AR) is lacking.In boys with suspected partial androgen insensitivity syndrome (PAIS), systematic evidence that supports the long-term prognostic value of identifying a mutation in the androgen receptor gene  who presented before the age of 16 years and had genetic analysis of AR mutations reported. At diagnosis, the median external masculinization scores were 7 and 6 in cases with and without AR mutation, respectively (P = .9), and median current external masculinization scores were 9 and 10, respectively (P = .28). Thirty-five men (67%) required at least one surgical procedure, and those with a mutation were more likely to require multiple surgeries for hypospadias (P = .004). All cases with an AR mutation had gynecomastia, compared to 9% of those without an AR mutation. Of the six men who had a mastectomy, five (83%) had an AR mutation.The median ages at presentation and at the time of the study were 1 month  and 22 years , respectively. Of the cohort, 29 men (56%) had 20 different AR mutation. Routine genetic analysis of AR to confirm PAIS informs long-term prognosis and management.Boys with genetically confirmed PAIS are likely to have a poorer clinical outcome than those with XY DSD, with normal T synthesis, and without an identifiable  Using the I-DSD Registry, a study of long-term outcome in young men suspected of having PAIS in childhood reveals that outcome is clearly worse in those with a confirmed AR gene mutation. AR)  . AlthougAR)  Registry , a studyhttp://www.gla.ac.uk/schools/medicine/research/childhealth/i-dsdproject/thei-dsdregistry/standardoperatingprotocol/. The registry is approved by the National Research Ethics Service in the United Kingdom as a research database of information that is collected as part of routine clinical care. All I-DSD Registry users who had registered an eligible case were approached for results of AR gene analysis, clinical characteristics at first and most recent presentations, biochemical characteristics, and subsequent management. The registry also collects information on the extent of certainty of diagnosis, and this is categorized as “clinical certainty,” “biochemical certainty,” and “genetic certainty.” In this study, all included cases had normal testes function and T synthesis. However, cases of suspected PAIS that did not reach genetic certainty because they did not have a genetic confirmation despite AR analysis were not referred to as having PAIS so that they could be differentiated from the cases of PAIS that actually had a mutation in AR. Details of the information collected are summarized in Supplemental Table 1. The external masculinization score (EMS) was calculated as previously described .Continuous variables were described as medians and ranges, and intergroup comparison for these variables was performed by Mann-Whitney AR mutation testing and clinical information were available in 52 of the 60 (87%) men. In this latter cohort, 29 men were identified as having 22 different AR mutations within exons 2 to 8  and one in exon 5 (T739C). The remaining 23 cases who did not have a mutation in AR were, for the purpose of this study, referred to as XY DSD.Among the 1892 records in the I-DSD Registry at the time of the study, there were 1225 (65%) cases with a 46, XY karyotype. Of these 46, XY DSD cases, 154 (13%) had been categorized as PAIS, and 60 of these 154 (39%) were identified as men over the age of 16 years. Details on s 2 to 8  and, thes 2 to 8 . One manAR mutation  and 6 , respectively. On excluding the cases who had presented with a positive family history or gynecomastia, the median EMS in the group with an AR mutation and those without was similar at 5  and 6 , respectively.Of the 52 men included in this study, the initial presentation was reported as hypospadias in 28 (54%), ambiguous genitalia in 11 (39%), and a positive family history in two (7%). In older boys, gynecomastia was another clinical presentation, but this was only encountered as a reason for presentation in those with an mutation ; none ofmutation . There wAR mutation, but the difference was not statistically significant  and 1.3 , respectively, the T:LH was significantly higher at 1.9  compared to 0.9  in those with a mutation (P = .02)  . In a seAR mutation status, undermasculinization was frequently observed at the most recent clinical presentation  and 9 , respectively. In the whole cohort of 52 cases, 25 (42%) had required T therapy at puberty, and the regimen that was used was very variable  and zero (0%) cases, respectively. Of the 29 cases who had an AR mutation, 20 had hypospadias at initial presentation and six (33%) continued to have a hypospadias despite surgical repair, whereas only one of the 20 with hypospadias and no AR mutation continued to have a hypospadias  and seven of 22 (32%) cases, respectively. Only one of these 18 men had a history of bilateral undescended testes. Of the men with no AR mutation, none of nine had a serum LH above 12 IU/L, and two of seven (29%) had a raised FSH. Both LH:FSH and T:LH ratios tended to be higher in men with a mutation, but the difference was not significantly different  had declined a psychological referral, eight (73%) were receiving psychological support at the last assessment, and two (18%) had previously received input but were no longer actively involved with this. Fertility data were available for 14 men with an AR mutation and four without a mutation who were between 18 and 34 years of age at the time of the most recent presentation. Among this cohort, two with an AR mutation had undergone sperm analysis that had demonstrated azoospermia . One of these men had one undescended testis requiring orchiopexy, and the other man had undergone bilateral orchiopexy. Only two men had offspring; one of these men did not have an AR mutation and had required assisted conception after sperm analysis demonstrated oligospermia, and the other, who had an AR mutation, had conceived naturally. None of the men in this study had any record of tumor-related events. Two of the 29 men with an AR mutation (7%) were noted to have obesity with body mass indices of 28 and 30 kg/m2; one had undergone assessment for possible bariatric surgery, and the other had refused any dietetic advice. None of the men had any recorded investigations for concerns regarding bone mineral density or cardiometabolic health.The provision of psychological input was reported in 11 of 52 (21%) cases, and seven of these 11 men had an AR in boys where there is a clinical suspicion of PAIS.The current report represents the largest study on clinical outcomes in young men with XY DSD and normal T synthesis and who were previously suspected of having PAIS in childhood and adolescence. The study not only raises concerns about the long-term outcome in those men with PAIS but also strengthens the clinical rationale for the routine genetic analysis of AR compared to the ones without a mutation. Although higher T concentrations and T:LH ratios were encountered in the cases with an AR mutation, there was a large overlap between the two groups and as observed previously (Access to the I-DSD Registry allowed the identification of a sufficiently large group of male cases of 46, XY DSD with a variable extent of undermasculinization at initial presentation and who had a clinical phenotype consistent with PAIS. The difficulty in diagnosis was illustrated by the fact that there was no clear difference in the physical and biochemical features that were assessed at presentation in those cases with an identifiable mutation in eviously , 14.AR mutation tended to have a worse prognosis for virilization as reflected by the gynecomastia. These men were also more likely to have hypospadias and micropenis, but this difference did not reach statistical significance. In addition, these men with an AR mutation were more likely to have had T therapy. A variable level of virilization after androgen therapy has been reported in PAIS (AR mutation, confirms previous reports (AR genetic status would allow the provision of informed guidance to the adolescent.As young men, those with an  in PAIS  and may  in PAIS . This va reports , 17. GivAR status and the outcome of hypospadias surgery. Most cases of hypospadias require one or two surgical procedures (AR mutation were more likely to have multiple procedures. Given that the relative proportions of the type of hypospadias was similar in the two study groups and that the cases were spread over all the centers, the different outcome cannot be due to case or operator bias. Thus, the poor results are more likely to be related to defective androgen signaling and its links to tissue healing (AR may guide the clinician toward a different approach to surgical management and may also allow improved preparation and counseling of the patient. One-fifth of the studied cohort was reported to have been offered clinical psychology input and, given the concerns raised previously about psychosexual outcome (AR.The current report has identified a clear association between ocedures ; however healing . Large-s healing , 20, but outcome  and gend outcome  in adultAR mutation. It is likely that suppression of LH secretion is partially dependent on both T and estradiol (Previous cross-sectional studies of the hypothalamic-pituitary-gonadal axis in genetically confirmed cases of PAIS, performed at a range of ages in childhood and adolescence, have reported concerns about testicular function, as reflected by a blunted T rise after human chorionic gonadotropin stimulation as well as raised gonadotropins , 7. The stradiol , and thestradiol , 27. ThiAR mutations have now been described in androgen insensitivity syndrome (AR mutation as well as the function of the defective AR protein may assist management (AR mutation in cases of XY DSD may itself be sufficient to predict poor prognosis and personalize long-term management. A recent survey of pediatric endocrine centers has revealed that 43% would routinely consider AR analysis in 46, XY infants with genital ambiguity (AR should be lowered in all cases of hypospadias that are proximal or associated with micropenis or undescended testes, especially when there are no associated concerns about T synthesis. In terms of EMS, this would be equivalent to a score of 11 or below and consistent with other recommendations (Although over 400 syndrome , the corsyndrome ; this vanagement . Howevermbiguity . Given tndations . Other fAR and with variable depth of coverage of the intronic region. The current study emphasizes the need for revisiting the genetic analysis of AR in those adolescents and young men who did not have a mutation identified before but who may have features such as gynecomastia, persistent hypogonadism, and poor outcome after hypospadias repair. With the availability of quality accredited laboratories offering analysis of a wide panel of genes associated with sex development as well as increased reliance on whole-genome sequencing (AR or mutations in other genes associated with DSD may be revealed. By targeting analysis in young men with a clearer phenotype of androgen insensitivity, it is also possible that future research can be more effectively directed at understanding the wider clinical relevance of the androgen-signaling pathway downstream of AR as well as the noncanonical pathways of androgen signaling (Knowledge of the biology and genetics of sex development as well as the technology employed for diagnostic genetics has progressed considerably since the period when the cases in this cohort were analyzed . Almost quencing , it is qignaling .AR may be severely hypogonadal, as suggested by the current findings, the need for careful monitoring of the wider cardiometabolic consequences of hypogonadism requires strong consideration. Successful fertility has been reported in men with PAIS using assisted conception techniques (AR mutation was reported to have an offspring naturally, indicating that spontaneous fertility is possible in young men with confirmed PAIS.In older men with hypogonadism, cardiovascular morbidity, impaired glucose tolerance, and osteoporosis pose the greatest health and economic burden , and the36–chniques , 40. In AR, and it is possible that a number of cases with no AR mutation have not been entered in the I-DSD Registry because PAIS and differences in the use of the registry may have introduced further bias. However, it is likely that this change in diagnostic practice would apply to younger cases than those reported here. Finally, a genotype-phenotype correlation was not explored in this study, but this will become more feasible as the number of cases with genetically confirmed PAIS that reach adulthood increases over the next decade.Although there are inherent weaknesses in performing a retrospective study that relies on data collected from multiple centers, the power of the study rests in the size of the cohort and the small number of clear and measurable outcomes that were collected. Although it is reassuring that all the participating centers routinely collected the information that was sought, the current study highlights the need for a standardized assessment of all boys who are suspected of having long-term hypogonadism, especially as they approach the age of puberty. It is possible that the cases entered into the registry suffered from selection bias and inclusion of the cases that were older at presentation also introduced further bias. However, this risk was mitigated by the discrete comparison of the group of children who presented at an early age. Increasingly, the diagnosis of PAIS is only reserved for those cases that have a confirmed mutation in AR are more likely to have a worse medical and surgical outcome as young men. Thus, routine genetic analysis of AR in boys suspected of PAIS is recommended to guide long-term prognosis and tailor management.In conclusion, this study represents the largest cohort to date of young men who presented in childhood with clinical features suggestive of PAIS. There is clear evidence that genetically confirmed cases of PAIS with a mutation in


This study investigated the network of genes that are co-expressed with androgen receptor (AR) to discover novel AR targets in breast cancer. Bioinformatics analysis of two datasets from breast cancer cell lines resulted in the identification of an AR-gene signature constituted of 98 genes that highly correlated with AR expression. Notably, C1orf64 showed the highest positive correlation with AR across the datasets with a correlation coefficient (CC) of 0.737. In addition, C1orf64 closely correlated with AR expression in primary and metastatic breast tumors and C1orf64 expression was relatively higher in breast tumors with a lower grade and lobular histology. Furthermore, there is a functional interplay between AR and C1orf64 in breast cancer. In this process, AR activation directly represses C1orf64 transcription and C1orf64, in turn, interacts with AR as a corepressor and negatively regulates the AR-mediated induction of prolactin-induced protein (PIP) and AR reporter activity. Moreover, the corepressor effect of C1orf64 results in a reduction of AR binding to PIP promoter. The other aspect of this interplay involves a cross-talk between AR and estrogen receptor (ER) signaling in which C1orf64 silencing intensifies the AR-mediated down-regulation of ER target gene, progesterone receptor. Therefore, the repression of C1orf64 by AR provides an underlying mechanism for the AR inhibitory effects on ER signaling. To elucidate the biochemical mechanisms of C1orf64 function, this study demonstrates that C1orf64 is a phosphothreonine protein that interacts with the chaperone protein 14-3-3. In summary, C1orf64 is a novel AR coregulator and a 14-3-3 binding partner in breast cancer. Recent advancements in the molecular profiling and cancer genomics have provided the opportunity for a better understanding of breast cancer biology and the discovery of novel therapeutic targets in this disease. In this process, androgen receptor (AR) has been identified as a key molecular marker in breast cancer that is expressed in 90% of estrogen receptor (ER)-positive and 50% of ER-negative breast tumors –4. The sStudies conducted by my group and others have revealed that AR as a transcription factor has an important function in the regulation of key signaling pathways in breast cancer , 8–12. TTo address these shortcomings, my group has recently examined gene expression data from a cohort of 52 breast cancer cell lines to identify a network of AR co-expressed genes . This stTherefore, the study of AR co-expressed genes in large genomic datasets would provide an innovative approach to investigate AR molecular functions and novel target genes in breast cancer. This approach ensures that different molecular subtypes of breast cancer are represented in the study and the identified genes are highly correlated with AR expression in a large non-biased model of this disease. Importantly, the identified AR co-expressed genes are likely to include biologically significant AR target genes and transcriptional coregulators that are present across different subtypes of breast cancer. In the current study, this approach has been further explored by a combined analysis of two large expression datasets in breast cancer to identify an AR-gene signature of highly co-expressed genes followed by the transcriptional and functional studies of some of the key findings. Notably, this study identifies a poorly-understood gene “C1orf64” as both a novel target gene and a coregulator of AR in breast cancer that interacts with 14-3-3 protein.et al. (study 1) and Kao et al. (study 2) as explained in methods . Next, the % Input was calculated as: % Input = 2 . Lastly, fold enrichment was calculated: % Input of antibody/ % Input of IgG [Amplification of 1% of input chromatin prior to immunoprecipitation was applied as the input control and ChIP assays using a non-specific antibody (rabbit IgG) served as a negative control . Fold ent of IgG .C1orf64-silencing by RNA interference in T-47D and MFM-223 cell lines was performed by the reverse transfection method using Lipofectamine RNAiMAX (Life Technologies), , 72. TwoC1orf64 open reading frame (ORF) clone in a pReciever-M02 plasmid was obtained from GeneCopoeia . Transfection of C1orf64 expression construct in T-47D and MFM-223 cell lines was carried out using TurboFect Transfection Reagent (Thermo Fisher Scientific) as instructed by the manufacturer. An empty pReciever-M02 plasmid was applied for the control experiments. The effect of C1orf64 overexpression was assessed 48h following transfections.T-47D and MFM-223 cell lines were cultured in solid opaque (white) 96-well tissue culture plates (Fisher Scientific) using phenol red-free media (Life Technologies) supplemented with 10% Charcoal/Dextran treated serum (Fisher Scientific) and cell lines were approximately 80% confluent on the day of transfection. AR Reporter Assay Kit  was obtained from Qiagen . AR reporter includes a mixture of an inducible androgen-responsive reporter construct and a constitutively expressing Renilla construct (40:1). This reporter encodes the firefly luciferase reporter gene under the control of a basal promoter element (TATA box) joined to tandem repeats of an Androgen Response Element (ARE). Negative control is a mixture of a non-inducible firefly reporter construct and a constitutively expressing Renilla construct (40:1).The co-transfections of reporter and expression constructs in each cell line were carried out using TurboFect Transfection Reagent (Thermo Fisher Scientific) as instructed by the manufacturer in the following four experimental groups: 1) Negative control for reporter + empty pReciever-M02 plasmid (Neg-Rep/Vec), 2) Negative control for reporter + C1orf64 expression construct (Neg-Rep/C1orf64), 3) AR reporter + empty pReciever-M02 plasmid (Pos-Rep/Vec), and 4) AR reporter + C1orf64 expression construct (Pos-Rep/C1orf64). Twenty-four hours following co-transfections, media were changed to phenol red-free media/10% Charcoal/Dextran treated serum containing DHT at 10 nM concentration. Forty-eight hours after the transfections, reporter activities were assessed using Dual-Glo Luciferase Assay System  as instructed by the manufacturer and measurements were carried out in an EnVision Multilabel Reader . The relative response ratio (RRR) for each reporter activity was measured relative to the internal control reporter followed by normalization to the respective Neg-Rep group , 9. All IP assays for endogenous AR and 14-3-3 were carried out using a non-denaturing lysis buffer as we previously published , 14. TheSubsequently, AR-IP was performed using 10 μg of rabbit polyclonal AR antibody (Active Motif) and 14-3-3 IP was carried out using 5 μg of rabbit polyclonal 14-3-3 (pan) antibody (Millipore). Control experiments were conducted with a non-specific rabbit IgG. The lysate-antibody mixtures were then incubated overnight under rotation at 4°C. Next, lysates were mixed with 100 μl of beads for 4h under rotatory agitation at 4°C and then centrifuged to remove supernatants. Beads were then washed three times with ice-cold IP lysis buffer. Following the removal of last supernatant, 50 μl of 2x Laemmli Sample Buffer  was added to each sample. Samples were then boiled at 100°C for 5 min and centrifuged at 14,000 xg for 10 min. Finally, supernatants were collected and applied for immunoblot analysis using C1orf64, AR, or 14-3-3 antibodies. In addition, for each sample, 5% of lysate was collected before IP to assess input by immunoblot analysis. All co-IP experiments were carried out in three replicates.Biostatistics was carried out using IBM SPSS Statistics 23. Unless specified, Student t-test and paired sample t-test were applied to calculate the statistical significance. For the analysis of association between C1orf64 expression and clinical and pathological features, p values were calculated using Wilcoxon Signed Rank Test. All error bars depict ± 2SEM.


An AR construct with the charged lysine substitution by arginine (AR-618R) reduces RNA Pol II binding, AR transcriptional activity, prostate cancer cell growth, and xenograft tumor formation due to attenuation of AR nuclear translocation, whereas, construct mimicking neutral polar substitution acetylation at K618 by glutamine (AR-618Q) enhanced these effects beyond that of the wild-type AR. Mechanistically, ARD1 forms a ternary complex with AR and HSP90 in vitro and in vivo. Expression of ARD1 increases levels of AR acetylation and AR-HSP90 dissociation in a dose dependent manner. Moreover, the AR acetylation defective K618R mutant is unable to dissociate from HSP90 while the HSP90-dissociated AR is acetylated following ligand exposure. This work identifies a new mechanism for ligand-induced AR-HSP90 dissociation and AR activation. Targeting ARD1-mediated AR acetylation may be a potent intervention for AR-dependent prostate cancer therapy.Prostate cancer is an androgen receptor (AR)-driven disease and post-translational modification of AR is critical for AR activation. We previously reported that Arrest-defective protein 1 (ARD1) is an oncoprotein in prostate cancer. It acetylates and activates AR to promote prostate tumorigenesis. However, the ARD1-targeted residue within AR and the mechanisms of the acetylation event in prostate tumorigenesis remained unknown. In this study, we show that ARD1 acetylates AR at lysine 618 (K618)  Androgen receptor (AR) plays a crucial role in prostate cancer (PCa) , 2. ActiIn prostate cells, prior to androgen binding, AR is inactively bound to a foldosome of chaperone proteins within the cytoplasm, including HSP90, preventing nuclear localization while maintaining ligand access , 8. Upon633 and is critical for AR activation, lncoRNA binding, and prostate tumor development [Posttranslational modification of AR, including acetylation, plays a crucial role for AR activation . To dateelopment , 14. Arrelopment , 16. It elopment –21. Prevelopment . Moreoveelopment . However633) [in vitro acetylation assays in the presence of in vitro synthesized His-ARD1 and acetyl CoA, respectively  targeted by ARD1 is different from that of p300 which targets lysine residues (KLKK633) . To deteectively . We demoectively . Since Dectively , we thenectively . The DBDt Figure . Motif Ion assay . Lysine on assay . These din vivo, we first silenced ARD1 by using shRNA in LNCaP and showed that the acetylation levels of the endogenous AR in these cells are greatly reduced . Cells were maintained in appropriate media per the ATCC guidelines. Where applicable, cells were treated with deacetylation inhibition cocktail (sc-362323) diluted in DMSO.Immunoblotting and immunoprecipitation were performed as previously described . The priThe FLAG-ARD1 construct was generated as previously described . The PSAin vitro acetylation assay on AR was performed using His-ARD1 as described previously with modification [E. coli cells, induced by .1 mM IPTG for 2 hours. GST-AR fragments were purified using glutathione beads , eluted with reduced glutathione, and dialyzed for 24 hours using Slide-A-Lyzer .5–3 ml dialysis cassettes (Thermo 66380). His-ARD1 was purified using a His SpinTrap Kit  per the manufacturer's recommendations. The in vitro acetylation assay was performed and proteins were separated by SDS-PAGE. Corresponding amounts of each fragment were separated by SDS-PAGE and stained with Coomassie blue stain. Cell proliferation and anchorage-independent growth assays. Cell proliferation MTT assays were performed with DU 145 cell pools stably expressing WT or mutant AR using CellTiter 96 Non-Radioactive Cell Proliferation Assay (Promega) kit according to the manufacturer's recommendations. Colonogenic growth of stable cells was analyzed by evaluation of growth in soft agar 17 days post-plating, as described previously [The fication . Brieflyeviously .n = 3 mice per group. 2.5 × 106 cells/injection) were injected into the hind flanks of nude mice (Charles River) as previously described [Stable DU 145 cell pools expressing WT or mutant AR , and serum-starved for 24 hours. Cells were then subjected to 24 hours of R1881 exposure. Analysis was performed using the Dual-Luciferase Reporter Assay system (Promega) per the manufacturer's instructions.Total RNAs were prepared using the RNeasy kit (Qiagen) and cDNA was synthesized using Superscript III First-Strand synthesis system (Invitrogen). Quantitative RT-PCR (qRT-PCR) was performed using SYBR Green detection method on a CFX96 Real-Time PCR detection system (BIO-Rad). All of the primers used for this study have been reported .ChIP assays were performed using SimpleChIP Plus Enzymatic Chromatin IP kit  as previously reported with a few modifications . BrieflyCos-7 cells were plated on chamber slides in phenol red-free RPMI 1640 media with 10% charcoal-depleted FBS. Cells were transfected after 24 hours with either AR-WT, AR-618R, or AR-618Q. Cells were treated with 1 nM R1881 for one hour 24 hours post-transfection. Cells were embedded on slides with paraformaldehyde, permealized with .25% triton-x, blocked with 5% goat serum, and probed with anti-AR antibody (sc-7305) overnight. Anti-mouse secondary antibody (Alexa Fluor 448) was applied, as well as Hoechst stain (Thermo 33342) specific for DNA.t test. P < 0.05 was considered statistically significant.Data are expressed as mean ± SD from at least 3 experiments. Statistical analyses were performed by Student's


Metastatic castration resistant prostate cancer is one of the main causes of male cancer associated deaths worldwide. Development of resistance is inevitable in patients treated with anti-androgen therapies. This highlights a need for novel therapeutic strategies that would be aimed upstream of the androgen receptor (AR). Here we report that the novel small molecule anti-androgen, galeterone targets USP12 and USP46, two highly homologous deubiquitinating enzymes that control the AR-AKT-MDM2-P53 signalling pathway. Consequently, galeterone is effective in multiple models of prostate cancer including both castrate resistant and AR-negative prostate cancer. However, we have observed that USP12 and USP46 selectively regulate full length AR protein but not the AR variants. This is the first report of deubiquitinating enzyme targeting as a strategy in prostate cancer treatment which we show to be effective in multiple, currently incurable models of this disease. Prostate cancer (PC) is one of the main causes of cancer-related mortality in men worldwide. In the early stages of the disease patients respond well to anti-androgen based therapies however, resistance invariably develops within approximately two years. Even in castrate resistant PC (CRPC) the androgen receptor (AR) signalling cascade remains the critical pathway. Currently, multiple novel second line anti-androgens have been discovered with abiraterone and MDV3100  introduced into the clinical regime. However, these therapeutics offer only a short life expectancy advantage and resistance to them is still inevitable , 3. ThisC-terminal loss  and and37] a50 values were comparable between LNCaP androgen sensitive cells and LNCaP-AI androgen independent cells , anti-H2A.Z , anti-MD2. VCaP cells kindly donated by Professor Guido Jenster  were cultured in DMEM medium with 2 mM L-glutamine (Invitrogen) supplemented with 10% (v/v) foetal calf serum (FCS) or steroid depleted serum (SDM) at 37° C in 5% CO2. Cell lines were never maintained for more than 30 passages or 3 months of continuous culturing. As per institutional policy, cell lines were tested for mycoplasma on a tri-monthly basis. Transfections were performed using TransIT-LT1 reagent (MirusBio) following the manufacturer's instructions. Luciferase assays were performed as previously described ; USP12: CAGAUCUCUUCCAUAGCAU[dTdT], WDR20: CGAG AAAGAUCACAAGCGA[dTdT] and UAF1: CAAAUU GGUUCUCAGUAGA[dTdT] [AR V7: GUAGUUG UGAGUAUCAUGA[dT][dT] and USP46: GUCUCAAU GGUCUGGCUGU[dT][dT]. For RNA sequencing, RNA was extracted using the QIAGEN RNeasy Plus Mini Kit and all samples were sequenced from three separate biological experiments using Illumina's total stranded RNA prep kit with ribozero gold for library preparation with 100 bp paired end reads on the Illumina HiSeq 2500 platform, performed by AROS.Cells were reverse transfected with siRNA using RNAiMax (Invitrogen) according to the manufacturer's instructions and incubated in culture medium for 96 h prior to cell lysis. siRNA sequences were as follows SCR: UUCUCCGAACGUGUCACGU[dT][dT]; GA[dTdT] ; AR V7: P values were adjusted to control for the false discovery rate (FDR) using the Benjamini-Hochberg method. Differentially expressed genes from each comparison were tested for functionally enriched pathways and gene ontology terms using GOseq with a gene length bias correction on pathways annotated in KEGG database.Reads were mapped to the reference human genome hg19 using STAR2-pass allowing up to two mismatches . Per genFor proliferation analysis cells were seeded 24 hours prior to treatment with either DMSO (control) or galeterone for 96 hours. IncuCyte measurements of cellular occupation of the wells were taken every 6 hours. Cell growth rate was normalised to the time point zero and additionally in a separate set of experiments cell numbers were counted at 96 h to assess cellular proliferation .®Green on 384-well optical reaction plates with the ABI 7900HT real-time PCR system. Results were normalised to HPRT1 expression. Primers were HPRT1 F: 5′-GAACGTCTTGCTCGAGAGATGTG-3′, R: 5′-CCA GCAGGTCAGCAAAGAATTT-3′; KLK2 F: 5′-AGCA TCGAACCAGAGGAGTTCT-3′, R: 5′-TGGAGGCTCA CACACTGAAGA-3′; PSA F: 5′-ACTGCATCAGGAAC AAAAGCGT-3′, R: 5′-TGTGGGAAGCTGTGGCTGA C-3′; NDRG1 F: 5′-ACAACCCCCTCTTCAACTACG-3′, R: 5′-GCCAATAATGCTTTTCAGCCCA-3′; AR F: 5′-G CAAAGCCTAAAGCCAGAT-3′, R: 5′-GAGTTCATGG GTGGCAAAG-3′; NKX3.1 F: 5′-AGCCAGAAAGGCA CTTGGG-3′, R: 5′-GGCGCCTGAAGTGTTTTCA-3′; TMPRSS2 F: 5′-CTGCTGGATTTCCGGGTG-3′, R: 5′-T TCTGAGGTCTTCCCTTTCTCCT-3′; FKBP5 F: 5′-GC AACAGTAGAAATCCACCTG-3′, R: 5′- CTCCAGAG CTTTGTCAATTCC-3′; USP46 F: 5′- TCCGGGAGA ATGTGTTGGC -3′, R: 5′- GTGTGGCAATGCTGTGGA AAA -3′; USP12 F: 5′-CAGCCTTCCAGTCATTGGCA-3′, R: 5′-ATCAATACGGCACAGATTCCG-3′; WDR20 F: 5′-TGCACCAGATCTAGAACTTGAAT-3′, R: 5′-TAT ACTCCCAGGAGGATGACTG-3′; UAF1 F 5′-GCT GATTGGTATGGACCGA-3′, R: 5′-TCTGCTTCCCT GGGGACAG-3′; AR V7 F: 5′-AACAGAAGTACCTG-3′, R: 5′-TCAGGGTCTGGTCATTTTTG-3′.For qPCR, RNA was extracted using Trizol (Invitrogen) according to manufacturer's instruction, and quantified using Nanodrop. Briefly, 1 μg of RNA was reverse transcribed with 1U of MMLV reverse transcriptase, 50 ng Oligo-dT and 630 μM dNTP (Promega). All qPCR was performed using the relative quantification method on three independent biological experiments and each sample was loaded in triplicate. qRT-PCR was conducted using SYBRt-test or ANOVA with Dunn's multiple comparisons where more groups where compared.All data was first tested for its Gaussian distribution. Normally distributed data was tested with


The nonclassical androgen signaling pathway translates signals into alterations in cellular function within minutes, and this action is proposed to be mediated by an androgen receptor (AR) localized to the plasma membrane. This study was designed to determine the mechanism underlying the membrane association of androgen receptor in TM4 cells, a mouse Sertoli cell line. Western blot analysis indicated testosterone-induced AR translocation to the cell membrane. Data from coimmunoprecipitation indicated that AR is associated with caveolin-1, and testosterone enhanced this association. Knockdown of caveolin-1 by shRNA decreased the amount of AR localized to membrane fraction and prevented AR membrane trafficking after being exposed to testosterone at physiological concentration. The palmitoylation inhibitor 2-bromopalmitate decreased AR membrane localization in basal condition and completely blocked testosterone-induced AR translocation to membrane fraction. These data suggested that AR localized to membrane fraction by binding with caveolin-1 through palmitoylation of the cysteine residue. This study provided a new evidence for AR membrane localization and its application for clarifying the nonclassical signaling pathway of androgens. Male fertility depends on the successful perpetuation of spermatogenesis . SpermatIn classical testosterone signaling, androgens exert their genomic effects via the AR . When boIt was reported that palmitoylation of a cysteine in the conserved region of estrogen receptor (ER) increases the physical association of ER with caveolin-1 , which iIn the present study, we further identified the association of AR with caveolin-1 in the membrane fraction. Knockdown of caveolin-1 decreased AR localization in the plasma membrane and inhibited AR translocation to the membrane, induced by testosterone. Preincubation with the palmitoylation inhibitor prevented AR membrane trafficking. These data suggested that testosterone induced AR membrane association with caveolin-1 by palmitoylation.Constructs for transient overexpression of shRNA targeted to caveolin-1 (GV-248) or control were obtained from Shanghai Genechem Co. Ltd. . The sequence of the shRNA is shown in Cav1 (132 bp) (Genebank: NM_007616): F 5′-ATG TCT GGG GGC AAA TAC GTG-3′, R 5′-CGC GTC ATA CAC TTG CTT CT-3′ and GAPDH (116 bp): F 5′-AGTGGCAAAGTGGAGATT-3′, R 5′-GTGGAGTCATACTCCAACA-3′.Primers used for the mouse gene v/v) and 100 μg/mL of penicillin and 100 μg/mL of streptomycin at 37°C with atmospheric conditions of 95% air and 5% CO2.TM4 cell, a mouse Sertoli cell line, was purchased from the ATCC biological resource center . The cells were cultured in Dulbecco's Modified Eagle's Medium (Gibco) containing 10% fetal bovine serum (μg plasmids by Lipofectamine 3000 (Life Technologies) when 40%–60% confluence was reached. The cell culture medium was changed to a culture medium containing 2 μg/mL puromycin 24 h later for 3 d. Subsequently, cells were kept in a culture medium containing 0.5 μg/ml puromycin.Cells plated in a 6-well plate were transfected with 3 μL aliquots containing 500–1000 μg of protein were subjected to immunoprecipitation using 5 μg of rabbit polyclonal antibody, anti-AR  and 5 μg of rabbit polyclonal antibody, anti-AR  or 10 μg of rabbit polyclonal antibody, and anti-caveolin-1 . After separation of immunoprecipitated proteins with the use of protein G Dynabeads (Life Technologies), pellets were resuspended in 30 μL of sodium dodecyl sulfate loading buffer for Western blot analysis.After the cells were harvested, cytoplasmic membrane and nuclear protein fractions were separated using the subcellular protein fractionation kit for cultured cells  as described . Proteinμg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% tricine-glycine gels and then transferred to polyvinyl difluoride membranes (Millipore). After blocking by incubation for 1 h at room temperature in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.1% Tween 20 ) containing 5% nonfat dry milk, membranes were incubated at 4°C with anti-AR antibody  overnight. Membranes were incubated for 1 h at room temperature with the second antibody in 5% nonfat dry milk in TBST after membranes were washed 3 times (5 min each) in TBST. Specific complexes were detected using the enhanced chemiluminescence system from GE Healthcare. After exposure, blots were stripped and assayed for histone deacetylase 1  in the nucleus, pan-cadherin  for the membrane, and glycerol glyceraldehyde-3-phosphate dehydrogenase  for the cytoplasm as loading controls.Proteins , incubated with primary anti-AR  antibody overnight, and rinsed and incubated with secondary antibody  for 2 h at room temperature. The slides were then rinsed in PBS for 3 × 5 min and incubated for 5 min at room temperature with Hoechst 33342 . Cells were washed with PBS for 3 × 5 min again and then mounted with Prolong Gold Antifade Reagent (Thermo Fisher) for imaging.t-test or a one/two-way ANOVA followed by a Fisher-protected least significant difference post hoc test. Statistical significance was considered when P < 0.05.Data were presented as the mean ± SEM. The statistical significance of the differences between groups was determined by the independent P < 0.01)   . A band  < 0.01) .To verify the translocation of AR under testosterone induction, which was exhibited by Western blot assay, we also performed immunofluorescence assay with the specific antibody against AR. As shown in To determine whether the membrane localized AR was associated with the caveolae protein caveolin-1, membrane fraction proteins from cells treated with vehicle  or testosterone for 30 min were immunoprecipitated with the anti-caveolin-1 antibody or anti-AR antibody. Consistent with To verify the immunoprecipitation data, caveolin-1 was knocked down by shRNA provided by Shanghai Genechem Co. Ltd. The results of Western blot analysis  and realP = 0.004, n = 4) and prevented AR translocation after exposure to testosterone . And the ratios  in NC- and shRNA-treated cells were 1.72 ± 0.037 and 1.56 ± 0.043, respectively (P = 0.030).In the cells transfected with negative control shRNA or caveolin-1 shRNA, knockdown of caveolin-1 does not affect AR nuclear translocation ; no signμM 2-bromopalmitate for 2 h before testosterone treatment. As shown in the P < 0.01, n = 3) and completely blocked AR membrane trafficking .Palmitoylation of the cysteine residue in ER increases the physical association of ER with caveolin-1 . We usedAndrogens affect male fertility in various ways. The action of androgens on Sertoli cells regulates claudin-3 expression and the formation and maintenance of the blood-testis barrier (BTB) , 14, as In the current study, using the subcellular fractionation kit and immunofluorescence, we found that physiological testosterone induced a rapid membrane association of AR within 30 min , and sigCav1 [Caveolin-1 is a protein that in humans is encoded by Cav1 . This scCav1 , is a poCav1 . Our immThe mechanism underlying the binding of AR to caveolin-1 needs further study. Nuclear receptors are ligand-activated factors sharing a common evolutionary history . A signahttp://csspalm.biocuckoo.org/) [μM) was also used to evaluate the effect of the inhibitor on AR translocation. We observed an inhibitory effect on testosterone-induced AR translocation, but the difference was not as significant as that of the 50 μM inhibitor. The palmitoylation inhibitor potentially exerts an inhibitory effect on other palmitoylated proteins that are important for AR trafficking. In the study by Pedram et al. [AR sequence was further confirmed to be palmitoylable by analysis with CSS-Palm 4.0 (free available from: oo.org/) . AR contoo.org/) . Our datm et al. , AR was Palmitoylation appears to be a common feature for some steroid hormone receptors. Palmitoylation may be involved in numerous signaling protein plasma membrane localization , 24, yetIn the present study, we found that testosterone induced AR translocation to the membrane fraction. AR was localized to the membrane by association with caveolin-1 via palmitoylation, and testosterone promoted this association. Knockdown of caveolin-1 decreased AR membrane localization and inhibited AR translocation to the membrane after exposure to testosterone. This proved AR membrane localization and helped clarify the mechanism of AR membrane anchoring and enriched the nonclassical pathway of androgens. Further studies should be conducted to identify and investigate the signal molecules and transport proteins involved in AR translocation to the membrane fraction. These data could potentially be used to address the challenges of treating infertility due to defects in testosterone signaling.


This study was conducted to investigate the recovery of motor function in rats through the silent information regulator factor 2-related enzyme 1 (Sirt1) signal pathway-mediated rehabilitation training. Middle cerebral artery occlusion (MACO) was used to induce ischemia/reperfusion injury. The rats were subjected to no treatment (model), rehabilitation training (for 21 days), resveratrol (5 mg/kg for 21 days), and rehabilitation training plus resveratrol treatment. 24 h later, They were assessed for neurobehavioral score and motor behavior score and expression of brain derived-nerve neurotrophic factor (BDNF) and tyrosine kinase receptor B (TrkB). Compared with sham group, models had significantly higher neurobehavioral scores, balance beam, and rotary stick scores. Compared with the model group, rats in rehabilitation training and resveratrol groups had significantly reduced scores. Compared with rehabilitation training or resveratrol treatment alone, rehabilitation plus resveratrol further reduced the scores significantly. The percentage of cells expressing BDNF and TrkB and expression levels of BDNF and TrkB were similar between the model and sham groups, significantly increased in rehabilitation training and resveratrol groups, and further increased in rehabilitation training plus resveratrol group. These results indicate that rehabilitation raining plus resveratrol can significantly improve the recovery of motor function in rats after cerebral ischemic injury, which is likely related to the upregulation of the BDNF/TrkB signaling pathway. Cerebrovascular disease is a major hazard to human health and life. Ischemia resulting from middle cerebral artery occlusion (MCAO) accounts for nearly 80% of cerebrovascular diseases, which have higher incidence, disability, and mortality rate and are heavy burden to the patient's family and society –3. At prγ-glutamylcysteine synthetase, and heme oxygenase-1 to protect endothelial cells [Silent information regulator factor 2-related enzyme 1 (Sirt1) is a member of the sirtuins family. It is an acetylated protein closely related to the age and aging. Studies have shown that Sirt1 is not only associated with inflammation, osteoarthritis, diabetes, cardiovascular disease, and cancer, but also associated with the progression of neurodegenerative diseases –10. A laal cells . HoweverIn this study, we investigate the effect of combined therapy of rehabilitation training plus resveratrol on the recovery of neural function, motor function in MCAO rats, and possible mechanisms underlying the effect. The findings would provide insights into additional role of resveratrol and rehabilitation for better clinical therapeutic use.Two-month-old male SD rats (SPD grade), weighting 200 ± 20 g, were purchased from Silaike Experimental Animal Co., Ltd., Shanghai (certificate number 2012-0002) and were hosted in animal rooms at 23 ± 2°C with free access to food and drinking water.Resveratrol was purchased from Sigma (lot number 34092-100 mg), USA; BCA protein assay kit, mouse anti-GAPDH monoclonal antibody, and horseradish peroxidase-labeled goat anti-rabbit IgG (H plus L) were purchased from Beytime Biotechnology , Beijing; rabbit anti-BDNF and TrkB polyclonal antibodies were purchased from Abcam (lots numbers ab108319 and ab18987), USA; Trizol kit and One-Step RT-PCR kit were purchased from Invitrogen (lots numbers 15596-018 and AM1005), USA. Electrophoresis apparatus, transfer apparatus, and gel imaging system were purchased from Bio-Rad, USA; plate reader was from TECAN, Swiss, and fluorescence microscope AF6000 was obtained from Leica, Germany. All animal experimental protocols were approved by the research ethic committee of Linyi Chinese Medicine Hospital.MCAO models were constructed as previously described , 5. Brien = 24), model (n = 24), rehabilitation training (n = 24), resveratrol (n = 24), and rehabilitation plus resveratrol groups (n = 24). Rehabilitation training started 24 h after the operation as described  at 37°C. The slides were developed in DAB solution and counterstained with hematoxylin. Dark brown or yellow-browed colored cells were considered positive, and the positive percentages were calculated using Image J software based on average light density.μL using One-Step RT-PCR kit according to manufacturer's recommendations. The amplified products were separated on 2% agarose gel by electrophoresis. RT-qPCR was performed on the 7900HT Fast Real-Time PCR system using TaqMan gene expression assays probes (Applied Biosystems). The primers used for BDNF were 5′-TATCCTTATGAACCGCCAGCC (forward) and 5′-TTCTGACATTAAGGGCCGTG (reverse) and for TrkB were 5′-CAAGTTGGCGAGACATTCCA (forward) and 5′-AGTCATCGTCGTTGCTGATGAC (reverse). Human glyceraldehyde-3-phosphate dehydrogenase, GADPH (Hs03929097_g1), was amplified using a pair of primers 5′-AGCCACATCGCTCAGACA (forward) and 5′-TGGACTCCACGACGTACT (reverse) as an internal control. The PCR was carried out in a total volume of 10 μL containing 1.5 μL of diluted and preamplified cDNA, 10 μL of TaqMan Gene Expression Master Mix, and 1 μL of each fluorescence TaqMan probe. The cycling conditions were 50°C for 2 min, 95°C for 10 min followed by 40 cycles, each one consisting of 45 s at 95°C and 45 s at 59°C with final extension at 72°C for 60 s. Samples were run in triplicate and the mean value was calculated for each case.BDNF and TrkB mRNA expression was analyzed by RT-qPCR on total RNA isolated from cortex tissues using the Trizol reagent according to the supplier's protocols. Reverse transcription was performed with 200 ng of RNA in a total volume of 10 −ΔΔCt) according to previously described protocol [The data were managed using the Applied Biosystems software RQ Manager v1.2.1. Relative expression was calculated by using comparative Ct method and obtaining the fold change value  to PVDF membranes . BDNF and TrkB protein expression levels were quantified using rabbit polyclonal antibodies specific for each protein. The expression levels of these proteins were standardized to human GADPH using a mouse polyclonal anti-GADPH antibody. Primary antibodies were detected using goat anti-rabbit or goat anti-mouse horseradish peroxidase- (HRP-) conjugated secondary antibodies. Immunoreactive bands were visualized using Western Lighting Chemiluminescence Reagent Plus  according to the manufacturer's instructions and then quantified by densitometry using a Bio-Rad gel imaging system and Quantity One software.To detect BDNF and TrkB protein expression, total proteins were extracted from the brain tissue in RIPA lysis buffer. The protein concentration was determined using the BCA kit. Sixty P value ≤ 0.05 was considered statistically significant.All data were expressed as means ± standard error of the mean (SEM) obtained from at least three independent experiments and analyzed using the statistical software SPSS 17. Means were compared using one-way ANOVA or repeated measures ANOVA. Tukey's test was used as the post hoc test to determine mean differences within groups. A P < 0.01), while the rats in rehabilitation training and resveratrol groups had significantly lower scores as compared to those in the model group (P < 0.01). Furthermore, the scores were further lowered in rehabilitation training plus resveratrol group (P < 0.01). These score decreases were statistically significant within 7 days of treatment and more remarkable as the time increased after the surgery .As shown in  surgery . In most surgery . ANOVA aP < 0.01) higher in the model groups than in the sham group and significantly (P < 0.01) lower in rehabilitation training group and resveratrol group than in model group. The scores were further reduced significantly (P < 0.01) in rehabilitation plus resveratrol group). These scores began to decrease significantly after 7 days of treatment throughout entire experimental period  and rotary stick scores .We then assessed the kinematics behavior scores of the rats and the results are shown in Tables d Tables  and 3. Id Tables  and 3. IP > 0.05). However, these parameters were significantly (P < 0.01) increased in rehabilitation training or resveratrol groups and were further increased significantly (P < 0.01) in rehabilitation training plus resveratrol group.To analysis changes at the molecular level, we assessed the expression of BDNF and TrkB in the brain tissues 21 days after the operation and the results are shown in Ischemic cerebrovascular disease or ischemic stroke is one of the major causes of disability in the elderly. The incidence of the disease is increasing rapidly as the population ages and recovery of motor functions after ischemic stroke has attracted increasingly attentions –3. While+-) dependent histone deacetylase. It regulates the transcription of a number of downstream genes through NAD+ to participate in various cellular processes such as apoptosis, cell cycle, cell energy metabolism, oxidative stress, and cell aging process and is involved in a number of diseases such as diabetes, tumor, inflammation, aging, and neural degenerative diseases [As a member of silence information regulator 2 (Sir2) family, Sirt1 is a nicotinamide adenine dinucleotide- (NADdiseases –10. Earldiseases –15. AlsoOur study shows that, from 7 to 21 days after operation, the recovery of motor functions in MACO rats was significant in a time-dependent manner as indicated by decreased neurobehavioral scores, balance beam, and rotary stick scores after rehabilitation training or resveratrol treatment. These results are consistent with early works , 19. Fur2+ imbalance, inhibit the toxic effect of excitatory amino acids on neurons, and stimulate the survival and regeneration of damaged neurons as well as differentiation and proliferation of neural stem cells [Research shows that the recovery of damaged nerve after cerebral ischemia may be promoted through the secretion of various neurotrophic factors, which bind to their receptors to activate downstream signaling systems , 21. BDNem cells –26. Rehaem cells  and in tem cells . On the em cells  in elderem cells . These rem cells . Howeverem cells , a proteIn conclusion, rehabilitation training and resveratrol can individually and synergistically improve the recovery of neurological function and motor function in rats after cerebral ischemic injury and this therapeutic effect is likely achieved through the upregulation of the BDNF/TrkB signaling pathway.


Recovery of Bell`s phenomenon after levator resection is unpredicatable. Delayed recovery can result in vision threatening corneal complications.To study the variability of Bell’s phenomenon and time taken for its recovery following levator resection for blepharoptosis and to correlate it with the amount of resection.A prospective observational study was conducted on 32 eyes of 32 patients diagnosed as unilateral simple congenital blepharoptosis who underwent levator resection at a tertiary care center between July 2013 and May 2015. Patients were followed up for 5 months and correction of ptosis, type of Bell`s, duration of Bell`s recovery and complications were noted.The study group ranged from 16-25 years with 15:17 male: female ratio. There were 9 mild, 16 moderate and 7 severe ptosis. Satisfactory correction was achieved in all cases. Good Bell`s recovery occurred in 13 eyes on first post-op day, in 2-14 days in 19 eyes and 28 days in 1 eye. Inverse Bell`s was noted along with lid oedema and ecchymosis in 2 patients. Large resections (23-26mm) were associated with poor Bell`s on the first postoperative day . However, the duration required for recovery of Bell`s phenomenon did not show any significant difference with the amount of resection. . Larger resections resulted in greater lagophthalmos . Patients with recovery of Bell`s delayed for more than 7 days were associated with greater number of complications (p=0.001 Fisher`s Exact Test).Close monitoring for Bell`s recovery is required following levator resection. Bell`s phenomenon is deviation of the eye, which accompanies an attempted closure of the eyelids . It is aLevator muscle resection is a standard procedure for ptosis correction and a good Bell`s phenomenon is a pre- requisite for ptosis surgery , 5. PostTo the best of our knowledge, except for a few case reports, none of the previous studies have correlated the amount of resection with the time required for recovery of Bell`s phenomenon. In this study, we shall study the effect of levator resection on recovery of Bell’s phenomenon and its association with post-operative complications.A prospective observational study was conducted on 32 eyes of 32 patients of simple congenital blepharoptosis attending the Oculoplasty clinic of Guru Nanak Eye Centre, New Delhi, who underwent levator resection between July 2013 and May 2015. Inclusion criteria included age >5 years, unilateral ptosis, levator action ≥4mm and good Bell’s phenomenon. Patients with dry eye, diminished corneal sensations, lid abnormalities, previous ptosis surgery, strabismus or uncontrolled illness were excluded from the study.Bell`s phenomenon was checked on attempted light closure of the lid after elevating the patient’s upper eyelids using the finger and thumb of one hand. Pre- operative work-up included general physical examination, best corrected visual acuity and cycloplegic refraction, marginal reflex distance, vertical palpebral aperture in upgaze, downgaze and primary position, horizontal palpebral aperture, ocular motility, corneal sensations and Schirmer Test for dry eye. A written informed consent, in accordance with the guidelines of the Declaration of Helsinki approved by the institute’s ethical committee was taken. Complete hemogram, bleeding and clotting time and random blood sugar was done.th post op week.All patients underwent LPS resection from the skin approach, under general or local anaesthesia depending on the patient`s age and were operated by a single surgeon (RG) with the amount of resection ranging from 18mm to 28mm. An inverse frost suture was passed in all patients using 5-0 silk at the end of the procedure. Satisfactory correction was taken to be ptosis correction within 1mm of the fellow eye at the end of 5Topical lubricant in the form of Polyvinyl alcohol 1.4% four hourly was prescribed in the operated eye till the recovery of Bell`s phenomenon after which it was reduced to three times daily till one month. Systemically, adults received Tab.Ciprofloxacin 500mg twice daily (children received Cap Amoxycillin 40 mg/kg/day in divided doses) for 5 days, and Tab Serratiopeptidase 10mg thrice daily (5mg for children) for five days. Povidone iodine was applied over sutures twice daily till suture removal. Frost suture was put up at night and while sleeping during the day and was removed after recovery of Bell`s phenomenon.Patients were followed up on day 1, 1 week and then every two weeks for a total of 5 months.Postoperatively type of Bell`s, complications such as lid oedema, dry eye, ecchymosis, exposure keratopathy and lagophthalmos if any, were noted. Lagophthalmos was assessed by asking the patient to extend his neck and gently close his eyes. The amount of lagophthalmos was measured as the distance between the upper and lower lids. Postoperative Bells’ phenomenon was recorded as normal or inverse. It was graded as good, fair and poor depending upon the amount of cornea visible, less than one third, one third to half or more than half respectively. For statistical evaluation, the fair and poor Bell`s was clubbed together. SPSS software 20 was used for data evaluation.1).There were 15 males and 17 female patients in an age group ranging from 6 to 25 years with a mean age of 16.26 years. The right eye was affected in 12 patients and the left eye in 20 patients. Nine patients had mild ptosis, 16 had moderate and 7 patients had severe ptosis (Table 1). Of the remaining, in 19 patients it took 2-14 days for good Bells’ to recover.  In majority of these patients, Bells recovered within the first week. In two of the patients inverse Bells was noted along with lid oedema and ecchymosis. One patient had poor Bells for 14 days and developed exposure keratopathy despite the presence of frost suture. He subsequently underwent paramedian tarsorrhaphy and the Bells’ phenomenon finally recovered on day 28. The patient had residual corneal opacity after healing of the keratitis. Large resections 23-26mm were associated with poor Bell`s on the first postoperative day.  .All the patients achieved satisfactory correction. In 13 patients Bells’ phenomenon was good on the first post-op day .The duration required for recovery of Bell`s phenomenon did not show any statistically significant difference with the amount of resection.  . Patients with recovery of Bell`s delayed for more than 7 days were associated with greater number of complications like ecchymosis, lid edema and exposure keratopathy.  .Larger resections resulted in greater lagophthalmos   contracts, the superior rectus and inferior oblique muscles (supplied by 3rd nerve) also contract and act synergistically to move the eye upward, causing normal Bell’s phenomenon [Levator resection acts as a surgical trauma and leads to delayed recovery of Bell`s phenomenon on eye lid closure, predisposing the patient to corneal complications. The Bell`s phenomenon is an oculogyric phenomenon first described by Sir Charles Bell in 1823 in a case of lower motor neuron facial palsy . It is athe eyes . Under tenomenon .In our study, the Bell`s recovered by 1 week in most of the patients with the longest recovery time being 4 weeks. Betharia et al found that even on moderate resection gross reduction in the Bell's phenomenon can occur in immediate postoperative period despite good cosmetic appearance and minimal lagophthalmos . We alsoth and 7th nerve nuclei though not as well established and numerous as those between 3rd and 7th nerve nuclei. But due to some pathologic conditions, connections between 4th and 7th nerve come into play and the mechanism of Bell’s phenomenon is reversed resulting in eye moving downwards due to action of superior oblique and inferior rectus. It probably occurs as a protective phenomenon where the up response fails to provide protection to the ocular surface [In Inverse Bell’s phenomenon, the eye moves downwards on lid closure. It has also been proposed that there are connections between 4 surface . Inverse surface , where sth postoperative day. This probably occurred due to improper taping of frost suture. The patient was managed by temporary paramedian tarsorraphy to facilitate recovery of Bell`s and healing of keratitis by preventing movement-induced micro trauma to the superior fornix by immobilization and stabilizing the tear film. Other available modalities of treatment for this complication are amniotic membrane grafts, soft bandage contact lenses and scleral contact lens [Lagophthalmos, is another concern especially in the presence of poor Bell`s. The combination of lagophthalmos and poor Bell`s has been known to make management of myopathic ptosis challenging . Howeveract lens . The occact lens . We did To conclude, in patients undergoing levator resection, a close watch is required on Bell`s recovery. Resections more than 23mm are especially prone to poor postoperative Bell`s and lagophthalmos, a combination that predisposes to exposure keratitis. Also, patients with delayed recovery of normal Bell`s warrant copious use of lubricants and if need be a temporary tarsorrhaphy to expedite recovery.


Pulmonary rehabilitation is an effective therapeutic intervention for people with chronic respiratory disease. However, fewer than 5% of eligible individuals receive pulmonary rehabilitation on an annual basis, largely due to limited availability of services and difficulties associated with travel and transport. The Rehabilitation Exercise At Home (REAcH) study is an assessor-blinded, multi-centre, randomised controlled equivalence trial designed to compare the efficacy of home-based telerehabilitation and traditional centre-based pulmonary rehabilitation in people with chronic respiratory disease.Participants will undertake an 8-week group-based pulmonary rehabilitation program of twice-weekly supervised exercise training, either in-person at a centre-based pulmonary rehabilitation program or remotely from their home via the Internet. Supervised exercise training sessions will include 30 min of aerobic exercise . Individualised education and self-management training will be delivered. All participants will be prescribed a home exercise program of walking and strengthening activities.Outcomes will be assessed by a blinded assessor at baseline, after completion of the intervention, and 12-months post intervention. The primary outcome is change in dyspnea score as measured by the Chronic Respiratory Questionnaire – dyspnea domain (CRQ-D). Secondary outcomes will evaluate the efficacy of telerehabilitation on 6-min walk distance, endurance cycle time during a constant work rate test, physical activity and quality of life. Adherence to pulmonary rehabilitation between the two models will be compared. A full economic analysis from a societal perspective will be undertaken to determine the cost-effectiveness of telerehabilitation compared to centre-based pulmonary rehabilitation.Alternative models of pulmonary rehabilitation are required to improve both equity of access and patient-related outcomes. This trial will establish whether telerehabilitation can achieve equivalent improvement in outcomes compared to traditional centre-based pulmonary rehabilitation. If efficacious and cost-effective, the proposed telerehabilitation model is designed to be rapidly deployed into clinical practice.ACTRN12616000360415). Registered 21 March 2016.Clinical trial registered with the Australian and New Zealand Clinical Trials Register at ( ChronicPulmonary rehabilitation is a proven, effective strategy to achieve clinically important gains in exercise and functional capacity, symptoms and quality of life  across aDespite compelling evidence for the benefit of pulmonary rehabilitation, only a very small percentage of eligible people ever attend a program . There aHome-based models of pulmonary rehabilitation have been proposed to increase the availability and accessibility of pulmonary rehabilitation services to patients –20. RecePreliminary studies have described the use of telerehabilitation in COPD, using a variety of program models. These studies suggest that telerehabilitation in COPD is safe, with no adverse events reported –28. HoweAnalysing the cost of telerehabilitation is critical to determine the economic viability of implementing such a model into clinical practice. To date, there is a lack of evidence to support the cost-effectiveness of telerehabilitation, despite apparent clinical benefits in a range of health conditions . A comprThis paper describes the protocol for the Rehabilitation Exercise At Home (REAcH) trial – a study of telerehabilitation in chronic respiratory disease. The aims of the study are to compare the: 1) clinical outcomes of telerehabilitation and traditional centre-based pulmonary rehabilitation for people with a chronic respiratory disease; 2) costs of telerehabilitation and centre-based pulmonary rehabilitation. We hypothesise that the clinical effects on symptoms, exercise capacity, and health-related quality of life (HRQoL) will be equivalent between pulmonary rehabilitation models; that the proportion of participants who complete pulmonary rehabilitation will be greater in the telerehabilitation group; and, that telerehabilitation, delivered using our low-cost model, will provide a cost-effective alternative to centre-based pulmonary rehabilitation  for people with chronic respiratory disease.anzctr.org.au (ACTRN12616000360415) on March 21, 2016.A randomised, controlled, assessor-blinded equivalence trial with an embedded economic evaluation will be conducted at two centres in metropolitan Melbourne , and one regional centre , Australia. Both regional and metropolitan sites have been included to increase the external validity of our model. The study was approved by the Human Research Ethics Committee at Alfred Health for all sites, and local governance approvals obtained from all participating sites . The trial was registered at 2 < 80% during exercise on cycle ergometer), to ensure safety of unsupervised training; 4) unstable or brittle asthma with a hospital admission or emergency department presentation within the preceding 3 months, to ensure safety of unsupervised training; 5) co-morbidities which preclude exercise training, such as neurological or musculoskeletal impairment; or 6) are unable to follow verbal instructions, suffer from cognitive impairment, or have language difficulties. Eligible participants will be provided with written and verbal information about the study from a clinician and/or a researcher. All participants will provide written consent. Trial participation will have no effect on routine management of their respiratory condition or any other healthcare requirements.Potential participants will be referred to pulmonary rehabilitation at the established centre-based programs of the participating sites, or those individuals admitted to hospital with an exacerbation of their respiratory disease. To be eligible for inclusion participants will: 1) have a primary diagnosis of a chronic lung disease; 2) be aged ≥40 years; and 3) be able to read and speak English. Potential participants will be excluded if they have: 1) a primary diagnosis of pulmonary hypertension or lung cancer, as rehabilitation is not yet a well established treatment for these diagnoses; 2) attended pulmonary rehabilitation within the previous 18 months and had no hospitalisations for a respiratory cause since rehabilitation completion, as these patients may not achieve further benefits; 3) oxygen desaturation resulting in cessation of cardiopulmonary exercise testing  to traditional centre-based pulmonary rehabilitation or telerehabilitation. A computer-generated, block randomisation scheme will be used with stratification for (i) recruitment in stable state vs post hospitalisation for respiratory exacerbation; (ii) site of recruitment; and (iii) diagnosis of ILD vs other diagnoses. Sequence generation will be performed by an individual who is independent of the research team and randomisation will occur using an online database. The randomisation sequence will be concealed from investigators. Participants will be allocated to groups after completion of baseline assessment. Given the nature of the intervention (exercise training) participants will not be blinded to the intervention, however all outcomes will be measured by an independent assessor blind to group allocation. The flow of participants through the study will be reported according to the recommendations of the Consolidated Standard of Reporting Trials (CONSORT) .2) on a cardiopulmonary exercise test (CPET) [Participants will undergo a standard outpatient pulmonary rehabilitation program at the centre where they were recruited, according to Australian best-practice guidelines . This int (CPET) . Intensit (CPET)  and walk2) on a cardiopulmonary exercise test (CPET) [Remotely supervised telerehabilitation at home will be conducted twice a week for 8 weeks, in groups of 4–6 participants at a time, supervised by an experienced physiotherapist. Exercise training will comprise 30 min of lower limb aerobic training and individualised strength training exercises. Participants will undertake a total of 30 min of cycle training, in two or more bouts, in each telerehabilitation session. Cycle ergometer intensity for telerehabilitation participants will be set with the same parameters as for centre-based participants, that is, at the work rate equivalent to 60% of the peak oxygen uptake (VOt (CPET) . Intensit (CPET) , based o2 and heart rate during training and at rest. The oximeter will not use Bluetooth but will be positioned such that the display is visible to the supervising physiotherapist throughout the session. Videoconferencing will be via Zoom videoconferencing software  that enables all participants to see and speak to each other. To ensure safety and understanding of equipment operation and the exercise program, the initial training session and establishment of the home exercise program will occur during a home visit by a physiotherapist.The telerehabilitation set-up will be based on our successfully piloted model  using reFidelity of the exercise training intervention for both groups will be facilitated through regular staff training, audit of exercise prescription and progression, and assessment of participant engagement .Disease-specific education and collaborative self-management training will be delivered to participants in both groups according to international guidelines . To stanDemographic details of age, gender, diagnosis, body mass index (BMI) and lung function will be collected at baseline. A maximal CPET using cycle ergometry with expired gas analysis will be completed as part of the baseline assessment to establish a work rate for an endurance cycle test (see secondary outcomes). Attendance rates at training sessions will be documented at the end of the intervention period and compared between the centre-based and telerehabilitation groups, with an a priori definition of pulmonary rehabilitation adherence (i.e. program completion) as undertaking a minimum of 70% of planned pulmonary rehabilitation sessions .Participants will undertake assessment of clinical outcome measures at baseline, end intervention and after 12-months follow-up  is a disease specific measure of HRQoL for people with chronic respiratory disease . The CRQExercise capacity will be measured using both the 6-min walk distance (6MWD) and endurance cycle time. The 6MWD will be measured using the 6-min walk test which is a validated measure of functional exercise capacity , and is The CRQ domains of fatigue, mastery and emotional function, reflecting HRQoL, will be reported as secondary outcomes. The SF-36v2, a validated generic quality of life measure will be used to derive a preference-based measure of utility, the SF-6D, for economic analyses .Self-efficacy will be measured using the Pulmonary Rehabilitation Adapted Index of Self Efficacy (PRAISE) tool, a reliable and sensitive measure of self-efficacy for patients attending pulmonary rehabilitation .Dyspnea will be measured using the Modified Medical Research Council scale .Anxiety and depression will be evaluated using the Hospital Anxiety and Depression Scale . Anxiety2) for offline analysis. Time (minutes) per day spent in sedentary, light, moderate and vigorous physical activity will be reported.Physical activity levels will be measured objectively using a wrist-worn activity monitor over seven days . The GeneActiv is a tri-axial accelerometer that is validated for the assessment of free-living activity in healthy people  and has Economic evaluation will take a societal perspective . EconomiDirect costs of telerehabilitation may be higher than those for centre-based pulmonary rehabilitation, due to capital expenditure for equipment, but these may be accompanied by savings in the form of averted expenditure on health service use. Thus we will also calculate the  rate of monetary return from investing in the intervention, which compares the difference in costs of health service use associated with telerehabilitation and centre-based rehabilitation to initial outlay for infrastructure . For thiIf there is truly no difference in the change in the dyspnea domain of the CRQ between telerehabilitation or traditional centre-based pulmonary rehabilitation at the end of the 8 week intervention, then 128 participants (64 in each group) are required to be 80% sure that the 95% confidence interval will exclude a difference in means of 2.5 points or more. This is the minimal important difference (MID) for the CRQ-D  and assu6-min walk distance (6MWD): if there is truly no difference in 6MWD at the completion of 8-weeks of centre-based pulmonary rehabilitation or telerehabilitation, a total of 108 participants will be required to ensure that the 95% confidence interval excludes a difference in group means of 30 m or more [ or more , assumin or more .Endurance Cycle Time: if there is truly no difference between telerehabilitation and centre-based pulmonary rehabilitation in change in constant work rate endurance time on cycle ergometer, then 52 participants are required to be 80% sure that the 95% confidence interval will exclude a difference in means of 150 s or more. This is the MID for endurance time for patients with COPD [ith COPD  and assuith COPD .Pulmonary rehabilitation adherence (program completion): data from our centres indicate that only 65% people who are referred to pulmonary rehabilitation take up the referral and complete the program. Previous studies have documented completion rates for telerehabilitation programs of over 90% [over 90% , 60. UsiThis sample size will also give sufficient power for secondary outcomes:Continuous variables will be analysed by fitting linear mixed models, controlling for recruitment centre and baseline values as required. The proportion of participants who attend for at least 70% of the program will be compared between groups using a chi-squared test and the relative risk of non-completion will be determined. All data will be analysed by intention-to-treat. A per-protocol analysis will also be conducted to reduce the risk of Type 1 error, as recommended in the CONSORT Extension for reporting of non-inferiority and equivalence trials . Alpha wwww.adeptrs.com), with encryption, password protection and restricted access. No identifying information will be stored in the online database.Data will be stored on a purpose-built online database  comprising a respiratory physician and two clinical research physiotherapists, with consultation with a statistician as required. The DSMB will review data relating to the primary outcome (CRQ-D) as well as 6MWD, endurance cycle time and Hospital Anxiety and Depression Scale scores. Data will be presented to the DSMB in a blinded fashion. The DSMB will initially review data at a time 6 months from the commencement of recruitment. Any serious adverse events will be notified immediately to the overseeing ethics committee  and the relevant site governance committee, as well as to the DSMB. If there are concerns about the safety of participants, the DSMB will make a recommendation to the trial steering committee about continuing, stopping, or modifying the trial.Pulmonary rehabilitation is a key recommended component of the non-pharmacological management of individuals with chronic respiratory disease , yet limThe telerehabilitation model under investigation directly addresses access barriers for both individual patients and the health system. By delivering pulmonary rehabilitation directly into the homes of people with chronic respiratory disease issues of transport, travel, their associated costs and weather could be negated , 62. By Unlike previous studies –25, 27 tThe purpose of this study conforms with policy statements to encourage the investigation of alternative models of pulmonary rehabilitation delivery in order to enhance implementation, access and delivery of pulmonary rehabilitation . If thisRecruitment commenced in August 2016 and is continuing.


Resist-free antireflective (AR) nanostructured films are directly fabricated on polycarbonate (PC) film using thermal-nanoimprint lithography (T-NIL) and the moth-eye shape of AR nanostructure is elaborately optimized with different oxygen reactive ion etching conditions. Anodic aluminum oxide (AAO) templates are directly used as master molds of T-NIL for preparation of AR nanostructures on PC film without an additional T-NIL resist. AR nanostructures are well arranged with a period of about 200 nm and diameter of about 150 nm, which corresponds to those of the AAO template mold. The moth-eye AR nanostructures exhibit the average reflectance of 2% in wavelength range from 400 to 800 nm. From the results, highly enhanced AR properties with simple direct imprinting on PC film demonstrate the potential for panel application in the field of flat display, touch screen, and solar cells. Recently, an antireflection (AR) property has been a great interest due to the demands for the enhanced performance in electronic devices such as solar cells and displays . EspeciaIn this study, we report a facile method for the preparation of AR nanostructure on PC film using AAO template as a master mold through the T-NIL process. Furthermore, the shape of AR nanostructures is elaborately optimized with different reactive ion etching (RIE) conditions and the optical properties of the fabricated AR films are investigated.2.To fabricate AR nanostructures on PC film, a commercial AAO template (Whatman Inc.) fabricated by a multi-step anodising process was used as template mold. As shown in Figure 1H, 1H, 2H, 2H-perfluorooctyl-trichlorosilane was used to form the self-assembled monolayer (SAM) based anti-adhesion layer. The formation of hydrophobic anti-adhesion layer on the surface of the AAO templates was confirmed by contact angle measurement which was determined to be 150° as shown in Figure Prior to T-NIL process, AAO templates were coated with the anti-adhesion layer to decrease the surface free energy of AAO for the easy separation of the AAO templates from PC film. A liquid phase deposition of T-NIL process was carried out on PC film with Nano-Imprinter  using AAO template mold under the various temperatures  and pressure  for 20 min. A home-made PC film was used and the thickness of PC film is about 1 mm. After removing the AAO template mold, oxygen RIE process was optimized for the proper formation of AR nanostructure on PC film with the various parameters, which were oxygen flow of 30 sccm under the power of 5 W at different etching time . Surface morphology of the imprinted AR nanostructures on PC film was investigated by field emission scanning electron microscope (FE-SEM) and the optical reflectance of the imprinted PC film was measured by UV-visible spectrophotometer (Hitachi U-3501). The schematic procedure for the preparation of the AR nanostructure on PC film is illustrated in Scheme Figure To evaluate the AR properties of nanoimprinted PC films, the reflectance was measured by UV-visible spectroscopy in the wavelength range from 400 nm to 1,000 nm, as shown in the Figure In order to optimize the shape of AR nanostructures and reduce the reflectance, AR nanostructures fabricated at 160°C by T-NIL was etched by the oxygen RIE process with the various etching time. It is observed that oxygen RIE process has great influence on the surface morphology of AR nanostructures, as shown in Figure Figure The reflectance difference between the bare PC film and PC film with oxygen RIE processed AR nanostructures is demonstrated visually in Figure We successfully demonstrate that the resist-free AR nanostructures are directly fabricated on PC film using T-NIL and the shape of AR nanostructures is elaborately optimized with different oxygen RIE conditions. AAO templates are directly used as master molds of T-NIL for preparation of AR nanostructures on PC film without an additional T-NIL resist. AR nanostructures are well arranged with a period of about 200 nm and diameter of about 150 nm, which corresponds to those of the AAO template mold. The average reflectance of the PC film with AR nanostructures was obtained to be about 2% in the broadband wavelength range of 400–800 nm. Comparing to bare PC film and the fabricated AR film, the average reflectance of AR film is reduced to approximately 11% from 13% to 2% in the visible light region. As a result, moth-eye AR nanostructured PC film using T-NIL and oxygen RIE can efficiently reduce the light reflectance at the visible light region. We suggest that the prepared AR film is useful for panel application in the field of flat display, touch screen, and solar cells.