Although hepatectomy and liver transplantation surgery for hepatocellular carcinoma (HCC) are effective treatment modalities, the risk of recurrence remains high, particularly in patients with a high number of circulating tumor cells (CTCs) expressing cancer stem/progenitor cell markers. Androgen receptor (AR) signaling has been shown to suppress HCC metastasis in rodent models of HCC. In this study, we investigated whether AR is associated with postoperative HCC recurrence.CTCs were obtained from patients with HCC who had undergone hepatectomy to investigate whether they are associated with disease outcome. AR knockout was introduced in two mouse models of spontaneous HCC (carcinogen- and hepatitis B virus-related HCC) to delineate the role that AR plays in HCC recurrence. Biological systems analysis was used to investigate the cellular and molecular mechanisms.We found that the expression of AR in CTCs was negatively associated with HCC recurrence/progression after hepatectomy. Our results suggest that AR-mediated suppression of HCC recurrence/progression is governed by a three-pronged mechanism. First, AR suppresses the expression of CD90 in CTCs by upregulating Histone 3H2A. Second, AR suppresses cell migration at the transcriptome level. Third, AR promotes anoikis of CTCs via dysregulation of cytoskeletal adsorption.The results indicate that AR expression may be the gatekeeper of postoperative HCC recurrence. Therefore, targeting AR in presurgical down-staging procedures may serve as a secondary prevention measure against HCC recurrence in the future. Hepatocellular carcinoma (HCC) is one of the most prevalent types of liver cancer worldwide , 2. The Although curative hepatectomy and liver transplantation surgery are effective treatments for HCC , the risIn this study, we found that AR expression was associated with a reduction in primary tumor CD90+ populations, a reduction in cancer cell migration, and an increase in CTC death, indicating that increased expression of AR might protect against postoperative HCC recurrence.In order to examine the role of AR expression in hepatic surgery HCC patients, in terms of its association with disease progression and the recurrence, we performed a single-cohort study as described in the Materials and Methods section; the demographic data are presented in Table 5 cancer cells to whole blood from healthy subjects. Peripheral blood mononuclear cells (PBMCs) were separated from the blood–cancer cell mixture as described in the Materials and Methods section. Approximately 20% (v/v) of the PBMCs were co-stained with AR, CD90, and CD45, and then subjected to flow cytometry analysis as shown in Figure Except for the detection of AR and CD90 in primary tumors, we also examined CTCs in the same cohort. Blood samples from 75 patients who had been followed for more than 900 days and passed the quality control check (> 90% of the blood cells were intact) were included in the study. Before measuring patient CTCs, we established the CD90-CTC detection method, which follows Yamashita et al.'s method . We dete2 = 1.62E-06; Figure We then examined whether differences in the levels of CD90 and AR expression in CTCs in blood samples from patients who had undergone surgery for HCC Table  correlatThe findings presented in Figures In order to characterize the relationship between AR and CD90 expression in HCC, we examined their expression levels at various stages of tumor development in a spontaneous HCC mouse model. We found that CD90 was rarely observed in the early 30 weeks) and middle (40 weeks) stages of tumor development (data not shown), but was sporadically detected in the late (60 weeks) stage, which supports the notion that CD90 is a marker of HCC progression . Further0 weeks aWe also investigated the effects that AR has on cancer cell migration. We established three SKAR clones with differential expression of AR Figure . We founIn order to understand how AR suppresses the mobility of HCC cells on a global scale, we used a cDNA microarray to observe the effect of AR on the transcriptome Figure . We founIn order to understand the transcriptome shift seen in cells with differential AR expression, we performed pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) platform and Gene Ontology database. KEGG analysis of the microarray data revealed that the levels of cancer-related pathways increased in an AR expression–dependent manner Figure . HoweverStudies have shown that the number of CTCs is positively correlated with the risk of cancer recurrence. We therefore tested whether AR facilitates anoikis of circulating HCC cells and found that the degree of anoikis of HCC cells in whole blood Figure  and in cThe cell anoikis mechanism could be related to cytoskeletal rearrangement , 24 or cEvidence has been put forward that amorphosis, a type of apoptosis induced by the disruption of cytoskeletal rearrangement, leading to a distorted cell shape, is one of the mechanisms governing anoikis . In thisThe data presented in Figures HCC patients at Barcelona Clinic Liver Cancer (BCLC) stage B (early cancer development) or stage C (intermediate cancer development) disease are normally eligible to receive hepatectomy , 31. TheAR has been reported to suppress HCC metastasis , 11. BoiOur finding that AR mediates the suppression of CD90 expression implies not only that AR is a potential biomarker of disease recurrence but also that AR could be a target for gene therapy, possibly using the CRISPR/Cas9-activator  genome editing system , 34. ForColonization of CTC CSPC  only partially explains the processes of the recurrence of HCC and other heterogeneous diseases. The second mechanism through which AR protects against HCC recurrence is its ability to suppress cell mobility at the transcriptome level. To the best of our knowledge, this is the first study to describe such a phenomenon in HCC.Although the presence of CTCs expressing CSPC markers partially explains the high rate of recurrence after hepatectomy, the number of CTCs in general is also responsible for HCC recurrence . TherefoIn this study, we have described three mechanisms through which AR suppresses HCC recurrence. Our results indicate that enhancing the expression of AR in CTCs might reduce the risk of HCC recurrence.In order to examine the association between AR expression and cancer recurrence, we prospectively enrolled patients who had undergone hepatectomy at the Organ Transplantation Center, China Medical University Hospital (CMUH), Taiwan, during the period 2010–2012. Primary HCC tumor specimens and 20 ml of whole blood were collected with informed consent from 110 patients, of whom 97 patients had been followed for up to 900 days. Of the blood samples collected, 87 patient samples passed the quality control check and were included in the CTC analyses. All paraffin-embedded tumor tissue blocks from the 110 patients were subjected to IHC studies. Detailed patient demographic data are presented in ° antibody IgG for 30 min and analyzed by flow cytometry .PBMCs from patients were used to detect the expression/positivity of AR and CD90 in CTCs. In brief, 10 ml of whole blood and 5 μM EDTA were mixed with FICO buffer  and then centrifuged to isolate the PBMCs. The CTCs were first defined by size; then the lymphocyte were removed by excluding CD45+ cells. The cells were then fixed with cold methanol (−20°C) and then stored at −80°C. For the detection of CTCs, PBMCs (200 μl) were washed and then incubated with CD90 and AR antibodies in the presence of 0.5% NB-40 (Sigma) for 1.5 h. Cells were then incubated with PE- or FITC-conjugated 25 cells/200 μl H2O), aspirating the mixture 30 times with a syringe (#30 needle), and centrifuging it at 1000 × g for 10 min. The insoluble fraction was then discarded. Total α-actin was isolated by adding lysis buffer  to the cells, aspirating the mixture 30 times with a pipette (200 μl tip), and centrifuging it at 1000 × g for 10 min. The insoluble fraction was then discarded.Since the definition of amorphosis is the change in solubility of cytoskeletal proteins between the attached and detached states, we measured cellular stress fiber solubility with different stringencies of lysis buffer, and detected α-actin  using an immunoblot assay. The amorphosis values were then quantified from densitometric data from the blots using BioRad QuantDot (BioRad) as shown in Figure Cells were maintained in DMEM with 10% FCS (Invitrogen), 1% L-glutamine, and 1% penicillin/streptomycin as described previously . The celThe following antibodies and chemicals were used: CD90, E-cadherin, phospho-RhoA, AKT, phospho-AKT ; RhoA, AR (N-20), vimentin, integrin β1, and actin (Santa Cruz); propidium iodine ; DHT ; flutamide ; FAK inhibitor ; Src inhibitor (4-(4′-phenoxyanilino)-6,7-dimethoxyquinazoline; Merck; 50 nM); Akt inhibitor (2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; LY294002; Sigma-Aldrich; 1.5 μM); ROCK1/2 inhibitor ((R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide; Y27632; Merck; 150 nM); Latrunculin B ; Epz ; suberanilohydroxamic acid ; TSA ; 5′-aza-2′-deoxycytidine ; CMFDA .shRNAs 1. shRNA toward β1-integrin and scramble siRNA were used as reported previously , 11. TheIn general, the histological studies were performed as described in previous studies, with slight modification , 41. ForAll of the animal experiments followed the Guidelines for the Care and Use of Laboratory Animals  and were approved by the China Medical University Committee of Laboratory Animal Welfare 101-38-N). Liver-specific ARKO (LARKO) and HBVtg-LARKO mice as well as the spontaneous HCC mouse model were generated using previously described protocols 8-N. Live.AR expression was analyzed using a previously described immunoblotting assay , 45. BriThe assay was performed as previously described , 47. BriThe RNAs were extracted from cells or tissue as previously described –50. The Cell morphology was studied by staining for α-actin, stress fibers, and nuclei. Cells were seeded on four-chamber glass culture slides and then fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS. After washing with PBS, rhodamine phalloidin, a high-affinity α-actin probe conjugated to the red–orange fluorescent dye tetramethylrhodamine , was mixed with DAPI  and then applied to cells for 5 min. The cells were then sealed with cover slides and observed under a fluorescence microscope . Images were recorded using a CCD camera (DS-Qi1Mc) and then analyzed with imaging software .Total RNA was extracted and then reverse transcribed to generate cDNA, which was then subjected to microarray analyses as previously described , 52, withttp://www.broadinstitute.org/gsea/msigdb/index.jsp), KEGG (http://www.genome.jp/kegg/kegg3a.html), and MetaCore pathway analyses (http://thomsonreuters.com/metacore/) with the support of the Bioinformatics Center at National Cheng-Kung University, Taiwan . A change of > 0.1 in Enrichment Score (ES) and an ES threshold > 0.4 were used as the selection criteria for the GSEA data  were observed and photographed every 15 min for 8 h using a Time-Lapse microscope . The series of images was stacked by ImageJ software  and saved in AVI film format. The cell travel distance was recorded by the track function of ImageJ, calculated, and imported to an Excel file for interpretation.The cell mobility assay was performed as previously described , with slThe colony formation assay was performed as previously described , 57. BriEach in vitro assay was performed in triplicate, and data were derived from at least three independent sets of experiments. Values were calculated and compared using Student's t-test. DFS was calculated using Kaplan–Meier survival curves and the log-rank test. In all analyses, a P-value < 0.05 was considered to represent statistical significance.


IL)-6 gene expression, indicating a positive interaction between AR-V7 expression and activated NF-κB/IL-6 signaling in castration-resistant prostate cancer (CRPC) pathogenesis. Importantly, both AR-V7-induced NF-κB activation and IL-6 gene transcription in LNCaP and 22Rv1 cells could be inhibited by melatonin. Furthermore, stimulation of AR-V7 mRNA expression in LNCaP cells by betulinic acid, a pharmacological NF-κB activator, was reduced by melatonin treatment. Our data support the presence of bi-directional positive interactions between AR-V7 expression and NF-κB activation in CRPC pathogenesis. Of note, melatonin, by inhibiting NF-κB activation via the previously-reported MT1 receptor-mediated antiproliferative pathway, can disrupt these bi-directional positive interactions between AR-V7 and NF-κB and thereby delay the development of castration resistance in advanced prostate cancer. Apparently, this therapeutic potential of melatonin in advanced prostate cancer/CRPC management is worth translation in the clinic via combined androgen depletion and melatonin repletion.A major current challenge in the treatment of advanced prostate cancer, which can be initially controlled by medical or surgical castration, is the development of effective, safe, and affordable therapies against progression of the disease to the stage of castration resistance. Here, we showed that in LNCaP and 22Rv1 prostate cancer cells transiently overexpressing androgen receptor splice variant-7 (AR-V7), nuclear factor-kappa B (NF-κB) was activated and could result in up-regulated interleukin ( Worldwide, prostate cancer is the fourth most common human cancer and the second most common cancer in men . DespiteIn the search for novel CRPC therapy, AR-mediated functions were noted to be incompletely abrogated by ADT. While prostate cancer cells may evolve mechanisms to bypass AR signaling, the growth of CRPC has in the majority of cases been shown to be dependent on sustained AR signaling despite a castration level of testosterone. Importantly, advances in the understanding of the mechanisms involved in CRPC pathogenesis, in particular aberrant AR signaling, have led to the recent successful development of new AR-targeting therapies, such as abiraterone and enzalutamide, to dampen sustained AR signaling in CRPC ,4,5,6. O1 receptor expression in human prostate cancer tissues, and showed the association of MT1 receptor expression with the antiproliferative action of melatonin in cellular and animal models of prostate cancer [1 receptors, was found to slow the progression of the patient’s relapsed CRPC [1 receptor-mediated down-regulation of dihydrotestosterone (DHT)-activated AR signaling, and inhibition of activated nuclear factor-kappa B (NF-κB) signaling in prostate cancer cells [Instead of focusing on AR and AR-Vs, a different research approach has been adopted by our laboratory in our search for novel CRPC therapy. We target the signaling pathway of melatonin, a pineal gland neurohormone, for prostate cancer growth modulation. Initially, we demonstrated G protein-coupled melatonin MTe cancer ,17,18. Ised CRPC . Furtherer cells ,21,22,23er cells ,25 and rer cells ,27 are iTo study the effects of AR-V7 on NF-κB, prostate cancer LNCaP and 22Rv1 cells were transfected with pEGFP, pEGFP-AR-FL, or pEGFP-AR-V7. As shown in p < 0.001) and 2.4-fold (p < 0.001), in LNCaP  in LNCaP cells (p < 0.001) in 22Rv1 cells . However, the stimulatory effects of AR-V7 on IL-6 expression could be significantly reduced by 10−6 M melatonin treatment (p = 0.039) (IL-6 (p = 0.393), indicating that melatonin could abrogate the increase in IL-6 gene expression induced by AR-V7 overexpression in LNCaP cells (p = 0.005) attenuate the AR-V7 induced 2.4-fold increase in IL-6 expression  A. It is aP cells A. On thepression B in 22RvAR-V7 mRNA expression [AR-V7 was then measured by Q-PCR. To observe the induction of AR-V7 by activated NF-κB, LNCaP but not 22Rv1 cells were used because 22Rv1 cells are already expressing highly elevated AR-V7 levels compared to LNCaP cells (−6 M betulinic acid significantly (p = 0.001) elevated the expression of AR-V7 by 3.5-fold, as compared to the DMSO-treated cells (control). Of note, the betulinic acid-stimulated expression of AR-V7 could be significantly reduced (p = 0.013) by co-incubation of the LNCaP cells with 10−6 M melatonin.It has been reported that activation of NF-κB could induce pression . To confaP cells C,D. As s1 receptor in LNCaP and 22Rv1 prostate cancer cells [1 receptor is responsible for mediating the inhibitory effect of melatonin on AR-V7 induced NF-κB activation observed in the present investigation. LNCaP and 22Rv1 cells were transfected with pEGFP or pEGFP-AR-V7 expression plasmid. The pEGFP-AR-V7-transfected cells were treated separately with 10−6 M melatonin, 10−6 M luzindole (a melatonin receptor antagonist), or 10−6 M melatonin plus 10−6 M luzindole. After 48 h of treatment with melatonin with or without luzindole, NF-κB activation in transfected cells was detected by immunoblot. As shown in −6 M melatonin. Furthermore, the inhibitory effects of melatonin on AR-V7-mediated NF-κB activation could be blocked by 10−6 M luzindole. In separate experiments, LNCaP and 22Rv1 cells were transfected with pEGFP expression plasmid and after 48 h of treatment with or without luzindole, NF-κB activation in transfected cells was detected by immunoblot. As shown in 1 receptor, which has been shown by us previously to play an important role in transducing the antiproliferative action of melatonin on prostate cancer cells [While it has been recently reported by us that melatonin can inhibit the constitutively active NF-κB via membrane G protein-coupled MTer cells , we wouler cells .Prostate cancer has become a major health problem of elderly men in the western world. Men with advanced or metastatic disease receive standard palliative castration or ADT . Sadly, It is worth noting that melatonin, which acts by inhibiting both activated AR and NF-κB signaling in prostate cancer cells, has been demonstrated to be a novel prostate growth inhibitor ,23. GiveIL-6 gene expression  (Life Technologies). To minimize the influence of hormones present in normal FBS on our experiments, charcoal stripped FBS  was used in all experiments involving 22Rv1. However, normal FBS was used in LNCaP cells whose growth was significantly compromised in charcoal stripped FBS. The expression plasmids which contain full-length AR (pEGFP-AR-FL) and AR-V7 (pEGFP-AR-V7) were kindly provided by Dr. J. Luo . The cDNAs encoding AR-V7 and full-length AR were inserted into the pEGFP-C3 vector to express the EGFP-AR-V7 and EGFP-AR-FL fusion protein respectively [Human prostate cancer cell lines LNCaP.FGC (CRL-1740) and 22Rv1 (CRL-2505) were purchased from American Type Culture Collection . LNCaP and 22Rv1 cells were grown and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium without phenol red  supplemented with ectively ,37. Mela® HD Transfection Reagent . After 24 h of incubation, any fluorescence signals from the recombinant EGFP or EGFP-tagged proteins in the transfected cells were observed under a fluorescence microscope.LNCaP and 22Rv1 cells were grown in 12-well plates until 50–60% confluency. The cells were then transfected with 0.5 µg of empty pEGFP vector, 0.5 µg of pEGFP-AR-FL, or 0.5 µg of pEGFP-AR-V7, using FuGENE−6 M melatonin, 10−6 M luzindole, or 10−6 M melatonin plus 10−6 M luzindole. After 48 h, activation of NF-κB was detected using a primary antibody against the active subunit of NF-κB (Millipore) and a secondary antibody against mouse immunoglobulin G . The signals were visualized by enhanced chemiluminescence Western blotting system . All blots were stripped in 25 mM glycine buffer (pH 2.0) for 30 min before re-probing with β-actin .LNCaP and 22Rv1 cells were transfected with pEGFP, pEGFP-AR-FL, or pEGFP-AR-V7 for 24 h. The cells were then lysed in SDS sample buffer, and the proteins were harvested for immunoblotting as described previously . Express4/mL) were seeded in 24-well plates (n = 6). After 24 h, the cells in each well were transfected with a mixture of 1.0 µL FuGENE® HD Transfection Reagent , 0.2 µg NF-κB reporter plasmid pNF-κB-TA-Luc , 0.04 µg Renilla luciferase reporter pRL-tk (Promega), and 0.1 µg pEGFP, 0.1 µg pEGFP-AR-FL, or 0.1 µg pEGFP-AR-V7. The NF-κB reporter activities were measured 24 h after transfection using the Dual-Luciferase® Reporter Assay System (Promega). Both the firefly and Renilla luciferase activities were measured according to the manufacturer’s instructions. The cells were washed three times with phosphate-buffered saline. Passive lysis buffer was added to each well and was shaken at room temperature for 15 min. Lysate (20 µL) from each sample was transferred to 96-well plate for firefly and Renilla luciferase activity measurements by a luminometer [LNCaP and 22Rv1 cells  for 48 h, before the levels of endogenous AR-V7 were measured by Q-PCR, using a specific forward primer 5′-CCATCTTGTCGTCTTCGGAAATGTTATGAAGC-3′ and a specific reverse primer 5′-TTTGAATGAGGCAAGTCAGCCTTTCT-3′. Briefly, 4 µL of diluted cDNAs, 1 µL of forward/reverse primers, and 5 µL of 2× iTaq™ Universal SYBR® Green Supermix (Bio-Rad) were added to form a 10 µL PCR reaction mixture. Q-PCR was performed using MyiQ (Bio-Rad). The conditions were 95 °C for 5 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 35 s. Relative quantities of specific RNAs were calculated using comparative CT method. The fold difference was normalized to endogenous β-actin, which was detected by Q-PCR using a specific forward primer 5′-GGACTTCGAGCAAGAGATGG-3′ and a specific reverse primer 5′-AGCACTGTGTTGGCGTACAG-3′.LNCaP and 22Rv1 cells were seeded in 6-well plates. At 50–60% confluency, the cells were transfected with 0.2 µg pEGFP or 0.2 µg pEGFP-AR-V7. After 24 h of transfection, the cells were treated with 10t test. The level of significance for all statistical analyses was set at p < 0.05.The data are presented as mean ± S.E. and analyzed by unpaired Student’s AR-V7 expression in prostate cancer cells. The data support the potential use of melatonin in CRPC therapy.In this study, we demonstrated that melatonin inhibits AR-V7-induced NF-κB activation and NF-κB activator-induced


Lythrypnus dalli, a bi-directional hermaphroditic teleost fish, both sexes produce agonistic approach displays, but reproductive behavior is sexually dimorphic. The male-specific courtship behavior is characterized by rapid jerky movements  towards a female or around their nest. Activation of the supracarinalis muscle is involved in dorsal fin contributions to both agonistic and sociosexual behavior in other fishes, suggesting that differences in goby sexual behavior may be reflected in sexual dimorphism in AR signaling in this muscle. We examined sex differences in the local distribution of AR in supracarinalis muscle and spinal cord. Our results demonstrate that males do express more AR in the supracarinalis muscle relative to females, but there was no sex difference in the number of spinal motoneurons expressing AR. Interestingly, AR expression in the supracarinalis muscle was also related to rates of sociosexual behavior in males, providing evidence that sexual selection may influence muscle androgenic sensitivity to enhance display vigor. Sex differences in the distribution and number of cells expressing AR in the supracarinalis muscle may underlie the expression of dimorphic behaviors in L. dalli.Androgen signaling, via receptor binding, is critical for regulating the physiological and morphological foundations of male-typical reproductive behavior in vertebrates. Muscles essential for male courtship behavior and copulation are highly sensitive to androgens. Differences in the distribution and density of the androgen receptor (AR) are important for maintaining dimorphic musculature and thus may provide for anatomical identification of sexually selected traits. In  The expression of male-typical reproductive behavior in vertebrates is often mediated by androgen signaling through the androgen receptor (AR) to activate both central and peripheral neural circuits that innervate sexually dimorphic muscle targets , 2. As aPorichthys notatus), AR expression is abundant in the sexually dimorphic vocal muscle of males and has been suggested to modulate aspects of muscle performance [Peripheral activation of muscle AR is necessary for the production of sexually selected display maneuvers in avian species . Similarformance . Aside fformance , 14–16. formance –19. GiveLythrypnus dalli, a bi-directionally hermaphroditic teleost fish, lives in small harems of one dominant male and multiple subordinate females. Reproductive behavior is sexually dimorphic, such that only males establish and aggressively defend their nest and court females [L. dalli, which are vital to reproductive success. females , 21. ‘Je females . In cont females . Activat females , 24. Act females , 25. In The current study investigates whether expression of AR is sexually dimorphic in supracarinalis muscle and spinal cord, relative to epaxial/hypaxial muscles used for general swimming. We expect that this muscle will be hypertrophied in males, because they utilize this muscle more frequently to produce both courtship and agonistic behaviors. Since AR is necessary to maintain aspects of male phenotype in the absence of dimorphic levels of KT and facilitates adult phenotypic transitions –28, elevL. dalli were collected in June and July 2001 near the Wrigley Institute of Environmental Studies , Santa Catalina Island, California (33°26'40.8"N 118°29'00.9"W) We made all collections by scuba diving and use of hand nets. Animals were transported back to a laboratory at the USC Wrigley Institute for Environmental Science where they were placed into 12 unique social groups consisting of one male and three females, which were at least 3mm smaller than males. All social groups were constructed on approximately the same date and no individual fish was used in multiple social groups. Each group was maintained in a 40 L aquarium containing a PVC nesting tube. Tanks were provided with a constant supply of Pacific Ocean water at 18.3 C, exposed to the natural light-dark cycle, and fish were fed frozen brine shrimp twice daily. For each fish, we measured standard length, body weight (g), and genital papilla length to width ratio. At the end of the study, all males (n = 12) and random subset of females (n = 8) from different social groups were then euthanized by exposure to excess tricaine methanesulfonate , and sex was confirmed by examining gonad and genital morphology [Male (n = 12) and female (n = 36) To allow for the formation of stable social groups and thereby minimize the effects of capture-induced stress on behavior and physiology, fish groups were housed together for three weeks prior to behavioral observations. Previous work has established that 3–5 days is a sufficient amount of time to form a stable social group in this species. We observed male behavior in these groups five times over the course of three days. In total, we had three morning (08–10:00 h) and two afternoon (14–16:00 h) observation periods. All behavioral observations lasted for 10 min per group. During these observation periods, we totaled the number of approach bouts (agonistic) and jerk swims (courtship) produced by the focal male and directed at females within the social group. The total amount of behavior produced by each individual was divided by observation length to produce approach or jerk rate per minute. Following the last morning observation period, animals were rapidly sacrificed as described above.Fish were immersed in Bouin’s fixative for 7 days, rinsed in 50% ethanol, and stored in ethanol until processed. Fish were then embedded in paraffin (46–48 C melting point) and sectioned at 20 μm. Since we were primarily interested in the role the supercarinalis muscle in the control of the dorsal fin movement, we intentionally chose sections that contained this muscle see .20, antigen retrieval was performed with citric acid buffer  as described in Muchrath and Hoffmann [L. dalli [™ DAB tablets in accordance with the manufacture’s instructions . The sections were then dehydrated in an ethanol series, cleared in Citrasolv, and mounted with permount. As a control, sections (n = 2 per sex) were processed in a similar manner, except for the omission of the primary antibody. Expression of gonadal AR was used as a positive control on sections where muscle and spinal cord expression was being evaluated.Sections were deparaffinized in Citrasolv and rehydrated through an ethanol-water series. After washing hydrated slides in dHHoffmann . FollowiHoffmann –33 and hL. dalli . Biotiny2) / standard length (mm)). In addition, two independent observers who were blind to the sex of the animal counted all AR positive supracarinalis muscle cells in each section. The number of AR cells was also counted within both the dorsal and ventral horns of the spinal cord. Additionally, the number of AR cells in swimming muscle was used as a control, as these muscles do not contribute to rapid starting and stopping or dorsal fin flexion. To perform these counts in epaxial and hypaxial muscles, we randomly chose a 400x300μm area of musculature and counted all AR+ cells within this area. This 120,000 μm2 area approximates the average area of the supracarinalis muscle . All cell counts and measurements were performed using ImageJ  on the previously acquired digital images.Images were acquired using a Zeiss Axioplan microscope and Axiovision image capture software (v. 4.1). For each fish, one section at approximately the same point within the body was selected. In this randomly chosen section, supracarinalis area was measured by tracing the outside of this muscle group. Given the pronounced size differences between males and females in this species, muscle area was standardized to the standard length of each individual  was used to examine the effect of sex and muscle type  on the number of AR positive cells. Similar two-way ANOVAs were used also to examine differences in number of AR cells in the dorsal and ventral horns of the spinal cord. When applicable, 15 = 0.91, p = 0.38; Relative muscle size was not significantly different between males and females, indicating that this muscle is not hypertrophied in males . Additionally, there were no differences in AR expression within the ventral or dorsal horns of the spinal cord . Similarly, males that approached other fishes more exhibited grater levels of AR expression within the supracarinalis .There was a positive association between jerk rate and AR expression within the supracarinalis muscle , male courtship behavior is associated with high circulating levels of testosterone, but not elevated local AR expression [L. dalli has demonstrated that both sexes maintain low and comparable androgen levels [L. dalli. Nevertheless, these two examples highlight the idea that behavioral flexibility can be attained via modulation of different aspects of androgenic signaling. To augment display performance, selection may act on local androgenic sensitivity and/or by mechanisms that regulate circulating levels of androgens.The current study and others , 9, provn levels , 40. ThuL. dalli, we did not observe any sex differences in expression. Although this result does not support our prediction, work in other vertebrates, investigating sex differences in AR expression in regions of the spinal cord that innervate the muscles responsible for controlling aspects of reproductive behavior, often finds contradictory results [L. dalli, a lack of sex differences in circulating androgens, coupled with rapid changes in local androgen biosynthesis might be important mechanism for exhibiting opposite sex reproductive behaviors during adult phenotypic transitions.While AR is abundantly expressed within the spinal cord of  results , 49, 50. results . Instead results . We simiL. dalli. Thus, we suggest that differences in androgenic sensitivity may augment display vigor in this species. Given that display vigor is thought to be important during assessment of mate quality, we expect that local differences in AR sensitivity is a response to sexual selection.In the current study, we show clear evidence that AR expression is sexually dimorphic and elevated in muscles that control sociosexual behavior. Additionally, individual differences in AR expression in the supracarinalis muscle are associated with differences in male display rates in S1 FileColumn labeled ‘ID’ is the individual fish for which behavior and AR expression was collected. The ‘muscletype’ column includes the supracarinalis and control muscles . The ‘SCarea’ column the two spinal cord that were assessed .(XLS)Click here for additional data file.


Overexpression of the androgen receptor (AR) characterizes a distinct molecular subset of triple negative breast carcinomas (TNBC). The role of AR as a prognostic/predictive biomarker in TNBC is controversial, but increasing evidence suggests that this subset may respond to therapeutic agents targeting AR. Evaluation of AR has not been standardized, and criteria for selection of patients for antiandrogen therapy remain controversial. In this study we determine the appropriate threshold of AR immunoreactivity to define AR positive (AR+) TNBC, describe the clinicopathologic features of AR+ TNBC, and discuss the utility of AR positivity as a prognostic and predictive marker in TNBC.135 invasive TNBC processed in accordance with ASCO/CAP guidelines, were immunostained for AR. Clinicopathologic features of AR+ TNBC were analyzed and compared to AR negative (AR-) TNBC. Patients’ age, tumor size, tumor grade, lymph node status, proliferation rate, immunopositivity for EGFR, CK5/6, Ki-67, and disease free survival (DFS) were evaluated statistically.A 1% cutpoint was confirmed as the appropriate threshold for AR positivity. Using this cutpoint 41% of 135 TNBC were AR+. AR+ TNBC occurred in older women, were larger, had lower mean proliferation rate and increased incidence of axillary metastasis than AR- TNBC. 76% of TNBC with apocrine morphology were AR+. A subset of AR+TNBC expressed basal markers (EGFR and CK5/6). A prognostic model was created.AR identifies a heterogeneous group of TNBC. Additional evaluation of EGFR expression allowed us to stratify TNBCs into 3 risk groups with significant differences in DFS and therapeutic implications: low-risk (AR+ EGFR-) which represents the LAR molecular subtype with the best prognosis and may benefit the most from anti-androgen therapies; high-risk (AR- EGFR+) which represents the basal molecular subtype with the worst prognosis and may benefit the most from chemotherapy regimens; intermediate-risk (AR+EGFR+ and AR-EGFR-) TNBC with an intermediate prognosis. Prospective trials are required to further validate this prognostic and predictive grouping. Triple negative breast carcinomas (TNBC) are defined by absence of expression for estrogen receptor (ER) and progesterone receptor (PR) by immunohistochemistry (IHC), absence of overexpression for human epidermal growth factor receptor HER2/neu (HER2) by IHC, and absence of amplification of HER2 by fluorescent in situ hybridization (FISH). TNBC accounts for 10–20% of newly diagnosed breast cancers .Molecular analyses have shown that TNBC is a heterogeneous disease. As reported by Lehmann et al., TNBC can be further classified into four molecular subtypes , each characterized by different clinicopathologic features and different driver signaling pharmacologically targetable pathways . In anotSignificant variability exists in the reported literature regarding the frequency of AR expression in TNBC with values ranging from 7–75% –19. The This study was designed to determine the threshold of AR for immuohistochemical evaluation (minimum staining required for a tumor to be considered AR+), to analyze the clinicopathologic features of TNBCs expressing AR, and to determine the utility of AR immunoexpression as a prognostic and predictive marker in TNBC. AR expression was also integrated into a prognostic model that stratified TNBC into risk groups that may be used to help guide personalized therapy.The study protocol was approved by the Institutional Review Board of Cedars-Sinai Medical Center. The committee waived the need for informed consent. 192 consecutive invasive TNBCs diagnosed from 2008 to 2012 were retrieved from our database. TNBC were defined by lack (<1% positivity) of ER and PR immunoreactivity and a HER2/neu score of 0 or 1+ by immunohistochemistry (IHC) and absence of amplification by FISH. Clinical parameters  and pathologic features  were recorded. All tissue samples were fixed in 10% buffered formalin and embedded in paraffin wax for routine histological examination in accordance with the 2007 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines . HematoxIHC stains were performed on whole sections using a Polymer and/or SA-HRP Detection System with appropriate positive and negative controls. ER, PR, Ki-67, p53, and HER2/neu labeling indices were determined using SP1, 1E2, K-2, DO7, and 4B5 antibodies , respectively. ER, PR, Ki-67 and p53 immunoexpression was evaluated as the percentage of cells exhibiting nuclear staining. Cell proliferation (Ki-67) was assessed by counting at least 500 tumor cell nuclei (depending upon the availability of tumor) and graded as low (<11%), intermediate (11–20%), and high (>21%) . ImmunosImmunohistochemical detection of Androgen Receptor was performed on 4-μm whole tissue sections with antibody clone F39.4.1  applied at 1:100 dilution and incubated for 45 minutes at room temperature. Pretreatment was performed by the Dako PT Link module with low pH buffer . Staining was done on the Dako Autostainer and visualized by the Dako Envision mouse detection system at 30-minute incubation using Dako DAB and subsequent counterstaining with Mayer’s hematoxylin. The percentage of tumor nuclei that stained was recorded.All cases were evaluated independently by two pathologists. Consensus results were recorded and discordances were resolved by review and discussion.Statistical analysis was performed using R software 3.1.1  and SPSS 17.0 statistical software . The Wilcoxon test was used to compare the clinicopathologic characteristics of AR+ and AR- TNBCs. Survival differences between the two groups were compared using log-rank tests and Kaplan-Meier curves. Disease-free survival (DFS), defined as the interval between period after curative treatment and the date of first recurrence or progression of disease, or the date of death from any cause, was used as the endpoint for survival analysis. Patients who were not reported to be dead at the time of the analysis were censored at the date they were last known to be alive.In order to determine the appropriate threshold for AR positivity the 135 cases were divided into 2 groups: Group A  and Group B . Tumor characteristics and DFS survival were similar in the two groups. Additionally, DFS survival of EGFR+ and EGFR- cases in the two groups was also similar . DFS wasPrognostic factors were determined using Cox proportional hazard regression analysis. The resultant significant biomarkers/variables were selected as candidate prognostic indicators, and the prognostic contributions of a combination of biomarkers were identified by multivariate Cox analysis . PatientThe 135 women ranged in age from 28 to 92  years at the time of TNBC diagnosis. Each patient had received lumpectomy or mastectomy as primary treatment followed by chemotherapy and radiation therapy. Follow-up ranged from 2 to 54 (median 22) months. Tumor size varied from 0.3 to 10.3  cm. 98% (132/135) of the TNBC were infiltrating ductal carcinomas of which 13% (17) showed apocrine differentiation  in at least 70% of the neoplastic cells . The remNo significant difference was observed in DFS within Group A or within Group B when 1%, 10%, 20%, 25% or 30% staining was used as the threshold for AR positivity . ClinicoWhen 1% was utilized as the threshold to define AR positivity, 41% (55/135) of the TNBC were AR+ . Among tWomen with AR+TNBC ranged from 29 to 89 years. The mean age of women in the AR+ group was significantly older than that of women in the AR- group .AR+TNBC varied in size from 0.6 to 10.3  cm. AR+TNBC were significantly larger than AR-TNBC .96% (53/55) of the AR+TNBC were ductal, one was lobular, and one was mixed ductal/lobular. 24% (13/55) of the AR+TNBC were ApoCA+  compared89% (49/55) of the AR+TNBC had a high histologic grade (Modified Bloom Richardson score III). No difference was noted in tumor grade when compared to the AR-TNBC. However, AR+ApoCA+TNBC showed a lower grade when compared to AR+ApoCA-TNBC (p = <0.001). 98% (41/42) of AR+ApoCA-TNBC demonstrated a high histologic grade. In comparison 62% (8/13) AR+ApoCA+TNBC demonstrated a high histologic grade and 38% (5/13) demonstrated an intermediate histologic grade.A statistically significant inverse correlation was observed between AR expression and proliferation rate (p = 0.014). Lower levels of AR expression were associated with higher levels of Ki-67 immunostaining . 78% . EGFR positivity was more frequently observed than CK5/6 positivity in AR+TNBC (76% vs. 20%). EGFR positivity was also more frequently seen in AR+ compared to AR- TNBC . 45% (42/94) EGFR+TNBC and 23% (11/34) CK5/6+TNBC expressed AR suggesting that a subset of AR+TNBC also expresses the basal phenotype .Axillary lymph node metastasis were more frequent in AR+ than AR- TNBC . This statistically significant difference was maintained when the AR+ApoCA+TNBC were compared with the AR+ApoCA- subgroup .The mean DFS in the AR+TNBC was 35.8 months. When compared to the mean DFS in the AR-TNBC (34.1 months), no significant difference was observed.Using ≥1% AR immunoreactivity to define AR+TNBC, 41% of our study cohort were AR+. When compared to AR-TNBC, AR+TNBC were larger and more frequent in older women, showed a higher incidence of apocrine differentiation, a higher incidence of axillary lymph node metastasis, and lower proliferation rates. No siginificant difference was observed in mean DFS between the two groups. 24% exhibited prominent apocrine differentiation. AR+ApoCA+TNBC were also more frequent in older women, were larger tumors with lower proliferation rates and increased lymph node metastasis when compared to AR+ApoCA-TNBC. A subset of AR+TNBC demonstrated EGFR and CK5/6 positivity suggesting a basal phenotype.We previously identified EGFR immunoreactivity in ≥15% of tumor cell nuclei as a negative correlate of DFS in TNBC . In the Group 1 (low-risk TNBC): AR+EGFR- tumors. This group had the best outcome with mean DFS of 43.7 months and only a single event (either disease recurrence or metastasis at one site).Group 2 (intermediate-risk TNBC): AR+EGFR+ and AR-EGFR- tumors. This group had a mean DFS of 35.9 months. 19% patients in this group had ≥1 events (disease recurrence and/or metastases at multiple sites).Group 3 (high-risk TNBC): AR-EGFR+ tumors. This group had a mean DFS of 29.8 months and the highest event rate with ≥1 events having occurred in 34% of patients. We also noted that the mean proliferation rate (Ki-67%) increased from 30% in the low-risk group to 57% in the high-risk group.AR is a nuclear steroid hormone receptor that is normally expressed in benign breast tissue where it is coexpressed with ER and PR in 5–30% of luminal epithelial cells . AR is aAR is reported in 7–75% of TNBC –19. ThisIn an effort to determine the most appropriate threshold, we analyzed a cohort of 135 TNBC using several different cutpoints and found no difference in DFS of patients with cutpoints >1%. Although similar clinicopathologic correlates  were noted when 25% and 1% were used as cutpoints, additional correlations with larger tumor size and increased incidence of node metastasis were observed only with 1% as the cutpoint. Therefore, 1% was considered the most appropriate cutpoint for evaluation of AR by immunohistochemistry. Adoption of 1% immunoreactivity to define AR+ TNBC would also allow the largest number of patients to benefit from targeted therapies.Several studies have reported AR immunoexpression as a favorable prognostic factor associated with lower clinical stage, lower histologic grade, lower mitotic score, and better outcome  –24, 40. AR expression in the LAR molecular subtype has been shown to be 10X that in non-LAR TNBC. LAR TNBC usually express low levels of basal biomarkers and are predominantly subclassified in the non-basal subgroup . ConversS1 Fig(TIFF)Click here for additional data file.S2 Fig1–24% of neoplastic cells are positive. 20x.(TIFF)Click here for additional data file.S3 FigMore than 15% of neoplastic cells are positive. 20x.(TIFF)Click here for additional data file.S1 TableA. group A, study set, n = 35 and B. Group B, validation set, n = 100.(DOC)Click here for additional data file.


The vast majority of prostate cancer deaths are due to castration-resistant prostate cancer (CRPC) – the lethal form of the disease that has progressed despite therapies that interfere with activation of androgen receptor (AR) signaling. One emergent resistance mechanism to medical castration is synthesis of intratumoral androgens that activate the AR. This insight led to the development of the AR antagonist enzalutamide. However, resistance to enzalutamide invariably develops, and disease progression is nearly universal. One mechanism of resistance to enzalutamide is an F877L mutation in the AR ligand-binding domain that can convert enzalutamide to an agonist of AR activity. However, mechanisms that contribute to the agonist switch had not been fully clarified, and there were no therapies to block AR F877L. Using cell line models of castration-resistant prostate cancer (CRPC), we determined that cellular androgen content influences enzalutamide agonism of mutant F877L AR. Further, enzalutamide treatment of AR F877L-expressing cell lines recapitulated the effects of androgen activation of F877L AR or wild-type AR. Because the BET bromodomain inhibitor JQ-1 was previously shown to block androgen activation of wild-type AR, we tested JQ-1 in AR F877L-expressing CRPC models. We determined that JQ-1 suppressed androgen or enzalutamide activation of mutant F877L AR and suppressed growth of mutant F877L AR CRPC tumors  Prostate cancer is the most commonly diagnosed and second-most lethal cancer in men in the United States . The vasPreviously, several groups identified mutations in the AR ligand binding domain (LBD) that convert first-generation anti-androgens such as bicalutamide and flutamide to AR agonists rather than antagonists , 8. ClinIn this study, we used cellular models to identify molecular mechanisms that contribute to enzalutamide agonism of mutant F877L AR. We determined that enzalutamide treatment of AR F877L-expressing cells led to greater agonistic effects when the cells were cultured in conditions with low androgen ligands. This suggests that the androgen content of the cell may determine whether enzalutamide has the capacity to activate mutant F877L AR. Further, we found that enzalutamide activation of mutant F877L AR leads to induction of a similar, albeit smaller, set of genes that are activated by the natural AR ligand dihydrotestosterone (DHT). This suggests that DHT is a more potent activator of AR function.in vitro and in vivo. Thus, BET bromodomain inhibition is a promising strategy to block AR F877L function irrespective of whether the AR ligand is androgens or enzalutamide.Finally, prior work showed that inhibition of the BET bromodomain family of chromatin readers is an effective strategy to suppress androgen ligand activation of wild-type AR . We detein vitro or in vivo with enzalutamide. One example is the MR49F cell line that was derived after LNCaP cells were implanted in castrated mice and treated chronically with enzalutamide [In order to study the problem of acquired enzalutamide resistance, several groups have chronically treated prostate cancer cell lines lutamide , 17. MR4lutamide  and a nelutamide . To conflutamide , 11.KLK3 and TMPRSS2 in V16D cells . Importantly, prostate cancer cells are capable of metabolizing the testosterone found in FBS into dihydrotestosterone (DHT) at concentrations similar to those found in CRPC tumors (~1–10 nM) that promote AR function and CRPC growth , 21. We s Figure . Howevers Figure .To more globally examine the effect of enzalutamide on gene expression, we performed RNA-seq on MR49F or V16D cells treated with vehicle or enzalutamide. Enzalutamide treatment of V16D cells led to 478 differentially-expressed genes  [To determine if the effect of androgen content on enzalutamide agonism of F877L was generalizable, we used LNCaP cells stably overexpressing F877L AR and also an AR-driven probasin promoter GFP reporter construct (LNCaP Pb. EGFP P-F877L) . We treaTo more globally examine the effects of DHT or enzalutamide on F877L AR activation, we grew MR49F cells in charcoal-stripped serum, treated the cells with vehicle, 10 μM enzalutamide, or 1 nM DHT, and then performed RNA-seq. Under androgen-depleted conditions, enzalutamide treatment led to 396 significantly differentially expressed genes compared to vehicle treatment. DHT treatment, on the other hand, induced expression changes in 2048 genes, again suggesting that DHT is a more potent agonist than enzalutamide in MR49F cells at the concentrations tested. Both the DHT- and enzalutamide-activated gene sets showed significant enrichment for the aforementioned signature of canonical AR target genes  [To more globally examine the effects of BET bromodomain inhibition on AR transcriptional function, we performed RNA-seq on samples from MR49F cells grown in androgen-depleted conditions and stimulated with vehicle, DHT, or enzalutamide with or without JQ-1. As described above, 358 conserved genes were significantly differentially expressed (at any fold change) in response to enzalutamide and DHT treatment Figure . JQ-1 co−9) . FurtherMR49F cells harbor both non-mutant and mutant F877L AR alleles. To determine if the JQ-1 treatment effect was independent of effects on wild-type AR, we transfected AR-null PC3 cells with ectopic mutant F877L AR and an ARE4 luciferase reporter of AR function, and then treated them with DHT or enzalutamide +/− JQ-1 , 24. JQ-in vivo. Prior work demonstrated that enzalutamide treatment of MR49F xenografts implanted in castrated mice leads to agonistic growth compared to vehicle treatment [Finally, we sought to determine the tolerability and preliminary anti-tumor efficacy of JQ-1 in an F877L CRPC model reatment . Since tDespite medical castration, CRPCs are still dependent on the AR . AR amplOne mechanism of resistance to newer AR-targeting agents is the expression of AR splice variants that lack the LBD entirely and promote ligand-independent growth . In partWhile not as common as AR-V7 induction, point mutations that result in functional changes to the AR LBD are another mechanism of resistance to anti-androgen treatment strategies . ExampleThe F877L mutation results in a conformational change to the AR LBD  that aboOur data shed new light on molecular mechanisms that contribute to mutant F877L AR activation by enzalutamide. While cells expressing the F877L mutation are resistant to enzalutamide, the enzalutamide agonistic effect on F877L AR is correlated with cellular androgen content. Indeed, we found that enzalutamide treatment of F877L-expressing MR49F cells only enhanced cell growth under low androgen conditions . Furthermore, enzalutamide activates a transcriptional program that strongly overlaps with that activated by DHT, but induction of this program only occurred when androgens were depleted from culture. The effect of androgen interference with enzalutamide agonism was confirmed in an additional model – LNCaP cells with ectopic overexpression of F877L.One explanation for the lack of enzalutamide agonism in androgen-replete conditions is that enzalutamide activation of mutant F877L AR is cancelled out by suppression of wild-type AR. Another possible explanation is that, in androgen-replete conditions, androgen ligands compete with enzalutamide for binding and activation of mutant F877L AR, thereby diminishing agonistic effects of enzalutamide. Indeed, a recent report demonstrates that intratumoral androgens persist or may increase in enzalutamide-resistant tumors . Our datIn this study, we also measured the global gene expression change induced by DHT or enzalutamide treatment in F877L AR-expressing cells. DHT treatment induced a greater number of gene expression changes versus enzalutamide treatment, and the magnitude of gene expression change for nearly all genes was also greater with DHT vs. enzalutamide treatment. This further demonstrates that DHT may more potently activate F877L AR than enzalutamide.Importantly, while DHT induced many unique gene expression changes vs. enzalutamide, nearly all the gene expression changes induced by enzalutamide were shared with DHT treatment. This demonstrates that enzalutamide agonism of F877L AR does not activate a distinct gene expression program vs. DHT. Further, there was a strong enrichment for a signature of canonical wild-type AR signaling  with eitc-Myc and others, by cooperating with transcription factors, including: AR, the estrogen receptor, GATA1, and p53 [Inhibition of BET bromodomain proteins was shown previously to block wild-type AR activation by androgens . BET bro and p53 , 44–47.  and p53 . For thiPrior work demonstrated that AR-dependent cell lines are more susceptible to JQ-1 treatment than AR-null cell lines, suggesting that AR is a critical BET bromodomain inhibitor target in CRPC. . We founin vivo. We determined that JQ-1 co-treatment with enzalutamide blocked tumor growth vs. enzalutamide treatment alone. Body weights were similar between the two treatment arms, and we did not observe any additional toxicity with JQ-1 + enzalutamide vs. enzalutamide alone. This further demonstrates the potential of BET bromodomain inhibition for the treatment of F877L AR-expressing CRPC tumors and the preliminary safety of combining BET bromodomain inhibitors with enzalutamide.We also measured the effect of JQ-1 on growth of MR49F xenografts implanted in castrated mice Altogether, our data demonstrate that the androgen content of the CRPC cell influences enzalutamide's ability to activate mutant F877L AR and suggest that enzalutamide agonism may be more pronounced in tumors harboring F877L mutations that have the lowest androgen concentrations. Finally, our results demonstrate that BET bromodomain inhibition is a promising treatment to block mutant F877L AR, irrespective of whether the AR ligand is androgens or enzalutamide. Clinical trials with BET bromodomain inhibition in CRPC that have recently begun (NCT02711956) will be necessary to confirm these results.F877L, ARWild-Type, or empty control vector were a kind gift from Dr. Charles Sawyers [PC3 cells were purchased from American Type Culture Collection . LNCaP Pb. EGFP cells stably overexpressing AR Sawyers . All celin vitro experiments, JQ-1 was obtained from BPS Bioscience (27402). For in vivo studies, JQ-1 was obtained from the laboratory of Dr. James Bradner, Dana Farber Cancer Institute Dept. of Medical Oncology/Harvard Medical School Dept. of Medicine. For in vitro studies, DMSO stocks of enzalutamide and JQ-1 and ethanol stocks of DHT were diluted to desired working concentrations in cell culture media  with a final vehicle concentration of 0.1%. Vehicle-only controls were used in all cases, and vehicle concentrations were normalized across all drug co-treatments. See below for information on administration of drugs for in vivo studies.Enzalutamide was obtained from MedchemExpress (HY-70002). Dihydrotestosterone (DHT) was obtained from Sigma (A8380). For nuFoxn1, Harlan Laboratories, strain code 069). Once tumors reached 100mm3, groups of mice were randomized to treatment with DMSO or JQ-1 (50 mg/kg) by intraperitoneal injection Monday through Friday. All animals were treated with enzalutamide (10 mg/kg) by oral gavage Monday through Friday. Tumor measurements and body weight measurements were recorded, and all animals were sacrificed on Day 28. Tumor volumes and final body weights are shown as mean ± standard error of the mean (SEM). 2-sided t-test with equal variance was performed using end point (week 4) data. Differential tumor wet weights at sacrifice between treatment groups was tested using a 2-sided 2-sample t-test with Welch's correction, as the distribution appeared to be approximately normal. P-values less than 0.05 were considered statistically significant. Mice were housed under specific pathogen-free conditions (5 mice per shoebox cage) and maintained by the OHSU Department of Comparative Medicine. All in vivo studies were conducted under an OHSU IACUC-approved protocol (protocol #IS00003757).MR49F xenografts were implanted in 7-8 week old castrated, immunocompromised male mice , TMPRSS2 (Hs01120965_m1), NKX3.1 (Hs00171834_m1). Human β-actin TaqMan probe (Hs99999903_m1) was used as an endogenous control. RT-qPCR raw Ct data were analyzed with 7500 Software v2.0.5 and DataAssist Software v3.0 (Life Technologies).Cells were lysed using Trizol reagent (Life Technologies), and RNA was isolated using chloroform extraction and alcohol precipitation per manufacturer's instructions. RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer. 1.0 μg RNA was reverse-transcribed using a High Capacity cDNA Reverse Transcription Kit (Life Technologies) with random primers. RT-qPCR was performed using a 7500 Fast thermocycler (Life Technologies) with the following cycling program: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 sec dissociation, 60°C for 1 min annealing/extension/read. 10 μL singleplex RT-qPCR reactions contained 1X TaqMan (Life Technologies) universal standard Master Mix, 1X TaqMan hydrolysis probe, and 10 ng RNA-equivalent cDNA template. TaqMan probes were used to detect human 3 (Life Technologies) according to the manufacturer's protocol. Two wells were combined for each replicate for RNA extraction in 1 mL Trizol, and the aqueous phase was put through the Qiagen RNEasy kit for cleanup. Sample preparation followed the Agilent SureSelect Strand-Specific mRNA Library Preparation Protocol . Poly-A RNA was purified from 1 μg total RNA per sample using two serial rounds of binding to oligo dT magnetic beads. The poly(A) RNA was chemically-fragmented, and first-stand cDNA was synthesized using the RNA-seq First Strand Master Mix (Agilent). After purifying the first strand cDNA using AMPure XP beads, second-strand cDNA was synthesized and ends were repaired. A second round of cDNA purification with AMPure XP beads occurred, and the 3′ ends were adenylated, followed by adapter ligation and AMPure XP beads purification. Ligated DNA was PCR-amplified for 14 cycles and purified again with AMPure XP beads. Quality of the resulting libraries was assessed with an Agilent 2100 Bioanalyzer DNA 1000 Assay. Libraries were sequenced on an Illumina HiSeq as single-end 50 bp reads in the OHSU Massively Parallel Sequencing Shared Resource.Total RNA was extracted with Trizol/CHClRNA-seq was conducted on three biological replicates of each cell line/drug treatment. RNA-seq data analysis was performed using the Tuxedo Suite –51. EachTo identify genes that were differentially-expressed compared to mock treatment, data was first filtered to remove genes with low variation across the sample set. For each cell line, we used a t-test on the filtered normalized expression values to identify genes whose expression was significantly changed by drug treatment . We used Benjamini-Hochberg false discovery rate to account for multiple comparisons, and deemed genes with q < 0.05 to be significant. Statistical analyses were performed in R (version 3.1.2) and displayed in RStudio (version 0.98.501).Gene Set Enrichment Analysis (GSEA)  was run Gene ontology (GO) enrichment and functional annotation was performed on the 358 genes concordantly differentially expressed with enzalutamide and DHT treatment from Figure Renilla luciferase, driven by the SV40 promoter. This plasmid was a kind gift from the laboratory of Dr. David Qian of the OHSU Knight Cancer Institute.The ARE-4 firefly luciferase reporter of AR transcriptional activation has been reported previously and was a kind gift from Dr. Xin Yuan , 24. ForTo transiently knock down AR, cells were transfected with AR siRNA oligonucleotides 5′-(GACCUACCGAGGAGCUUUCdTdT-3′) (Dharmacon) described previously . Control® Aqueous One Solution Cell Proliferation Assay System (Promega) per the manufacturer's instructions. Counts of viable cells were performed using the trypan blue exclusion method as calculated by the Countess instrument (Life Technologies) according to the manufacturer's instructions.MTS assays of viability were performed using the CellTiter 96Wild-Type or ARF877L overexpressing cells were cultured in either full-serum or charcoal-stripped medium as described above for one day prior to drug treatment. Drug treatments were then performed in the appropriate serum  as described above for three days. Cells were dissociated with Accumax reagent (Stem Cell Technologies). GFP reporter activity in live cells was measured with flow cytometry Core using a BD FACS Canto II instrument (BD Biosciences) in the OHSU Flow Cytometry Core. The resultant FCS files were analyzed and plotted using FlowJo Single Cell Analysis Software.LNCaP Pb. EGFP ARPC3 cells were transiently co-transfected with equal amounts of an AR F877L mutant expression construct and a 10:1 ratio of ARE4-Luc AR firefly luciferase reporter , 24 and in vivo experiments is also described in detail above, and values of error bars and number of replicates is indicated in the Figure Statistical analyses of RNA-seq data are described in detail above, and the number of replicates is described in the above methods or the respective figure legends. Statistical analyses of


Allergic rhinitis is a frequent presenting problem in primary care in the UK, and has increased in prevalence over the last 30 years. When symptomatic, patients report significant reduction in their quality of life and impairment in school and work performance. Achieving adequate symptom control is pivotal to successful allergic rhinitis management, and relies mostly on pharmacotherapy. While it is recognised that most mild-moderate allergic rhinitis symptoms can be managed successfully in primary care, important gaps in general practitioner training in relation to allergic rhinitis have been identified. With the availability of new effective combination therapies, such as the novel intranasal formulation of azelastine hydrochloride and fluticasone propionate in a single device , the majority of allergic rhinitis symptoms can be treated in the primary care setting. The primary objective of this consensus statement is to improve diagnosis and treatment of allergic rhinitis in primary care, and offer guidance on appropriate referral of difficult-to-treat patients into secondary care. The guidance provided herein outlines a sequential treatment pathway for allergic rhinitis in primary care that incorporates a considered approach to improve the management of allergic rhinitis symptoms and improve compliance and patient satisfaction with therapy. Adherence with this care pathway has the potential to limit the cost of providing effective allergic rhinitis management in the UK by avoiding unnecessary treatments and investigations, and avoiding the need for costly referrals to secondary care in the majority of allergic rhinitis cases. The fundamentals presented in this consensus article should apply in most health-care settings. The aim of any disease management plan is control of the disease. In 2015, a multi-professional group of clinicians met in Stirling, Scotland to discuss the development of a simplified allergic rhinitis (AR) treatment algorithm for use in primary and secondary care, incorporating current knowledge and recently available treatments. This consensus statement is a result of that initial meeting and subsequent collaboration among the working party. The primary objective is to promote better management of AR patients, improve diagnosis and treatment in primary care and to facilitate transition of difficult-to-treat patients into secondary care.2 and is increasing in prevalence.4 It is associated with considerable morbidity; significantly reducing quality of life,6 interfering with attendance and performance at school and work,9 and resulting in considerable health-care and indirect costs.13 Around 50–80% of AR patients report that their condition interferes with sleep, resulting in daytime fatigue, decreased alertness, inability to concentrate, depression and irritability.14 AR is also associated with a number of coexisting conditions, including asthma, sinusitis, upper respiratory tract infection, otitis media with effusion and nasal polyposis.16AR a common condition that affects >20% of the UK population, is a frequent presenting problem in primary care,18 Most mild-moderate AR symptoms can be managed successfully in primary care. However, one-third of general practitioner (GP) specialty training programmes do not provide allergy training.19 Initial AR monotherapy is failing many patients in the UK.20 A database survey of 22,000 AR patients living in the UK showed that the majority of seasonal AR (SAR) and perennial AR (PAR) patients received monotherapy as their initial prescription of the season, but such monotherapy  proved insufficient for many . Other patients started the season on a multi-therapy regimen  and this proportion increased during the season. As the number of therapies prescribed increased, so too did the number of return GP consultations. By the end of the season around half of all AR sufferers were prescribed multiple therapies, most commonly oral AH plus INCS. However, this combination is not supported by clinical trials of combination regimens vs. monotherapy.22 The Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines state that “combination between drugs has been tested, but insufficient data are available to make a recommendation concerning the combined use of oral AH and INCS”.23Management of AR is generally not allergen-specific and relies in the main on pharmacotherapy to control symptoms.24 Both AR and non-AR are risk factors for the development of asthma; co-morbid rhinitis affects up to 75% of those with asthma.25 Poor control of rhinitis predicts poor control of asthma; asthma patients with significant rhinitis are four-times more likely to have poorly-controlled asthma than those without,24 with a negative impact on asthma control equivalent to that of smoking.26 Moreover, failure to treat AR appropriately results in increased asthma medication use for co-morbid patients.27The link between AR and asthma is well-established.28 and Rhinitis Control Assessment Test.30 Others advocate a multi-factorial assessment incorporating an assessment of (i) severity and/or frequency of daily or nocturnal symptoms, (ii) impairments in social, physical, professional and educational activities, (iii) respiratory function monitoring and (iv) exacerbations .31 Although useful in clinical trials these are unlikely to be used routinely in day-to-day practice. ARIA has recently recommended a simple visual analogue scale (VAS) as the new language of AR control, with a score of 5/10 cm used to assess control and guide treatment decisions as part of a simple algorithm called the AR clinical decisions support system (CDSS).32 This VAS has also been incorporated into an app for patients (Allery Diary). A companion app for physicians is currently under development , thus linking key stakeholders with a common language of AR control.Some sophisticated AR control questionnaires have been developed including the Control of Allergic Rhinitis and Asthma Test33 AR patients have high expectations from their treatment,34 but also a high degree of dissatisfaction with AR treatment,35 for reasons that include: symptom breakthrough, lack of 24-hour coverage, lack of coverage of both nasal and ocular symptoms, and unwanted side effects.15 Patients want faster and more complete symptom control.27 Insufficient symptom control with INCS and oral AH is a major concern, and a situation, which has not improved over time.15 Many physicians underestimate AR severity and consequently fail to issue adequate treatment,36 a situation which appears to have changed little in a decade.37Living with symptomatic AR means coping with any or all of the symptoms of nasal congestion, headache, postnasal drip, repeated sneezing, runny nose and other symptoms on a near-daily basis.23 and the diagnosis of AR by symptom assessment is outlined in Fig. 23 Confirmation of the diagnosis may be made through questions concerning family history, social history , and visual examination. The common triggers for AR are listed in Table 1The definition, aetiology and classifications of AR are described in Table 1 At greatest risk are those with a personal or family history of atopy .2 The clinical history should determine whether allergy testing is required, which may be useful to identify or exclude an allergic trigger that may influence management, e.g., aeroallergen avoidance. GPs should perform either skin prick testing or serum total/specific IgE depending on local availability. The former has the advantage that it is cheaper and the patient gets instant feedback, but is time consuming and requires stopping anti-histamines for a week. Ideally, allergy testing would be performed in all patients with rhinitis not only for advice about potential allergen avoidance but also because it will alter the management pathway. Allergy testing will not, however, identify non antigenic chemical irritants which can cause nasal airway hyper-reactivity through a non IgE-mediated pathway. Indeed in many cases exogenous chemical irritants may aggravate underlying AR. In reality, the majority of patients presenting in primary care with AR can be managed without formal identification of the specific allergic trigger18 with pharmacotherapy.AR is the predominant form of rhinitis in children, but also accounts for one-third of adult cases.1 in order to simplify treatment in primary care and incorporate recently available treatment options.Figure Patients should be educated on the nature of AR, allergen avoidance, available treatments , and be given realistic expectations of the results of therapy. Treatment failure may be related to poor technique in the use of nasal sprays and drops and, therefore, appropriate training is essential.39 It is more commonly used in mainland Europe than in the UK. In our opinion it has only a limited role in the management of AR, and there is little objective data to support its use.Saline douching is a safe, inexpensive treatment, demonstrated to reduce symptoms in children and adults with seasonal rhinitis.40 with the additional benefit of a faster onset of action .41 Patients responsive to AH therapy should be instructed to continue therapy during periods of allergen exposure. Those non-responsive to therapy  should be stepped up.Oral and topical non-sedating AHs are recommended as first-line therapy for mild-moderate intermittent and mild persistent AR. The therapeutic effect of intranasal AH is superior to that that of oral AH,43 Responsive patients should be instructed to continue therapy during periods of allergen exposure. Those non-responsive to therapy  should be stepped up.INCSs are recommended as first-line therapy for moderate-severe persistent symptoms and treatment failures with AHs alone. For patients who do not tolerate INCSs or prefer not to use them, adding a leukotriene receptor antagonist to an oral or topical AH may be considered.44 In primary care, the consensus group considers that INCS/AZE (single device) may be used second line after failure of topical INCS after checking for the correct use and compliance with medication. If incorrect use or non-compliance is found, re-education followed by repeat trial of topical INCS is recommended before INCS/AZE (single device) prescription. Alternatively, the addition of an oral AH to INCS may be considered in the case of INCS monotherapy failure, particularly for children <12 years for whom INCS/AZE (single device) is not yet indicated. However, it should be noted that the evidence to support this practice is lacking.45The combination of intranasal azelastine hydrochloride (AZE) and fluticasone propionate (FP) in a single device (Dymista® (INCS/AZE); Meda, Solna, Sweden) is a novel formulation that is indicated for adults and adolescents ≥12 years with moderate/severe SAR or PAR if monotherapy with either INCS or AH is not considered sufficient.44 This recommendation is based on an extensive review of existing clinical trial evidence,49 and data obtained in routine clinical practice in several countries in Europe including Germany, Sweden, Denmark, Norway and Romania.50 INCS/AZE (single device) is recommended first-line in the recently published AR CDSS from ARIA for those patients with a VAS score >5/10 cm.32 The speed of onset with INCS/AZE (single device) for both nasal and ocular symptom relief and the use of a single spray may improve compliance and treatment outcomes by avoiding administration and delivery issues.51INCS/AZE (single device) may be used first line in certain cases in which the GP considers that monotherapy is more likely to fail, if they consider it warranted in their professional opinion, in accordance with the labelled product indication.1This consensus group does not recommend routine early use of oral corticosteroids (OC), which should be reserved for short-term acute severe symptoms, in combination with a topical INCS or AZE/INCS (single device). A suggested regime for adults is 0.5 mg/kg given orally in the morning for 5–10 days.In the event of treatment failure on second-line INCS/AZE (single device) in primary care, checking for the correct use of medication, compliance, and whether the correct diagnosis was made is advised. Following that, referral to secondary care is recommended for further investigations.In secondary care, the consensus group considers that INCS/AZE (single device) could be used as a first-line alternative to topical INCS in patients with moderate-severe symptoms Fig. . This is45 In the event of INCS/AZE (single device) failure, the consensus group recommended adding OC for 7–14 days for control of acute severe symptoms, for example during the peak of the pollen season. If treatment failure continues, the diagnosis should be revisited in the first instance, while surgery or immunotherapy may be subsequent treatment options.Symptom-specific investigations for refractory symptoms should be completed in secondary care following INCS/AZE (single device). Add-on therapy with symptom-specific treatment such as ipratropium for watery rhinorrhoea, or oral AH for nasal itching/sneezing may be reserved for use in secondary care. It should be noted that currently there is no scientific evidence to support this practice.It seems likely that the use of INCS/AZE (single device) may be an additional treatment option prior to immunotherapy. Immunotherapy may be appropriate for some patients with persistent symptoms predominantly due to one allergen such as grass pollen. However, many patients are polysensitised and will remain symptomatic despite immunotherapy; these patients should receive symptomatic treatment in addition to immunotherapy. Relapse following successful immunotherapy treatment may occur; for these patients further treatment options including symptomatic treatment may be considered.47 In addition, switching INCS is also not recommended as all have comparable efficacy.54 The panel recommends prescription of INCS/ AZE (single device) as the evidence-based option,49 rather than adding oral AH to INCS, as there is lack of consistent evidence for the additive efficacy of the latter.45 The consensus panel judge that positioning INCS/AZE (single device) as a second-line intervention in the primary care pathway may provide value to the health service by averting repeat GP visits (at £45 per consultation)27 or costly secondary care referrals .55 The fundamentals presented in this consensus article should apply in most health-care settings, although obviously different patterns of allergen exposure and allergens will occur, and cost considerations and pharmacotherapy availability may differ.Achieving adequate symptom control is pivotal to successful AR management and may be attained though the stepwise treatment algorithm outlined here. Correct administration technique for INCS and good compliance with treatment regimen are essential to achieve symptom control, and should be checked in the event of early treatment failures. In primary care, following treatment failure on INCS the consensus panel does not recommend increasing the dose of INCS, for which there is little evidence due to the recognised ceiling effect.56 There are patients who do not tolerate the first-generation oral AHs available OTC , and the safety of drivers who self-medicate on escalating doses of additive first-generation oral AHs is a concern. In addition, there is a need for alternatives to OCs for symptom relief for major life events, such as exams and weddings. The panel considered that OCs are positioned too early in the 2008 BSACI algorithm,1 and should be recommended only for short-term use to gain control of acute severe symptoms. The forthcoming update to the BSACI guidelines is awaited with interest.Achieving adequate symptom control through efficacious prescription medication will avoid patients resorting to self-medication with add-on over-the-counter (OTC) products, which can lead to adverse effects with prolonged use, e.g., rhinitis medicamentosa with repeat topical nasal decongestants such as oxymetazoline.27 A proportion of patients will not be adequately controlled on initial therapies, and should be referred to secondary care for confirmation of diagnosis and further investigation. They may also receive symptom-specific add-on therapy. There is some advocacy for a step down approach to AR management; for example, starting with INCS/AZE (single device) in patients with previous treatment failure or resistance to monotherapy. After a few weeks of achieving complete control, consideration could be given to treatment reduction e.g., stepping down to INCS alone. However, it is acknowledged that the step-down approach is based on consensus and more data are needed. Nonetheless, positioning INCS/AZE (single device) first line in secondary care with an integrated check of compliance and technique has the potential to avert costly add-on treatments, repeat visits and possibly the need for immunotherapy or surgery.It was noted that all current guidelines take a patient down a route of escalating treatment without taking a step back and assessing whether treatment can be stepped down and when the patient should be referred. Up to 50% of AR patients in primary care are receiving step 3 treatment with polypharmacy, perhaps inappropriately.With the availability of effective therapy, the majority of AR symptoms can be treated in the primary care setting. This guidance provides a clear pathway for AR treatment in primary care practice that outlines a considered approach to improve the management and control of AR symptoms and improve compliance and patient satisfaction with therapy. Adherence with this care pathway has the potential to limit the cost of providing effective AR management in the UK by avoiding unnecessary treatments, and investigations and avoiding the need for unnecessary repeat GP visits and costly referrals to secondary care in the majority of AR cases.


All the variables were analyzed taking into account both the absolute values accumulated over the week and the normalized values in relation to individual mean competition values. Neuromuscular fatigue was measured objectively using the countermovement jump test and subjectively via the Total Quality Recovery (TQR) scale questionnaire. Analytical correlation techniques were later applied within the general linear model. There is a correlation between the fatigue experienced by the player, assessed objectively and subjectively, and the load accumulated over the week, this being assessed in absolute and relative terms. Specifically, the load relative to competition correlated with the physical variables TD (-0.279), PL2D (-0.272), TDD < -2 (-0.294), TDA >2 (-0.309), and sRPEmus (-0.287). The variables related to heart rate produced a higher correlation with TQR. There is a correlation between objectively and subjectively assessed fatigue and the accumulated TL of a player over the week, with a higher sensitivity being shown when compared to the values related to the demands of competition. Monitoring load and assessing fatigue, we are closer to knowing what the prescription of an adequate dose of training should be in order for a player to be as fresh as possible and in top condition for a match. Normalizing training demands with respect to competition could be an appropriate strategy for individualizing player TL.The aim of the study was to assess the relationship of external and internal training load (TL) indicators with the objective and subjective fatigue experienced by 15 semi-professional football players, over eight complete weeks of the competition period in the 2015–2016 season, which covered microcycles from 34th to 41st. The maximum heart rate (HRmax) and maximum speed (Vmax) of all the players were previously measured in specific tests. The TL was monitored via questionnaires on rating of perceived exertion (RPE), pulsometers and GPS devices, registering the variables: total distance (TD), player load 2D (PL2D), TD at >80% of the V The main aim of training is to provide a stimulus which will optimize the player/team’s performance during competition whilst minimizing the negative consequences of that training such as lack of freshness, fatigue, over-training, or injury . The loaHowever, appropriate doses of stimulus could improve performance and protect against possible injury . It is tBy monitoring load , informaWellness   , The Prood State .There is an increasing tendency to study  the relaAccumulated load values are usually assessed in absolute terms . To dateTo that end, the aim of this research is to study the relationship between external and internal training load indicators (TL), in absolute values and relative to competition, with respect to the fatigue experienced by semi-professional football players measured using objective and subjective values. The results of this research could increase knowledge about how to manage the load imposed on players with a view to adjusting its prescription in order to optimize physical performance in competition.A total of 15 semi-professional male football players  took part in the study  percentage of body fat  was 11.6All the training sessions and competition matches were monitored during the microcycles of the study. In total, 250 recordings were made in 20 training sessions (16.7 ± 3.6 per player) and 72 recordings from eight matches (4.9 ± 2.1 per player). The individual session or match recordings were grouped into microcycles, with a total of 69 weekly recordings (4.6 ± 1.3 per player). In order to calculate the mean value of the demands in competition, all the values were normalized to a 90 min match (mean match duration recorded per player ±SD).max = 1, 60–70% HRmax = 2, 70–80% HRmax = 3, 80–90% HRmax = 4, 90–100% HRmax = 5) which are later added together.In all the training sessions and competition matches heart rate (HR) was recorded via a short range telemetry system . The reliability of the devices used in this study has been reported in previous studies . To quan-1 which was increased at a rate of 1 km/h-1 every minute until the point of physical exhaustion was reached  next to the unit of assessment. The assessment was carried out 10 min after the end of the session or match . AfterwaThe players’ external load was monitored using GPS devices  which function at a sampling frequency of 10 Hz and contain a 100 Hz triaxial accelerometer. The reliability and validity of the devices used in this study have been reported in previous studies . The meamax) in m; (c) PL2D, player load 2D [in arbitrary units (AUs)]; (d) TD80%, percentage of distance covered at more than 80% of Vmax (in %); (e) TDD < -2, TD in deceleration under -2 m/sec-2 (in m); and (f) TDA >2, TD in acceleration over 2 m/sec-2 (in m).The following physical variables were studied: (a) TD, total distance (TD) in m; (b) TD80, distance covered at more than 80% of maximum speed  of each player. Furthermore, in each microcycle the level of absolute fatigue (FATabs) of each player was calculated by discovering the percentage of the value obtained by the player on the pre-match session day  with respect to the CMJmax value. The following formula was used to calculate the absolute fatigue value: CMJpre/CMJmax. To calculate relative fatigue (FATrel) the formula CMJpre/CMJpost was used. This second fatigue value was calculated in order to know specifically whether the load accumulated in the week prior to the one studied had any repercussion on the freshness or objective fatigue of the player. The coefficient of variation (CV) for each of the CMJ tests was of between 0.0 and 7.7%.The best value obtained in the 14 tests of the trial was used to calculate the maximum CMJ  of the 2015–2016 season during the microcycles from 34 to 41°. All training and matches were monitored via pulsometers and GPS. Two of the microcycles were excluded from the analysis as not all of the sessions were present. Before beginning the trial, the players underwent a maximal progressive resistance test on a treadmill (in laboratory) to calculate the maximum HR of each player and a 40 m speed test on the training ground whilst wearing the GPS devices. Furthermore, where higher values in peak speeds were detected, these were taken as the VDuring the study period, performance in a CMJ test was recorded both on the first training day of the week (with a minimum of 48 h with respect to competition) and the last (24 h prior to the next competition). The test was always carried out indoors and the players previously familiarized with it. It was decided not to include the CMJ test for matches for two reasons – one was the lack of adequate facilities in away matches and the second was due to the difficulty in getting the players to carry out maximal tests on competition days, despite these having a low impact on fatigue. Furthermore, before beginning the first or the last training session of the week or pre-match warm-up, the players completed the TQR questionnaire . Finally, after the training session or match they completed the RPE questionnaire.For the correlation analysis between load and fatigue, both the absolute values accumulated through the week and the accumulated values normalized to individual competition were used. The competitive reference values were obtained from the competitions recorded during the same trial. For this, the mean values of each player in competition were used as reference.r ≤ 0.3), moderate (0.3 < r ≤ 0.7) to high (r >0.7). The statistical analysis was conducting using SPSS v.23 . Significance level was fixed at 0.05 (p < 0.05).Starting from the relative values of the different physical, physiological and perception of exertion variables, correlation analysis techniques were implemented within the general linear model. The results are shown as mean and standard deviation (±SD). The Pearson correlation coefficient was calculated to determine whether there was a relationship, and if this was significant among the analyzed variables. To interpret the results, threshold values for the Pearson correlation coefficient used by Table 1 shows the mean and ±SD values for each of the variables obtained by the players in the matches played during the trial. It also shows the mean and (±SD) values of the load accumulated by microcycle, normalized to the demand of competition. It can be seen that in all the analyzed variables, except that of TD80, the accumulated weekly load was higher than the mean load in competition .Table 2 shows the values obtained in the different CMJ tests carried out during the trial together with assessment of neuromuscular fatigue (FATabs and FATrel) and the subjective assessments of the state of fatigue . As it can see when TQR scale are closed to the matches the values of subjective fatigue are higher, that is, player finished the week with better wellness.Table 3 shows the correlations between objective fatigue with the 13 load variables studied, both in absolute values and relative to the competitive demand for each player. The values show that the objectively measured fatigue (FATabs and FATrel) correlated only with some of the internal and external load variables, when the load was assessed in absolute and relative terms. Among the physical variables, only TDA >2 is moderately correlated with the FATabs variable, while the other variables that are significantly correlated with FATabs present low correlations. There were higher correlations between relative values of the match demands than when were used absolute values. Subjective assessment of fatigue (TQRpre and TQRcomp) obtained correlations in a moderate range and were significantly high (p < 0.01) for the three HR variables.The aim of this research was to study the relationship between the TD, from external and internal indicators, and the objective and subjective assessment of fatigue in semi-professional football players. This is the first piece of work which relates the load placed on semi-professional footballers in terms relative to the demands of competition with the fatigue accumulated in each microcycle of the competitive period, calculated using an objective CMJ test and also via the subjective perception of recovery quality (TQR).The main results of the study can be summarized as follows: (1) normalizing training demands with respect to competition could be an appropriate strategy for individualizing player TD, (2) both the use of objective (from CMJ) and subjective (from TQR) fatigue indicators proved to be related to the load borne by players in the weekly microcycle.training status  do not suppose the same percentage in relation to that which competition demands of each player (% of the match). This is not only because of differences in imposed demands on the players depending on their position on the pitch , but alsThis gives rise to a new hypothesis regarding the need to individualize the variables which can affect a player’s fatigue or recovery, which will require further research. It is known that each player assimilates loads in a different way, due to his/her past and present characteristics , which pIn order to normalize the weekly load, in this work it was decided to take as a reference the mean values of each player in competition, whilst being aware that these competition demands present a moderate-elevated variability  in respoHowever, the quantity of load placed on the players should be conditioned by what they are able to assimilate, in order not to avoid over-training or increasing the probability of injury . To avoiAssessment of neuromuscular fatigue from CMJ may not be sensitive when the aim is to compare acute fatigue from a football training session (difference in a test of jump over height reached in pre with respect to post-training)  perhaps Along the same lines, This innovative study has also analyzed variables connected to accelerations and decelerations in which correlations with fatigue have also been found. To be more specific, the variables related to the neuromuscular dimension (PL and accelerations and decelerations) showed a greater sensitivity (correlation) with this objective jump test.The use of questionnaires such as the TQR has allowed us to discover the player’s degree of subjective recovery at the end of the microcycle, which provides information on the fatigue generated in the player during the week. Despite there being practically no differences in the TQRpost with respect to the end of the week (TQRpre), it is worth pointing out that in the variables related to HR, and therefore the cardiovascular energy system, it was the TQR questionnaire which showed a higher sensitivity to the changes.This suggests that it may be interesting to consider that just as different dimensions of TD are monitored, it could be relevant to have various tools available with which to assess the state of player fatigue or recovery, which would deal with different dimensions of the fatigue generated.One of the limitations of the study was the relatively low number of recordings of training session load and match load. A higher number of recordings would have provided more information about each player and therefore established particular load-fatigue relationships for each one, in the search for the adequate dose and typology of TD for each player.We should also highlight that using a higher number of players in the study, apart from providing information about their physical condition, would have shown how far players with different ability or fitness present a particular load-recovery relationship. This would thus allow attention to be paid to the capacity for bearing load and/or being fresher in competition or having better recovery after the match .Finally, it should also be underlined that the reduced sample group did not allow the incorporation of variables with which to differentiate the players who played a match in the week prior to the studied microcycle. It is probable that different states of recovery at the beginning of the week could have conditioned some of the results of this work.With the information obtained from the monitoring of TD and assessment of fatigue (objective and subjective), we are closer to knowing what the prescription of an adequate dose of training should be in order for a player to be as fresh as possible and in top condition for a match.The main conclusion of this study is that in those microcycles where the players accumulated a greater TD or high values in the load indicators normalized to those demanded in competition, the players showed a higher level of neuromuscular fatigue, measured with CMJ. However, the players were able to recover practically the same CMJ values (measured with the FATrel) as at the beginning of the week prior to competition. This research provides a better understanding of the load-fatigue relationship with respect to competition demands. Information about external objective load , internal objective load (HR) and internal subjective load (RPE) on the one hand, and the objective (CMJ) and subjective (TQR) indicators for fatigue assessment on the other, can help trainers to better understand and adequately manage training status and player freshness throughout the training process. Finally, it would be necessary to know whether the load borne by the players in the weekly training process maintains or improves their fitness, and thus discover whether management of the load-fatigue binomial produces an improvement in the players’ physical performance.UZ: design, data getting, analysis, redaction; JC: design, analysis, redaction; IE: data getting, redaction; DC: design, redaction.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There is an error in the second sentence of the Abstract. The correct sentence is: Thus, our aim was to test two null hypotheses: SM and microradiography result in similar correlations between ICDAS and histology; SM and microradiography result in similar positive (PPV) and negative predictive values (NPV) of ICDAS cut-off 1–2 (scores 0–2 as sound) with histological threshold D4 .There are errors in the penultimate sentence of the Abstract. The correct sentence is: For ICDAS cut-off 1–2 and D4, PPV from MRC (0.373) was higher than that from SM (0.196) , and NPV from MRC (0.73) was lower than that from SM  .There are errors in the last sentence of the Introduction. The correct sentence is: The aim of this study was to test two null hypotheses: (i) the correlation between the visual aspect of occlusal caries in permanent teeth detected using ICDAS and lesion depth determined with SM. is similar to the same correlation when lesion depth is determined using microradiography with contrast (MRC); and (ii) the number of both sound and caries occlusal surfaces diagnosed with ICDAS cut-off 1–2 (0–2 as sound) presenting deep dentin demineralization  is similar when lesion depth is measured by both SM and MRC.The first sentence of the third paragraph beneath the “Data analysis” heading of the Materials and Methods section is incorrect. The correct sentence is: We also tested the null hypothesis that the positive predictive value  and the negative predictive value  using ICDAS cut-off 1–2  with histological threshold D4 from both SM and MRC are similar.The final and penultimate sentences of the Results section are incorrect. The correct sentences are: The PPVs for sound occlusal surfaces  and histological threshold D4  were the following: 0.196 using SM, and 0.373 using MRC. The corresponding NPVs were: 1.00 for SM and 0.73 for MRC. Both PPV and NPV differed statistically (p < 0.0002), with an effect size h of 0.56 for PPV and 1.55 for NPV, both with statistical power > 96.0%.The final sentence of the second paragraph of the Discussion section contains a typographical error. The correct sentence is: This is the rational of the use of MRC for measuring lesion depth as described here.The first sentence of the fourth paragraph of the Discussion section contains errors. The correct sentence is: Both the PPV and the NPV of ICDAS cut-off 1–2 with histological threshold D4 differed significantly from SM to MRC. The low PPV of 0.196 from SM is close to the PPV of 0.162 from SM calculated with data available in the literature [3].MRC = 0.373) with a medium effect size (h = 0.56), and NPV decreased (NPVMRC = 0.73), also with a large effect size (h = 1.55). The PPV for ICDAS 1–2 cut-off and D4 threshold is the proportion of carious occlusal surfaces with deep dentin demineralization, while the NPV for ICDAS 1–2 cut-off and D4 threshold is the proportion of sound occlusal surfaces with deep dentin demineralization.The third and fourth sentences of the fourth paragraph of the Discussion section contain errors. The correct sentences are: When MRC was used for the histological examination, PPV increased  than the correlation between ICDAS and lesion depth measured with MRC.The penultimate sentence of the final paragraph of the Discussion section contains errors. The correct sentence is: The proportions of both sound and carious occlusal surfaces diagnosed with ICDAS 1–2 cut-off presenting deep dentin demineralization detected with MRC increased significantly  compared to the same proportions when SM was used to detect dentin demineralization.