetic contribution of the disease. Also, the present study identifies an excess of rare variants in the


NMR relaxation dynamics analysis confirmed the intrinsic disorder of the cross-over loop. Hydrogen deuterium exchange analysis further revealed a positive correlation between the length of the cross-over loop and the degree of local fluctuations, despite UCH-L5 being thermodynamically and kinetically more stable than the shorter UCHs. Considering the role of UCH-L5 in removing K48-linked ubiquitin to prevent proteasomal degradation of ubiquitinated substrates, our findings offered mechanistic insights into the evolution of UCH-L5. Compared to its paralogs, it is entropically stabilized to withstand mechanical unfolding by the proteasome while maintaining structural plasticity. It can therefore accommodate a broad range of substrate geometries at the cost of unfavourable entropic loss.Human ubiquitin C-terminal hydrolyase UCH-L5 is a topologically knotted deubiquitinase that is activated upon binding to the proteasome subunit Rpn13. The length of its intrinsically disordered cross-over loop is essential for substrate recognition. Here, we showed that the catalytic domain of UCH-L5 exhibits higher equilibrium folding stability with an unfolding rate on the scale of 10 Ubiquitin C-terminal hydrolases (UCHs) are papain-like cysteine proteases that deubiquitinate by hydrolysing the isopeptide bond between the C-terminus of ubiquitin and the lysine side-chain of its adduct, countering ubiquitination in protein homeostasis1456While the UCH domain of UCH-L5 shares limited sequence homology with UCH-L1 (23%) and UCH-L3 28%) compared to the much high sequence homology between UCH-L1 and -L3 (55%), their three-dimensional structures are essentially identical. The few differences in local dynamics result in some disordered secondary structures 1891011121% compare910111213129KM value) at a low nM range161718N240)kcat) for which UbAMC that is over three orders of magnitude higher than that of UCH-L1 and slightly higher than that of UCH-L3Enzyme kinetics analyses of UCH-L1, -L3, and -L5 using a fluorogenic ubiquitin derivative, ubiquitin C-terminal 7-amido-4- methylcoumarin (UbAMC), as a model substrate indicate that UCH-L1 and -L3 form a productive Michelis-Menten complex with ubiquitin  is higher than that of UCH-L1 −1)N240, [D]N-D,50%, is about 0.5 M higher than those of UCH-L1 and -L3 with comparable m-values , indicating its association with global folding events  and the associated free energy of unfolding of the phase 1 are essentially the same as those derived from equilibrium unfolding  that is five and four orders of magnitude slower than those of UCH-L1 and -L3. The folding rate of UCH-L5N240 in water (−1) is comparable with that of UCHL1 and is about 100 times slower than that of UCH-L3.To compare the folding kinetics of UCH-L1, -L3, and -L5H-L5N240 . While ttage) in the presence of different urea concentrations (open blue circles for k3 and open green triangles for k4 in N240 as a function of the aging time (tage) can be best fit to a linear three-state reaction with the microscopic forward and back reaction rates extracted through global fitting, although the results are noisy and a lag phase in the amplitude build up of the second fastest phase is barely visible. The results therefore suggest the presence of transiently populated intermediate corresponds to the fastest kinetic phase of UCH-L5N240  interrupted refolding fluorescence analyses to obtain additional refolding rates through global fitting of the kinetic traces obtained with different aging times  and the C-terminal extension beyond the UCH domain (residues 230–240) for which we did not have complete assignments (2) reflecting fast local motions on the ps to ns timescale (N240. We therefore resorted to NMR hydrogen-deuterium exchange (HDX) analysis to probe molecular dynamics on a much slower timescale by determining the rate of HDX for a specific amide group and comparing it with the expected value for a random coil to derive the corresponding PF that reflects the stability of the corresponding hydrogen bond Having established the slow global folding kinetics of UCH-L5ignments . The strimescale . This tiN240 compared to UCH-L1 and -L3 based on the fluorescence-based equilibrium and kinetic data  is about two orders of magnitude slower than that of UCH-L3. We repeated and confirmed the previously reported parameters for UCH-L1, -L3, and -L5N240 , we did not observe appreciable changes in melting temperature (data not shown). We next carried out NMR titration analyses with 0.1 mM UCH-L5N240 and up to 0.15 mM ubiquitin to evaluate the binding processes under conditions that should significantly increase the population of ubiquitin-bound UCH-L5N240. The end NMR titration points nonetheless showed severe line-broadening of UCH-L5N240 backbone amide and side-chain methyl resonances as opposed to the well-defined backbone and side-chain resonances of ubiquitin-bound UCH-L1Having established that our recombinant UCHs are enzymatically active, we subsequently carried out isothermal titration calorimetry at four different temperatures  to gain further insights into the ubiquitin binding process . For UCHN240 . The lacN240 and ubiquitin in solution, we focused on the global analysis of the variable temperature ITC data to extract the heat capacity ΔCp in addition to the enthalpy, entropy, and thus free energy associated with ubiquitin binding for the UCH-L1 and -L3 in order to extrapolate the findings to correlate with potential characteristics of ubiquitin binding events for UCH-L5N240 (KD) of 0.26 μM, while that of UCH-L3 is significantly weaker (Cp) associated with ubiquitin binding is significantly larger for UCH-L3  compared to that of UCH-L1 . ΔCp is associated with the changes in solvent accessible surface area (ΔSASA) upon binding. Indeed, UCH-L3 has a larger ΔSASA value (2691 Å2) compared to that of UCH-L1 (2372 Å2), according to their respective crystal structures in complex with ubiquitin . This is accompanied by a large conformational change and cross-over loop ordering in UCH-L3 manifested in the large entropic penalty associated with ubiquitin binding. It is plausible to speculate that for UCH-L5N240, the entropic penalty associated with mono-ubiquitin binding is far greater that that for UCH-L3, hence the much weaker binding that is not accessible to ITC analysis.Despite our inability to obtain a stable binary complex of UCH-L5H-L5N240 . Under sy weaker . While Uy weaker . As a reN240, in comparison with previously reported results on UCH-L1 and -L3. Despite the apparent two-state folding equilibrium behaviour (27k3 and k4) are connected sequentially rather than separately with two slower kinetic phases (k1 and k4)  . SubsequHuman UCHs are important DUBs for maintaining cellular proteostasisN240 highlighted the large dynamic range of the intrinsic unfolding rates . The resulting recombinant protein construct contains a glutathione S-transferase (GST) fused at the N-terminus of UCH-L5N240. The expression vector was transformed into a BL21 (DE3) E. coli strain and cultured in LB medium containing 0.1 mg/mL ampicillin for antibiotic selection. Recombinant protein over-expression was induced by adding 0.5 mM isopropyl thio-β-d-galactoside (IPTG) when the optical density of bacterial culture at 600 nm (OD600) was close to 0.8, followed by overnight incubation with shaking at 16 °C. The cells were harvested and purified by a standard protocol for GST-based affinity column purification, followed by fusion tag removal using PreScission protease , as described previouslyThe pGEX-6P1 vector-based expression construct of UCH-L5N240 was monitored by exciting the samples at 260 nm and recording the emission spectra between 300 and 450 nm at 25 °C using a temperature-controlled fluorimeter . Urea-induced equilibrium unfolding was monitored by generating 41 aliquots of protein solution containing a gradient of urea (0–6 M) concentrations with a linear increment step of 2.5% made by a two-channel liquid dispenser  to minimize manual pipetting errors.The intrinsic fluorescence of UCH-L5N240 was monitored by analysing the CD signal changes at 215 nm between 25 and 80 °C while the changes at 222 nm were monitored for UCH-L1 and -L3. In all cases, 5 μM of protein was used and 1.2 molar equivalent of ubiquitin was added to assess the effect of ubiquitin binding. The resulting isotherms were normalized and fitted to a two-state equilibrium unfolding model to extract the thermodynamics parameters, i.e., melting temperature (Tm), enthalpy (ΔH), entropy (ΔS) and changes in heat capacity (ΔCp) as described previouslyUrea-induced equilibrium unfolding was also monitored by far-UV CD between 200 and 260 nm at 25 °C using a temperature-controlled CD spectrometer . The spectra were collected with a bandwidth of 1 nm, a data interval of 0.5 nm, and an averaging time of one second. Thermal denaturation of UCH-L5m × n matrix, M, as inputs for SVD analysis, where m corresponds to the number of recorded wavelengths and n corresponds to the number of titration points. SVD reconstruction generates an m × m unitary rotation matrix Ur, an n × n unitary rotation matrix Vr, and an m × n diagonal singular value matrix S to reconstruct the input matrix M as followsAll equilibrium unfolding data were subjected to singular value decomposition (SVD) analysis using an in-house written script for MATLAB  as described previously2930Ur corresponds to the basis function of the observed spectral patterns, and Vr corresponds to the basis coefficient as a function of denaturant concentration. To determine the number of significant components necessary for reconstructing the input matrix, the normalized correlation coefficients of individual singular values were calculated and a threshold of 0.8 was used, leading to the conclusion that UCH-L5N240 unfolds in a two-state model.where The fluorescence and CD data were subjected to a two-state equilibrium-unfolding model as described previously282930N240 was monitored using a stopped-flow spectrometer in fluorescence detection mode  as described previously282930μM UCH-L5N240 was buffered in 20 mM Tris-HCl (pH 7.6), 100 mM NaCl with 8 M urea for unfolding or without urea for refolding measurements. For single-jump kinetic measurements, the folding reactions were triggered by rapidly mixing the folding or unfolding buffer with protein solution at an asymmetric mixing ratio of 10:1. After the 11-fold dilution, the final protein concentration was 1.8 μM. For double-jump kinetic measurements, three different modes were employed: interrupted refolding, interrupted unfolding, or sequential refolding kinetic measurement.The folding kinetics of UCH-L5tage).For the interrupted refolding measurements, 7.7 M urea-denatured protein solution was first mixed with refolding buffer to initiate refolding over different aging periods, followed by the second mixing step with denaturing buffer containing 8 M urea immediately before we took kinetic fluorescence measurements under denaturing conditions. The first mixing step was achieved by mixing protein solutions with unfolding or refolding buffer with a 1:5 mixing ratio. After a specific aging period, a second mixing step was carried out by mixing the aged protein solution with an equal amount of unfolding or refolding buffer (1:1 mixing ratio) immediately before kinetic fluorescence measurements. The resulting kinetic traces were subjected to global fitting in which the folding rates were shared amongst all the kinetic traces of different aging times, and the amplitudes corresponding to different kinetic phases were shown as a function of aging time, and the rates at which the corresponding amplitudes built up were extracted by single exponential fitting to the observed amplitude as a function of aging time . The choice of model, kobs), with a linear refolding arm and a linear unfolding arm, were fit to the following equation:The observed reaction rates in the stopped-flow fluorescence measurements were fit to a simple two-state folding model to extract the associated kinetic and thermodynamic parameters. The observed reaction rates  and 13C-labelled D-glucose (2 g/mL) followed by the same purification procedure as described above. For backbone resonance assignments, a suite of triple resonance experiments were collected, including 15N-1H heteronuclear single quantum correlation (HSQC) spectroscopy, HNCO, HNCA, HNcoCA, HNCACB, and CBCAcoNH at 25 °C. All NMR data were collected using a Bruker AVIII NMR spectrometer, equipped with a cryogen-cooled TCI probe head, operating at 14.1 or 20.0 Tesla, corresponding to a proton Larmor frequency of 600 or 850 MHz, respectively. The data were collected using Topspin 3.0 , processed by NMRPipehttps://www.cgl.ucsf.edu/home/sparky/). Near complete backbone  assignments were achieved through the use of an iterative computer-aided procedure as described previously52Uniformly 13C- and 15N-labelled UCH-L5N240 with unlabelled ubiquitin (5 mM stock solution concentration) in a 3 mm MATCH tube at 25 °C. and 20.0 Tesla. Two-dimensional 15N-1H SOFAST-HMQC and 13C-1H HSQC spectra were collected for individual titration points  as described previously54The ubiquitin titration experiment was carried out using 0.1 mM of uniformly 15N spin relaxation dynamics of UCH-L5N240 was characterized by measuring the longitudinal (R1) and transverse (R2) relaxation rates and 15N-1H heteronuclear nuclear Overhauser effects (hetNOEs) of individual backbone amide groups at 37 °C and 14.1 Tesla as described previously2) using model-free analysis by TENSOR2The N240 were carried out in 20 mM Tris-HCl (pH 7.6), 100 mM NaCl at 25 °C, as described previously283315N-1H correlations. The amide 15N-1H cross-peak intensity as a function of HDX time was fit to a single exponential function using the built-in relaxation analysis module of the software package Sparky, and the rate constants were subsequently used to derive the protection factors (PFs) together with the protein sequences using the Excel spreadsheet with pre-defined functions from the Englander laboratory at University of Pennsylvania, USA (http://hx2.med.upenn.edu/download.html).NMR HDX analyses of UCH-L1, -L3, and -L5A Microcal 200 instrument  was used for the ITC measurements. The protein concentrations of UCHs and human ubiquitin were set to 27.5 and 275 μM, respectively. The latter was used as the titrant to be injected into the UCH solutions. The ITC experiments were carried out at 20, 25, 30, and 37 °C, and the resulting ITC data were individually analysed using NITPICThe enzyme kinetics of UCH variants were assessed by UbAMC as a model substrate as described previously161718How to cite this article: Lee, Y.-T. C. et al. Entropic stabilization of a deubiquitinase provides conformational plasticity and slow unfolding kinetics beneficial for functioning on the proteasome. Sci. Rep.7, 45174; doi: 10.1038/srep45174 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Early psychological researchers proposed a link between intelligence and low-level perceptual performance. It was recently suggested that this link is driven by individual variations in the ability to suppress irrelevant information, evidenced by the observation of strong correlations between perceptual surround suppression and cognitive performance. However, the neural mechanisms underlying such a link remain unclear. A candidate mechanism is neural inhibition by gamma-aminobutyric acid (GABA), but direct experimental support for GABA-mediated inhibition underlying suppression is inconsistent. Here we report evidence consistent with a global suppressive mechanism involving GABA underlying the link between sensory performance and intelligence. We measured visual cortical GABA concentration, visuo-spatial intelligence and visual surround suppression in a group of healthy adults. Levels of GABA were strongly predictive of both intelligence and surround suppression, with higher levels of intelligence associated with higher levels of GABA and stronger surround suppression. These results indicate that GABA-mediated neural inhibition may be a key factor determining cognitive performance and suggests a physiological mechanism linking surround suppression and intelligence. Subjects had normal or corrected to normal vision. Subjects were post-graduate students or post-doctorate researchers at the Psychology department of Royal Holloway, University of London. All subjects except one (one of the authors) were naïve to the purpose of the experiment.2.2The Matrix Reasoning subtest of the Wechsler Abbreviated Scale of Intelligence (WASI) was administered in accordance with the standardised procedures outlined in the WASI user’s manual 2.3Estimates of GABA concentration in the visual cortex were obtained using the methods of Edden et al. A T2 anatomical scan was performed to collect images in 3 planes for MRS voxel placement . Following visual inspection of this anatomical image the MRS voxel was placed over the primary visual cortex, identified by the calcarine sulcus. The voxel was placed so as to exclude as much cerebrospinal fluid as possible and care was taken to ensure the voxel did not extend into the dura.Acquisition of MRS data was performed using the MEGA-PRESS sequence 20) and one relative to creatine (GABA+/Cr). GABA/H20 was used due to the superior signal to noise ratio MRS data was processed using the Gannet toolbox 2.4http://www.pc.rhul.ac.uk/staff/J.Larsson/softare.html) run on a Linux operating system.Visual stimuli were displayed on an EIZO 660-M monochrome CRT monitor at a viewing distance of 57 cm. A chin rest was used to ensure head stabilization. Presentation of stimuli and acquisition of responses was carried out using Matlab 7.4.0 (R2007a) and MGL , in 2 quadrants of the visual field (upper left and upper right). Within each block of trials , the stimuli were always shown at a single location. Within each surround suppression testing session, the stimuli were always of the same experimental condition (control or surrounded). Throughout each trial, a white 1° fixation cross was displayed at the centre of the screen. The background was a uniform gray with a luminance of 26.1 cd/mFirst-order grating stimuli consisted of luminance-defined sinusoidal gratings  within a 3° wide circular aperture. Second-order grating stimuli consisted of contrast-modulated sinusoidal gratings generated as described in Larsson, Landy and Heeger S1₁ was calculated by subtracting the ratio of the matching contrasts (averaged across blocks and hemifields) of the surround and control conditions from 1 An index of first-order surround suppression magnitude SI₂ calculated in the same way as for first-order suppression. For the statistical analyses below, first-order and second-order suppression indices were normalized by transforming to z-scores and averaged within subjects to yield a single measure of surround suppression strength for each participant.A value of 0 would indicate no surround suppression , 1 complete suppression (zero perceived matching contrast). The magnitude of second-order surround suppression was quantified by an index 3We found a strong and significant positive correlation between visual intelligence and cortical GABA concentration , such that subjects with high levels of GABA in primary visual cortex performed better on the matrix reasoning IQ sub-test A. Higher4These results demonstrate an association between cortical GABA levels and measures of intelligence, and provide evidence of a relationship between GABA and surround suppression magnitude in typical individuals. A link between GABA and first-order surround suppression has previously been shown for schizophrenic patients, and typical individuals to a lesser extent The observed correlation between GABA and surround suppression is consistent with the notion that visual surround suppression is mediated by inhibitory synaptic connections in the visual cortex Combined with our finding of a strong positive correlation between surround suppression strength and intelligence, and between intelligence and GABA, these results provide a plausible candidate neural substrate for Melnick et al.’s Although our results provide evidence for a link between GABAergic neural inhibition and visual intelligence, the specific mechanisms and nature of this link remain to be elucidated. The generalisability of our findings is necessarily limited by the relatively small sample size, as is common with much of the MRS literature


Constitutively active androgen receptor (AR) variants have been involved in the expression of mesenchymal markers such as N-cadherin in prostate cancer (PCa). However, the underlying molecular mechanisms remain elusive. It remains unclear, whether N-cadherin gene (CDH2) is a direct transcriptional target of AR variants or whether the observed upregulation is due to indirect effects through additional regulatory factors. Moreover, the specific contribution of full-length AR and AR variants in N-cadherin regulation in PCa has never been explored deeply. To investigate this, we artificially mimicked the co-expression of AR variants together with a full-length AR and performed miRNA-seq, RNA-seq and ChIP assays. Our results were in favor of a direct AR variants action on CDH2. Our data also revealed a distinctive mode of action between full-length AR and AR variants to regulate N-cadherin expression. Both wild type AR and AR variants could interact with a regulatory element in intron 1 of CDH2. However, a higher histone H4 acetylation in this genomic region was only observed with AR variants. This suggests that full-length AR may play an occluding function to impede CDH2 upregulation. Our data further highlighted a negative effect of AR variants on the expression of the endogenous full-length AR in LNCaP. These differences in the mode of action of AR variants and full-length AR for the control of one key gene for prostate cancer progression could be worth considering for targeting AR variants in PCa. The androgen receptor (AR), a member of the nuclear receptor superfamily, is a ligand-dependent transcription factor that controls the development and the normal function of the prostate gland . The AR in vitro and in vivo in an androgen-depleted environment and lead to resistance to novel therapies such as enzalutamide and abiraterone acetate [Constitutively active AR variants are due to nonsense mutations in exon 4 e.g. AR-Q640X) 40X 6, 1–12 or al or al12  acetate , 20–23. v567es induces autonomously prostate tumorigenesis and furthermore leads to invasive adenocarcinoma after castration [In addition to their role in castration resistance, a number of studies suggest that constitutively active AR variants are involved in PCa progression. In the first place, ARstration . Moreovestration . Besidesstration . An incrstration . EMT prostration , 27–29. stration , 30. HowCDH2 gene [CDH2 gene followed by histone H4 acetylation, but also from a decrease of endogenous AR-FL. These data emphasize the role of AR variants in the progression of CRPC.N-cadherin is involved in cell adhesion and promotes tumor progression owing to its role in cell migration, invasion and survival . As mentDH2 gene , we postp-value for multiple testing < 0.05). First, biological functions of deregulated genes were studied using IPA software. Interestingly, deregulated genes in the presence of AR-V7 in LNCaP cells were associated with functions such as cellular movement, cell morphology or cellular assembly and organization (CDH2 gene using SABiosciences proprietary database DECODE (DECipherment Of DNA Elements)  and AR variants to upregulate N-cadherin expression in PCa cells . We invenization . As expe) Figure . Among t) Figure . The inc) Figure . Neverth) Figure . Finallyp-value ≤ 0.05 . Taken together, these results reveal that neither miR-221–3p nor miR-26b-5p is involved in N-cadherin upregulation in our model.CDH2 [CDH2  to induce N-cadherin expression. Besides, AR-FL could occlude the AR binding sites and prevent AR variant activity. Since the ligand-binding domain (LBD) is an important platform for the interaction of cofactors, the loss of this region in AR variants could partly explain the distinct transcriptional programs in the presence of AR-FL or AR variants [Our data from ChiP-qPCR evidenced that both AR-FL and AR variants were able to bind to the AREs present in intron 1 of the  of CDH2 . Our obs of CDH2 . Furthered AR-FL . Howevervariants . Consistvariants .CDH2 gene. Further analyses need to be performed to validate this model and to understand how AR variants induce a decrease of endogenous AR-FL in prostate cancer cells. In the present study, we focused only on N-cadherin expression, but, it may be interested to study if the increased expression of other mesenchymal markers observed in the presence AR variants occurs following the same mechanism. Indeed, in our previous study, we showed an increased expression of vimentin, ZEB1 and SNAIL [In our model, as observed in human CRPC samples, we have a co-expression of AR-FL and AR variants. As suggested in our previous work, AR-FL and AR variants could compete each other to regulate N-cadherin expression . Here, wnd SNAIL . Interesnd SNAIL . These dCDH2 promoter [CDH2. Here, in our model the increased expression of ETV5 revealed in the RNA-seq was confirmed by qRT-PCR, but at the protein level, ETV5 expression was not detectable in immunoblots. Moreover, ETV5 downregulation by siRNA was not associated with N-cadherin downregulation, suggesting that ETV5 could not be the leading transcription factor induced by AR variants to upregulate N-cadherin expression in our model. The upregulation of SOX9 expression observed in our RNA-seq data was not confirmed at mRNA and protein levels. Anyway, the association between ETV5 or SOX9 expression level and the presence of AR variants in prostate cancer cells is not clear. In a first study, a DNA microarray analysis performed in LNCaP cells overexpressing AR-V7 revealed an upregulation of ETV5, but not SOX9 [Using RNA-seq analysis, we identified ETV5 and SOX9 as potential regulators of N-cadherin. Indeed, previous studies have reported a link between N-cadherin and ETV5 or SOX9 expression. For example, the overexpression of ETV5 in endometrial cancer cells was associated with an increased expression of N-cadherin . Moreovepromoter  There arnot SOX9 . Conversnot SOX9 . Likewisnot SOX9 . HoweverCDH2. From this analysis, we have identified two microRNAs, miR-221–3p and miR-26b-5p, able to target 3′UTR extremity of CDH2 with a high score of prediction. However, the decreased expression of miR-221–3p and miR-26b-5p observed in the miRNA-seq analysis was not confirmed by qRT-PCR. The link between these two microRNAs and N-cadherin was only based on bioinformatics prediction. Indeed, the direct regulation of N-cadherin by miR-221–3p or miR-26b-5p was never described in previous reports. Nevertheless, in hepatocellular cancer cells, miR-26b-5p regulates Epithelial Mesenchymal Transition. Indeed, its overexpression is associated with an increase of E-cadherin expression and a decrease of vimentin expression. However, the authors have not analyzed N-cadherin expression in the presence of miR-26b-5p [Since Epithelial Mesenchymal Transition (EMT) was generally associated with a particular miRNA expression profile, we wondered whether N-cadherin induced by AR variants could be also regulated by microRNAs. So, using an integrative analysis between miRNA-seq and RNA-seq, we have examined microRNAs with a decreased expression in the presence of AR-V7 compared to AR-WT and selected the ones that were known to target the 3′UTR extremity of R-26b-5p . In contR-26b-5p , 49. In CDH2 gene followed by histone H4 acetylation, but also from a decrease of endogenous AR-FL. These data emphasize the role of AR variants in the progression of CRPC and highlight the importance to develop drugs targeting these variants or their mode of action.In summary, in this study we bring evidence that N-cadherin upregulation in prostate cancer cells appears to result from the binding of AR variants to AREs in intron 1 of the LNCaP cells, clone FGC (ECACC) and C4–2B cell line  were maintained in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin (Sigma-Aldrich) and 1 mM pyruvate (Invitrogen) (complete medium). HEK 293T cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich).TM Tet-ON® 3G Inducible Expression System (Clontech) was used. Briefly, cDNA of EGFP-AR-WT, EGFP-AR-Q640X and EGFP-AR-V7 were amplified from previously described pEGFP-AR using specific primers (® HD Cloning Kit (Clontech).For lentiviral infection, the full-length wild type androgen receptor (AR-WT) and the constitutively active AR variants, AR-Q640X and AR-V7, were excised from previously described pEGFP-AR , 12 usin primers . Each cD6 HEK 293T with 9 μg of packaging plasmids  and 3 μg of pLenti-PGK-AR or 1 μg pLenti-PGK-GFP as control using respectively 36 μl or 30 μl of lipofectamine 2000 (Invitrogen). Cells were transfected in antibiotic-free medium (DMEM containing 10% FBS and 2 mM L-glutamine). Culture media were changed the day after and 72 h after transfection, culture media were recovered, centrifuged at 3000 rpm for 15 min to pellet debris. The viral supernatants were filtered through a Millex-HV 0.45 μm, concentrated 10x with Amicon® Ultra-15 Centrifugal Filters Ultracell®-100K (Millipore). Then, LNCaP cells were incubated with 1:10 of concentrated viral supernatant in LNCaP complete medium supplemented with 10 nM DHT and 6 μg/mL of polybrene (Sigma Aldrich) for maximal transduction efficiency. These conditions lead to almost 100% of transduced cells. For miRNA-seq and RNA-seq experiments, a pool of transduced cells was used and the transduction efficiency was determined by analyzing EGFP level by fluorescent microscopy and qRT-PCR.Lentivirus expressing GFP, AR-WT, AR-Q640X and AR-V7 were prepared by co-transfecting 6 × 10To establish Tet-3G-expressing stable LNCaP and C4–2B clones, Tet-3G lentiviral particles produced in HEK 293T cells were used to transduce LNCaP and C4–2B cells. Stable clones were selected with 400 μg/mL geneticin (Life Technologies). For an inducible expression of AR variants, Tet-3G stable LNCaP and C4–2B cells were transduced with pLVX-TRE3G-EGFP-AR-WT, pLVX-TRE3G-EGFP-AR-Q640X and pLVX-TRE3G-EGFP-AR-V7 lentiviral particles, and transduced LNCaP and C4–2B cells were selected with 300 μg/mL geneticin and 400 ng/mL or 100 ng/mL puromycin (Life Technologies) respectively. AR expression was induced using 20 ng/mL doxycycline (Life Technologies).Two days before transfection, AR doxycycline-inducible LNCaP cells were plated into 12-wells plates. Prior to transfection, medium was refreshed with RPMI-1640 supplemented with 10% Tet System Approved FBS , 10 mM HEPES, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin (Sigma-Aldrich), 1 mM pyruvate (Invitrogen), 10 nM DHT and 20 ng/mL doxycycline (Life technologies). Cells were transfected with 50 nM siRNA against ETV5  using 3 μL of JET-PRIME . AllStars Negative Control siRNA  was used as control. After 48 h, total RNA was extracted and ETV5 and CDH2 expression levels were analyzed by qRT-PCR.® RNA II assay (Macherey-Nagel) according to the manufacturer's procedure and 400 ng of total RNA were reverse transcribed using iScript kit (Bio-Rad). Real-time PCR was conducted using GoTaq® qPCR Master Mix (Promega) and validated primers  and transferred onto a nitrocellulose membrane. Membranes were blocked with PBS/0.1% Tween/4%nonfat dry milk and probed with primary antibodies against EGFP , ETV5 , AR (clone G122–434) , AR-C19 , N-cadherin , b-actin  or GAPDH  at 4°C overnight. Blots were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse , goat anti-rabbit , rabbit anti-goat  or rat anti-mouse IgG2a  secondary antibodies for 1 h. Immunoreactive proteins were visualized by chemiluminescence . For Stain-Free gels, total protein normalization was performed using Image LabTM Software (Bio-Rad Laboratories).Transduced cells were lysed in RIPA buffer  supplemented with 1x phosphatase inhibitor, 1x protease inhibitor (Sigma Aldrich) and 250 U/mL Benzonase (Millipore). Protein concentration was quantified using BCA Protein Assay (Pierce Biotechnology) according to the manufacturer's protocol. An equivalent quantity of total proteins was separated by 7.5% SDS-PAGE or by TGX Stain-FreeCDH2 gene, we have performed a ChiP using ChiP-IT® High Sensitivity (Active Motif) according to the manufacturer's protocol. Briefly, 24 h or 72h after doxycycline induction, LNCaP and C4–2B overexpressing AR-WT, AR-V7 or non-induced cells as control were crosslinked with 1.1% of paraformaldehyde and fixation buffer for 15 min at room temperature and the reaction was quenched with Stop Solution. After cell lysis, chromatin was sonicated 2.5 h using qSONICA Q800R (20sec ON/40sec OFF). Then, 30 μg of chromatin was immunoprecipitated with 5 μg of anti-EGFP antibody  or goat IgG isotype . Immunoprecipitated DNA was incubated with agarose beads for 3 h, extensively washed, eluated, reverse-crosslinked and purified according to the protocol. To analyze histone H4 acetylation in CDH2 gene, 10 μg of chromatin was incubated overnight with 10 μg of Histone H4ac (pan-acetyl) antibody  or rabbit IgG isotype control .To analyze the occupancy of AR in ® qPCR Master Mix (Promega). The primers used for DNA amplification are listed in ChiP DNA was analyzed by real-time PCR using GoTaqLNCaP cells were transduced with lentivirus expressing AR-WT and AR-V7 in complete medium containing 10 nM DHT. Three days after transduction, total RNA was extracted using TriPure Reagent (Roche) according to the manufacturer's protocol. For RNA-seq analysis, three samples per condition were analyzed. The library of cDNA and the sequencing were performed by IGBMC Microarray and Sequencing platform .p-values were adjusted for multiple testing by using the Benjamini and Hochberg (1995) method [® Pathway Analysis tool .RNA-seq reads were mapped onto the hg19 assembly of the human genome using Tophat v2.0.10  and the ) method  . Only trimmed reads with a length between 15 and 40 nucleotides were kept for the further analysis. Data analysis was performed according to published pipeline ncPRO-seq [For data analysis, adapters were trimmed from total reads using FASTX_Toolkit  software  and the putative targets of DEM were identified using microRNA Target Filter. Briefly, this tool is the combination of four databases based on prediction binding (TargetScan) and experimentally validated interaction . For further analyses, only pairs of mRNA-miRNA with anticorrelated expression were selected.Integrative analysis between RNA-seq and miRNA-seq was performed by comparing a list of differentially expressed genes (DEG) and differentially expressed miRNA (DEM). The list of DEG comprised all the genes with a fold change ≤ or ≥ 1.5 and adjusted