AR) is a common genomic event in metastatic castration-resistant prostate cancer (mCRPC). To evaluate the prognostic value of the amplifications of specific loci in the AR gene in cell-free DNA, we developed a multiplex digital PCR (dPCR) assay that targeted AR enhancer (AR-En), AR exon 1 (AR-E1), AR exon 8 (AR-E8) and OPHN1 (downstream of AR). We selected three relatively stable genes, C2orf16, FAM111B, and GRIA3, as reference controls for copy number normalization. One hundred and eight mCRPC patients were recruited to test the association of specific AR loci amplification with clinical outcome. Using a normalized ratio ≥ 1.92 as cutoff, amplification of AR-En, AR-E1, AR-E8 and OPHN1 was observed in 28, 25, 24 and 19 of 108 mCRPC patients, respectively. Among the 41 patients with AR region amplification, 9 (21.9%) showed amplification at all four selected regions and 15 (36.6%) showed amplification at AR-En, AR-E1, and AR-E8. Six (14.6%) patients showed independent AR-En amplification, while the remaining 3 (7.3%) demonstrated AR-E8 amplification only. Kaplan–Meier analysis showed overall survival’s association with the amplification of AR-En ), AR-E8 ) and AR-En-E8 ). Multivariate models that included AR-En-E8 amplification and clinical factors significantly improved prognostic performance (p = 0.0001). With further validation, the multiplex dPCR assay may assist in prognostication of mCRPC patients.Amplification of androgen receptor ( Circulating cell-free DNA (cfDNA) has become a promising tool in molecular oncology, allowing the detection of molecular alterations associated with cancer biology, treatment response ,2 and ovAR) gene is amplified in nearly half of mCRPC cases [AR activity can drive therapeutic resistance in advanced prostate cancer [AR enhancer region, 650 kb upstream of AR, drives the progression of mCRPC [OPHN1 gene, which is downstream of AR, is also amplified in mCRPC [Prostate cancer is one of the leading causes of cancer death in males in the Western world , which oPC cases ,18,19,20e cancer ,22, rendof mCRPC ,24. We rin mCRPC .AR gene including AR-En and OPHN1 to determine the impact on survival. For this, two multiplex dPCR assays were developed that targeted four selected loci including two targets genes and two normalization controls in each assay by adjusting concentrations of primers and probes and the type of fluorophores. We then applied the assays to analyze the amplification status of AR-En, AR exon 1 (AR-E1), AR exon 8 (AR-E8), and OPHN1 in mCRPC patients receiving chemotherapy as a clinically useful molecular biomarker.In this study, we systematically determined the frequencies and association of amplifications in multiple loci in and near the One hundred and eight mCRPC patients were enrolled between September 2009 and July 2013 and followed until the date of cut off for analysis (1 October 2018). After failure of androgen-deprivation therapy (ADT), 59/108 patients received systemic chemotherapy (majority being with docetaxel). One hundred and two died during follow-up with a median follow-up of 92.2  months. At the time of study enrollment to the mCRPC cohort, the median prostate specific antigen (PSA) and serum alkaline phosphatase (ALP) levels were 16.7 ng/mL  and 94 IU/L , respectively. The median time for ADT failure prior to progression to mCRPC state of the study cohort was 19.7 months . Clinical characteristics of the study cohort are listed in AR-E1, AR-E8, and control FAM111B, GRIA3; Assay II included OPHN1, AR-En and control FAM111B, C2orf16. Both assays shared a common control to normalize potential sample loading bias  for AR-En, 0.92 (Range 0.69–1.30) for AR-E1, 1.08 (Range 0.86–1.41) for AR-E8, and 1.12 (range 0.96–1.29) for OPHN1, indicating the consistency of CN detection for these targets (We first established the multiplex dPCR assay in prostate cancer cell line DNA (LNCaP and 22RV21) using duplex qPCR by combining single fluorophore FAM and HEX-labeled probes. To separate two targets with the same fluorophore, we adjusted the probe ratio, with one using normal dilution (1×) and another using 40% of normal dilution (0.4×). To balance the signal density of different fluorescence, we applied 1:1.5 ratio between FAM and HEX-labeled probes. Each sample was analyzed by two assays. Assay I included target genes ing bias A,B. To t targets .AR loci, we first examined existing databases [C2orf16 at 2p23.2, FAM111B at 11q12.1, and GRIA3 at Xq25. We tested the stability of these three genes in the 108 plasma cfDNA dPCR data using software RefFinder and found their stability in order of FAM111B > GRIA3 > C2orf16. However, the most stable reference control was found by the combination of FAM111B, GRIA3, and C2orf16  for AR-En, 0.29 (range −1.69–5.45) for AR-E1, 0.36 (range −0.92–5.45) for AR-E8, and 0.14 (range −1.54–5.04) for OPHN1  had AR-E8 amplification; 25 (23.1%) had AR-E1 amplification; and 19 (17.6%) had OPHN1 amplification (AR loci amplified patients (at least one of the targets amplified), we observed that 9 patients shared AR-En, AR-E1, AR-E8 and OPHN1 amplifications; 15 patients shared AR-En, AR-E1, and AR-E8 amplification; 6 patients had independent AR-En amplification; 3 patients had independent AR-E8 amplification; and 2 patients had independent OPHN1 amplification  showed clear amplification of AR region in 12 of the 13 samples , less than or greater than the median time to ADT failure (<19.7 months), and above or below the median ALP (>94 IU). We found statistically significant correlation between plasma AR amplifications and clinical indices of time to ADT failure (p = 0.0052), and Gleason score (p = 0.0086). No significant association between AR amplification and PSA and ALP level (p > 0.1) was found. To test the association of the CN with overall survival (OS), we performed K–M analysis using the defined amplification of AR-En, AR-E1, AR-E8 and OPHN1 gene loci. Significant association of amplification in AR-En ), AR-E8 ) with OS were observed. Patients harboring AR-En and AR-E8 amplification had a significantly shorter OS. The median OS was 19 vs. 27 months for AR-En amplification-positive and -negative patients and 21 vs. 27 months for AR-E8 amplification-positive and -negative patients, respectively (AR-E1 (p = 0.08) and OPHN1 (p = 0.23)  )   was obse with OS . Conside5–2.73)) C.p = 0.004), and greater than median ALP levels (p = 0.005) were statistically associated with poor OS, but not Gleason score, age and presence of bone metastasis (p > 0.1). We then constructed a multivariate model, which included molecular (CN of AR-En-E8) and clinical prognostic factors significantly associated with survival at the univariate levels. The results indicate that AR-En-E8 amplification, shorter time to ADT failure, and higher ALP were all independently significantly associated with poor OS , and level of ALP, where the weights were the regression coefficients from the multivariate Cox model. We found that the risk group based on a median risk index cut point was significantly associated with OS )  with OS. At the univariate Cox-regression level, shorter time to ADT-failure ( poor OS . We creao 2.88)) D.AR amplification has been previously reported in several independent studies to prognosticate survival in mCRPC state, and our group recently also reported AR-En amplification was associated with primary resistance to abiraterone acetate treatments [AR-En, AR-E1, AR-E8, and OPHN1 genes using cfDNA from plasma of mCRPC patients. By varying the concentrations of primers and probes and the type of fluorophores, accurate, precise, and absolute quantification of the specific nucleic acid sequences and the high sensitivity of dPCR, we were able to detect low frequency of tumor DNA based on alterations among the excessive background of wild-type DNA in cfDNA samples. We also observed that the increased number of potential targets per test significantly improved dPCR clinical utility, by reducing the cost and also improving the CN alteration output information from a single plasma specimen. These results suggest that a multiplex dPCR assay can be used as a potentially valuable molecular biomarker assay to detect genomic alterations in mCRPC patients.eatments ,25,30. WAR- and OPHN1 in a different cohort of mCRPC patients by using low-pass whole genome sequencing (WGS) [AR-En, AR-E1, AR-E8, and OPHN1 by multiplex dPCR. We observed that the amplification frequencies of AR-En was more frequent than AR itself and OPHN1. While most of the cases with AR enhancer, AR-E1, AR-E8 and/or OPHN1 were co-amplified, six patients with AR-En and three with AR-E8 were independently amplified, possibly indicating an independent interplay of metastasis driver for both AR and AR enhancer, which is consistent with previous reports [We have previously reported amplification frequencies in plasma cfDNA for ng (WGS) . In the  reports ,23,24. IAR amplification [AR and AR-En amplifications were determined in cfDNA by using targeted sequencing [AR and AR-En amplifications was detected to be 45% and 40%, respectively, and was correlated with poor survival. Using a dPCR-based assay, we observed lower amplification frequency (about 20–25%) in our cohort. There may be several factors for this difference which could contribute to the low detectable amplification in our study. One key factor is the cutoff call to define AR amplification. Because the cutoff value  determines amplification status, the number of patients with AR amplification can be significantly increased if lower cutoff value is applied. In fact, if, in our study, the cutoff value is set to 1.8, the number of patients with amplification at AR loci is observed to increase to 46 (42.5%), which is similar to other reports [p value from 0.08 was changed to 0.02). However, to stay consistent with the recently published cutoff call rates on amplification status [Previous studies have shown that 30–50% mCRPC patients have fication ,31, and quencing . The rat reports ,31. We on status , we seleAR-En and AR-E8) potentially improves the sensitivity of the test and also informs about inherent tumor heterogeneity in the mCRPC patient population. We expect that applying multiplex dPCR to other genomic loci, such as ZFHX3 and PIK3CA, in the mCRPC stage will show significant associations with treatment response [The reference controls selected in our study were based on the gene loci that are relatively stable in prostate cancer based on published databases. Our results show that the average of the three controls was more stable than any individual one. By combining the three reference loci, the assay can further minimize bias caused by potential CN changes at these regions. Additionally, the information derived from combination of target loci -approved hospital-based cohort study from September 2009 to July 2013, following androgen-deprivation therapy (ADT) failure, as was previously described [escribed ,34. All escribed ,34.AR loci, including AR-En, AR, and OPHN1 in mCRPC patients [C2orf16 at chr2p23.3, FAM111B at chr11q12.1, and GRIA3 at chrXq25—were selected as controls for CN normalization based on the CN stability of these genes in four different prostate cancer databases [AR-En, AR-E1, AR-E8, OPHN1, C2orf26, FAM111B, GRIA3) and 3μM of probes.cfDNA was extracted from plasma and pre-amplified by using a ThruPlex DNA-Seq Kit  as previously described . The tarpatients . Three gatabases ,27,28. TAR-E1 and AR-E8 in Assay I, and AR-En and OPHN1 in Assay II, respectively. Two CN control primer/probe pools were added for CN determination, which were FAM111B and GRIA3 in Assay I and FAM111B and C2orf26 in Assay II, respectively. QuantStudio 3D Digital PCR System (Life Technology) was used for the dPCR. For each chip, reactions were performed in 15.5 μL volume using 7.25 μL of 2 × 3D Digital PCR master mix, 1.5 μL High Hex 20 × primers/probes master mix, 0.6 μL low Hex 20 × primers/probes master mix, 1 μL high FAM 20 × primers/probes master mix and 0.4 μL low FAM 20 × primers/probes master mix, respectively. Reactions were performed under universal cycling conditions: 96 °C for 10 min, followed by 45 cycles at 58/60 °C for 2 min and 98 °C for 30 s with a final extension at 60 °C for 2 min.We performed two dPCR assays for each individual sample. Each assay included two target genes, The chip signal image was captured by the QuantStudio 3D Digital PCR system. Data analysis was performed using the AnalysisSuite Software (Life technology). The number of target molecules was calculated using the Poisson distribution, which provided the CN per μL reaction mix. The CN ratio of each target at AR loci was estimated by normalizing to the average of three reference genes. The formula is: CN on chrX = target copies/μL*2 divided by control copies/μL.Thirteen of 108 samples were selected for low-pass whole genome sequencing to confirm AR amplification. cfDNA libraries (ThruPlex DNA-Seq Kit) were sequenced in Illumina HiSeq2500 for single-end 50 bp read. Sequencing reads from FastQ files were first mapped to human genome (hg19) and then summarized into 1 Mb genomic bins, which were further normalized to a group of 15 healthy individuals by log2 ratio transformation. The log2 ratios with 1Mb bin size at AR and its flanking regions were analyzed for CN changes. Detail methods for CN analysis have been published previously [AR loci CN aberrations and overall survival (OS). OS was calculated from the time from the date of study enrollment at the time of progressing to mCRPC state to the date of death or the date of the last follow-up (30 October 2018). Association of CNs with clinical factors were tested with Fisher’s exact test. Association between CNs and OS was assessed using Kaplan–Meier (K-M) survival curves. Tests of significance for amplification and association with OS were performed using log-rank test with statistical significance set at p ≤ 0.05. To determine the effect of multiple factors on survival, a multivariate Cox regression model was utilized to assess association of several covariates measured at study enrollment with OS. These included AR loci amplification, PSA, serum ALP, and time to ADT failure. To avoid over-parameterization during the multivariate modeling process due to a relatively small number of patients and large number of potential covariates, the final multivariate model only fitted factors with an entry threshold of p ≤ 0.05 in univariate analysis.The primary aim of the study was to identify associations between plasma AR loci in plasma cfDNA and highlight its potential clinical use as non-invasive molecular testing tool for mCRPC prognosis. We were able to successfully use three relatively stable genes as reference controls to minimize potential error by single reference gene assays and were able to probe a combination of AR target regions to increase the detection sensitivity of these AR loci. We anticipate that further development of the easy-to-use and low-cost liquid biopsy assay will facilitate its clinical application in the highly heterogeneous mCRPC patients.We reported a multiplex dPCR assay to detect CN changes at


Podospora anserina has been appreciated for its targeted carbohydrate-active enzymatic arsenal. As a late colonizer of herbivorous dung, the fungus acts specifically on the more recalcitrant fraction of lignocellulose and this lignin-rich biotope might have resulted in the evolution of ligninolytic activities. However, the lignin-degrading abilities of the fungus have not been demonstrated by chemical analyses at the molecular level and are, thus far, solely based on genome and secretome predictions. To evaluate whether P. anserina might provide a novel source of lignin-active enzymes to tap into for potential biotechnological applications, we comprehensively mapped wheat straw lignin during fungal growth and characterized the fungal secretome.The ascomycete fungus 13C lignin internal standard py-GC–MS analysis showed substantial lignin removal during the 7 days of fungal growth (24% w/w), though carbohydrates were preferably targeted . Structural characterization of residual lignin by using py-GC–MS and HSQC NMR analyses demonstrated that Cα-oxidized substructures significantly increased through fungal action, while intact β-O-4′ aryl ether linkages, p-coumarate and ferulate moieties decreased, albeit to lesser extents than observed for the action of basidiomycetes. Proteomic analysis indicated that the presence of lignin induced considerable changes in the secretome of P. anserina. This was particularly reflected in a strong reduction of cellulases and galactomannanases, while H2O2-producing enzymes clearly increased. The latter enzymes, together with laccases, were likely involved in the observed ligninolysis.Quantitative P. anserina and expand the view on its enzymatic repertoire beyond carbohydrate degradation. Our results can be of significance for the development of biological lignin conversion technologies by contributing to the quest for novel lignin-active enzymes and organisms.For the first time, we provide unambiguous evidence for the ligninolytic activity of the ascomycete fungus  Podospora anserina has long been studied for its lifecycle characteristics, particularly regarding its short growth phase, but more recently also has sparked biotechnological interest [P. anserina enzymes were shown to be of biotechnological potential, being able to enhance the saccharification efficiencies of enzymes commonly used in industry [With the increasing interest in biological lignocellulose valorization strategies, there is a quest for novel lignocellulose-acting enzymes and organisms producing such enzymes . The ascinterest –6. The finterest –8. Indeeindustry , 10.P. anserina is expected to specifically degrade the more recalcitrant fraction of lignocellulose [P. anserina [2O2 levels [P. anserina in biomass upgrading approaches [2O2-producing oxidoreductases [P. anserina is, amongst ascomycetes, characterized by the highest number of genes encoding auxiliary activities (AA), encompassing oxidoreductases only, as accommodated in the carbohydrate-active enzymes database  [P. anserina remains to be further elucidated.As a late colonizer of herbivorous dung, ellulose . Furtherellulose . The latanserina . The fun2 levels . Consideproaches . The sugductases , 12, 14.azy.org) . Secretoazy.org) , 16. Furazy.org) . HoweverRecently, we have demonstrated that quantitative pyrolysis-GC–MS analysis is exceptionally useful for mapping the extent of fungal plant biomass delignification as well as for unravelling the underlying ligninolysis mechanisms –20. ImpoP. anserina was unambiguously confirmed through comprehensive substrate analysis after growth of the fungus on wheat straw lignin. Quantitative py-GC–MS analyses revealed substantial lignin removal and an increase in Cα-oxidized moieties indicative of oxidative degradation. HSQC NMR analysis further substantiated these findings by showing a decrease of intact β-O-4′ aryl ether linkages concomitant with the increase of Cα-oxidized substructures. In addition, p-coumarate and ferulate moieties were decreased, which suggests feruloyl esterase activity during fungal growth. Secretome analysis indicated that ligninolysis likely involved laccases and H2O2-producing enzymes.In this work, the ligninolytic activity of P. anserina, we used cultivation conditions that would allow us to evaluate changes in both lignin structure and in the fungal secretome [P. anserina could be compared to growth on GAX alone. The isolated lignin was structurally representative of wheat straw lignin and of high purity (90% w/w)  besides GAX (58% w/w) during growth  and resinol (β-β’) structural elements appeared to be more resistant against degradation by P. anserina and as a result they relatively accumulated in the fungal-grown residues, albeit slightly  could be assigned to CAZymes  with a putative plant cell wall-active function  were strongly increased  Fig. b and thiPaMan5A) and was able to enhance the degradation of spruce polysaccharides [The overall decrease in galactomannanases could be attributed to endomannanases, β-mannosidases and α-galactosidases Fig. c and moscharides . Mäkelä charides .P. anserina. Alternatively, the secreted LPMOs might have depended on other electron transfer systems involving the other H2O2 producing enzymes that were strongly induced in the presence of lignin, as will be elaborated below [Overall, LPMOs did not decrease with the cellulase system in the presence of lignin Fig. . Interesed below , 46.The total xylanase machinery was not significantly affected by the presence of lignin Fig.  and the P. anserina was grown on wheat straw, the latter GH62 arabinofuranosidase was produced in greater abundance than when grown on cottonseed and soybean hulls [Likewise, the presence of lignin induced a 2.9-fold reduction in two predicted GH43 arabinofuranosidases (#438134 and #300046), while the GH51  and GH62  arabinofuranosidases, respectively, increased by sixfold and 4.8-fold  was not produced in the presence of lignin, while a putative CE12 rhamnogalacturonan acetylesterase (#381424) was decreased by a factor 6.1  and feruloyl esterases (FAEs) was not significantly different in the presence and absence of lignin Fig. . Still, 2O2-producing enzymes (2.9-fold) was particularly due to a single putative AA5_1 glyoxal oxidase , which increased 7.3-fold  belong to other subclades of the family of BBE-like enzymes -like enzymes , 49. Howbod1, but it was later found to actually correspond to a ‘new’ thermotolerant laccase after heterologous production and biochemical characterization [bod1 enzyme, furthermore, showed a mildly decreased ability to grow on lignin next to a decreased ability to resist various phenolics and H2O2 [P. anserina was grown on other lignocellulosic substrates [The presence of lignin caused a 20-fold overall increase in laccases, which was primarily due to a 63-fold induction of an AA1_3 laccase #430065)  was only produced in the presence of lignin, albeit in relatively low amount  [2O2 producers to enzymes involved in ligninolysis seems unlikely. However, the produced H2O2 might have been involved in LPMO action and, consequently, co-regulation between (some) H2O2 producing enzymes and LPMOs cannot be excluded [Knowing that specific enzyme activities are not taken into account, the extensive induction of Hsis Fig. . Althoug and VP) . As suchexcluded , 53.2O2 might have been involved in Fenton chemistry. The Fenton reaction describes the conversion of H2O2 to hydroxyl radicals in the presence of iron at acidic conditions. These highly reactive radicals are expected to result in ‘untargeted’ or ‘nonselective’ ligninolysis, a mechanism that is often associated with the action of brown-rot basidiomycetes [P. anserina biodegradation reactions. Although these secretome analyses cannot provide a definite answer on the underlying ligninolysis mechanisms, they do highlight potentially involved enzymes that are interesting candidates for follow-up gene-knockout and enzyme production/characterization studies.Alternatively, the produced Homycetes , 55. Theomycetes , 56, 57.Podospora anserina through comprehensive substrate analyses of wheat straw lignin after growth of the fungus. Fungal growth resulted in substantial lignin removal and the accumulation of oxidatively degraded substructures. The insights obtained through substrate characterization agreed well with complementary proteomics analysis. Most importantly, the analysis showed that the secretome of P. anserina was considerably altered in the presence of lignin, with a strong reduction of cellulases and galactomannanases in particular, and suggested the potential involvement of laccases and H2O2 producing enzymes in ligninolysis reactions. Our work highlights several interesting candidate enzymes for further biochemical characterization and employment in biotechnological applications.In summary, we have for the first time, provided unambiguous confirmation of the ligninolytic activity of the ascomycete fungus All chemicals and solvents were obtained from commercial suppliers and used as supplied. Water used in all experiments was purified via a Milli-Q water system .g, 10 min, 20 °C) and washed two times with water before being suspended in 50 mM sodium acetate buffer pH 5 at a concentration of 5% (w/w). The suspension was incubated with the commercial enzyme cocktail ViscoStar 150 L  (0.125 mL/g biomass) at 50 °C for 24 h to degrade the cell wall polysaccharides. The insoluble material after incubation was obtained through centrifugation , resuspended in buffer, loaded with fresh enzyme and incubated for another 24 h. After incubation, the suspension was centrifuged and the insoluble residue was washed three times with water before freeze-drying. The freeze-dried material was subsequently suspended in 80% (v/v) aqueous dioxane at 5% (w/w) dry matter loading and extracted at room temperature under nitrogen atmosphere for 24 h. The supernatant was recovered by centrifugation  and the residue was extracted again. The supernatants were combined, freeze-dried, washed with water to remove traces of dioxane and freeze-dried again to yield the wheat straw lignin isolate.Lignin was isolated from wheat straw by modifications of the classical lignin isolation procedures , 59. ExtP. anserina were based on previously described procedures [P. anserina strain S mat + (CBS 143333) was grown on Luria–Bertani agar at 27 °C for 5 days before 0.5-cm plugs were taken from the formed mycelium to inoculate the samples. Flasks were statically incubated at 27 °C for 3 or 7 days. All experiments were performed in triplicate. After incubation mycelium was scooped out with a sterile loop and stored at − 20 °C. Culture samples (2.5 mL) were taken and centrifuged  to separate the solid fraction from the supernatant for proteome analysis. Both fractions were stored at − 20 °C. The residual substrate in the flasks was removed by dispersion in water. Residual substrate and mycelium were water-extracted for 1 h at 4 °C under continuous stirring before centrifugation  to separate water-soluble and insoluble fractions. The obtained fractions were flash-frozen in liquid nitrogen and freeze-dried.Media and growth conditions for ocedures , 60. Wat13C wheat straw lignin internal standard (IS) solution (1 mg mL−1 ethanol/chloroform 50:50 v/v) was added and dried prior to analysis [13C labeled). Pyrograms were processed by TraceFinder 4.0 software. Lignin contents and relative abundances of lignin-derived pyrolysis products were calculated as described previously [Analytical pyrolysis coupled to gas chromatography with high-resolution mass spectrometric detection  was performed as previously described , 61. To analysis . All sameviously , 19.For NMR analysis, biological triplicates were mixed in equal dry matter amounts to a single replicate. HSQC NMR spectra were recorded on a Bruker AVANCE III 600 MHz NMR spectrometer as previously described . SpectraConstituent monosaccharides released after acid hydrolysis were determined in duplicate as previously reported by Englyst & Cummings with reported modifications , 62.p < 0.05 from an unpaired t-test were used, as based on Daly et al. [Podospora anserina mat + v1.0 database from JGI was used. Functional annotations were assigned on the basis of InterPro descriptions (https://www.ebi.ac.uk/interpro/), Pfam predictions (https://pfam.xfam.org/) and Signal P prediction from JGI. CAZymes in the P. anserina mat + v1.0 genome were annotated on the basis of dbCAN (http://bcb.unl.edu/dbCAN2/) and Pfam predictions.For secretomic analysis, triplicate samples after 3 days of fungal growth were used. A 900-μL aliquot of water-soluble culture fraction was mixed with two volumes of methanol and proteins were left to precipitate overnight at − 20 °C. The precipitates were pelleted by centrifugation, washed by 60% (v/v) cold aqueous methanol, pelleted again and resuspended with 30 μL of anionic acid labile surfactant II  in 200 mM ammonium bicarbonate. Twenty microliters of the resolubilized proteins were mixed with 5 μL 5X SDS-PAGE loading buffer , heated at 95 °C for 5 min before being loaded onto a 12% w/v acrylamide SDS-containing separating gel. Proteins were separated by SDS-PAGE until the 250-kDa marker of the BLUelf prestained protein ladder  entered the separating gel. After the gel was stained with Coomassie brilliant blue, about 1 cm of the gel lane was cut out and polypeptides were in-gel digested with trypsin as described previously . Peptidey et al. . The PodAdditional file 1: Substrate characterization data: Figure S1. Py-GC-HR-MS pyrograms (TIC) of wheat straw, wheat straw lignin and glucuronoarabinoxylan (GAX). Table S1. Py-GC-HR-MS relative abundance of lignin compounds in wheat straw lignin isolate used for fermentations with P. anserina. Table S2. Semiquantitative 1H–13C HSQC NMR structural characterization of wheat straw lignin isolate used for fermentations with P. anserina.Additional file 2: Proteomic analysis: Figure S2. Proteomic analysis of secreted proteins during growth of P. anserina on glucuronoarabinoxylan (GAX) and GAX + Lignin. Grouped according to functional annotations. Figure S3. Proteomic analysis of secreted individual proteins during growth of P. anserina on glucuronoarabinoxylan (GAX) and GAX + Lignin. Accession numbers according to JGI database (P. anserina mat + v1.0).Additional file 3: Proteomic analysis data. Raw and processed proteomic analysis data of secreted proteins during growth of P. anserina on glucuronoarabinoxylan (GAX) and GAX + Lignin. Table S3. Unique/induced proteins other than plant cell wall active CAZymes during growth of P. anserina on GAX or GAX + Lignin. Minimal relative abundance 0.2% of total protein and > twofold increase in absence/presence lignin.Additional file 4: Figure S4. Phylogenetic analysis of lignin-induced P. anserina AA7 oxidoreductases.


Professor Li-Pin King  is a famous physiologist in China. He devoted his entire life to the researches on Pharmacology of traditional Chinese medicine, Experimental Biology and Modern medicine. He made tremendous contributions to the development of Physiology in China. Prof. King is also the first scientist who applied the scientific methods to researches on traditional Chinese medicine and one of the earliest generations of Chinese researchers who studied modern medical science Luo, .In 1895, Prof. King was born in Zhejiang, China. He studied abroad at the University of Lyon in France and graduated with degrees in both science and medicine. After graduation, he served as a teaching assistant there. Later on, he returned back to China and became a professor at the Agricultural College of Peiping University and also was appointed as the director of the Department of Biology. In October 1929, Institute of Biology, National Academy of Peiping  was established and Prof. King was appointed as the director. He was also a professor of Peiping University, Peiping Women’s College of Arts and Sciences and Sun Yat-sen University. Unfortunately, Peiping was occupied by the invaders in 1937. Prof. King moved to Kunming, Yunnan together with the National Academy of Peiping and later served as professor in biology at Yunnan University and the director of the Chinese Medical Academy  to compile Physiological Hygiene, a junior high school textbook, boosting biology and physiology education in China.Prof. King engaged in physiological researches all his life and compiled many books such as Mai Jing (Pulse Classic) written by Shuhe Wang (王叔和) to the National Yunnan University during his stay in Yunnan.Prof. King was not only an outstanding researcher, but also an excellent educator who has cultivated many young talents. In September 1925, the Department of Biology of Peiping University was established and Prof. King was recruited as a lecturer to teach Physiology and Experimentation for the first three students of the Department: Fengying Zhang (张风灜), Yuangao Shi (石原皋) and Jingsheng Hao (郝景盛). One of them, Yuangao Shi, joined the research team led by Prof. King to study the pharmacological effects of Chinese medicine and the food nutrition in northern china and later on became a famous medical scientist in China. In addition, to support the students’ studies, Prof. King donated the book Chinese Journal of Experimental Biology , which still has international influences and advances the development of Experimental Biology in China.Prof. King was also one of the founders of Chinese Zoological Society. Together with other 29 scientists including Profs. Ti-Chow Tung (童第周) and Tchou Su (朱洗), Prof. King signed to establish the Chinese Zoological Society. In August 1934, Chinese Zoological Society was officially established in Jiangxi province, aiming to promote the development of Zoology in China. Prof. King was the second director and the fourth vice president of the society. Two years later, Profs. King, Tsung-Lê Loo (罗宗洛), Shitsan Pai (贝时璋) and Ti-Chow Tung set up Prof. Li-Pin King is a great physiologist and made outstanding contributions to the development of physiology in China in terms of academic researches, cultivation of young talents, establishment of academic journals and research groups, especially in the areas of Chinese medicine pharmacology, experimental biology and modern medicine, etc. His rigorous research style, cooperative research spirit and collaborating research personality are worthy of appreciation and learning by scientific researchers.


Adults living with hearing loss have highly variable knowledge of hearing aids, resulting in suboptimal use or nonuse. This issue can be addressed by the provision of high-quality educational resources.This study aims to assess the everyday experiences of first-time hearing aid users when using a newly developed, theoretically informed cocreated mobile health  educational intervention called m2Hear. This intervention aims to deliver greater opportunities for individualization and interactivity compared with our previously developed multimedia intervention, C2Hear.A total of 16 first-time hearing aid users trialed m2Hear for a period of 10-weeks in their everyday lives, after which individual semistructured interviews were completed. The data were analyzed using an established deductive thematic analysis procedure underpinned by the Capability, Opportunity, Motivation-Behavior model. The model stipulates that to engage in a target behavior, an individual must have physical and psychological capability, physical and social opportunity, and automatic and reflective motivation.Capability—m2Hear was viewed as a concise and comprehensive resource, suitable for a range of digital literacy skills. It was stated that m2Hear could be conveniently reused to provide useful reminders that facilitate knowledge of hearing aids and communication. Opportunity—m2Hear was simple and straightforward to use, enabling greater individualization and independence. The availability of m2Hear via mobile technologies also improved accessibility. Motivation—m2Hear provided greater support and reassurance, improving confidence and empowering users to self-manage their hearing loss.Overall, this qualitative study suggests that m2Hear supports first-time hearing aid users to successfully self-manage their hearing loss postfitting. Furthermore, this study demonstrates the utility of employing a combined theoretical and ecologically valid approach in the development of mHealth educational resources to meet the individual self-management needs of adults living with hearing loss.ClinicalTrials.gov NCT03136718; https://clinicaltrials.gov/ct2/show/NCT03136718 Chronic health conditions, including hearing loss, are a leading cause of morbidity and mortality worldwide . EmpowerHowever, knowledge about hearing loss and hearing aids is often poor. First-time users, for instance, experience difficulties using their hearing aids because they struggle to remember all of the information given to them by their audiologist at the time of fitting ,8. Similchunks of multimedia learning that contain highly visual components, such as animations, video clips, and patient testimonials. The RLOs that we have developed cover a range of topics, prioritized by hearing health care professionals, and include both practical  and psychosocial  aspects of the adult aural rehabilitation process  available to them all the time are those who haven’t got a smartphone, a tablet or a computer. I would say they’re going to be non-existent, very shortly. I mean, even Granny’s got a smartphone now.M065The short mRLOs enabled participants to locate personally relevant information with ease:I think the value is that you can just go to the subjects you want. For bits that you're not interested in, well, what's the point of going to that? What you want is information, and to be able to go straight to that bit of information, I think, is a valuable part of [m2Hear].M064The succinct mRLOs also encouraged access and re-engagement, “They’re concise and logical, and that’s why I went back to them a couple of times” (M065). Furthermore, the mRLOs were a good use of time and effort: M062 said, “It meant best use of my time. I didn’t have to spend ages getting bored and wondering where it was, I could clearly see what it was I needed.” The concise content also held participants’ interest, which improved information retention:It’s better because you only have a certain amount of attention span, and I think because they are only like a minute, that’s about right…there’s so much more information in longer ones, whereas if it’s a little bit of information it makes it easier to take in.M067Taken together, the mRLOs were concise and subsequently viewed favorably.Although participants reported that they initially decided to view all mRLOs within the first week, they would regularly reuse m2Hear throughout the 10-week study period to improve their knowledge and understanding of specific topics. For example, participants consistently commented that they would rewatch the mRLOs for troubleshooting advice, particularly when they experienced a problem with their hearing aid or communication:I’d click on the “communicate with others” one, and “looking after my hearing aid”. I’d click on those two if I’d had a bad day about something, so I’d click on there just to have a look, and then make sure I was looking after them properly. Just to refresh what I should do with them.M067It was consistently reported that m2Hear supplemented and expanded upon the information provided by the audiologist during their hearing aid fitting appointment, as well as providing useful reminders of the information they had been given:Although the audiologist had probably given me all the right information, you don’t store it. You don’t remember it because there’s so much coming in…Because I’d got [m2Hear], I could go back, and I could go and check on something.M062Participants commented that it was often difficult to retain all the information provided during audiological appointments and that re-using m2Hear during the study period provided a means to refresh their memory, without feeling overwhelmed or confused:You can only take in so much at any one time…It’s nice to have a reference to go back to it, just to check it on your own…The information is there if you need it. That’s what a [m2Hear] is all about.M065In addition, the quizzes and interactive features also ensured that the content of the mRLOs were successfully retained and remembered, “Yes, it was like a little test to work on my memory. Even though I was watching the [mRLO] I was probably not watching it, whereas a quiz it was making me remember stuff” (M067). Nevertheless, some participants stated that they did not think it necessary for them to reuse m2Hear once they had viewed all the mRLOs, as they understood the information and were confident using their hearing aids:Well, in the first instance of course it was very good because it told me about the hearing aid. After a while, no, I don't think there's much more you need to know.M068One of the main reported advantages of m2Hear was that it enabled participants to self-manage their hearing loss. This was summarized by M060:The thing is that everyone is responsible for their own wellbeing. You shouldn’t rely on other people. For me, I don’t feel that I should be relying on other people. Other people are there in a desperate need, which is why [m2Hear] is good. As I keep saying, it’s a prop.Participants stated that managing their hearing loss independently was crucial to their self-esteem:I haven’t got to rely on anyone else. I haven’t got to ask someone else; I don’t like asking people…I’d rather just be able to do it myself...If I’m constantly asking people, I just feel helpless, and I don’t like that.M067In addition, participants were often reluctant to telephone and arrange an appointment with an audiologist due to limited health care resources and stated that m2Hear empowered them to be more self-sufficient:I think [m2Hear] is very useful. I mean, in audiology, they tell you things and that, but the limited time. Yes, it just seems a useful reference instead of having to keep phoning someone up or go back… You're straight there for as many times as you want it.M064m2Hear was not only relevant to people living with hearing loss, but also to frequent communication partners. Indeed, several participants stated that they shared m2Hear with friends and family members to improve their communication. For example, M060 said:I showed my wife…to make sure that she’s aware of the subtleties of not talking to me in a different room. That if she’s talking to me in a different room then we’re back to square one.The added value of m2Hear was that it provided an objective voice that was external to the participant’s immediate social network:[husband] was concerned.I think because it’s produced by somebody professionally, rather than me just giving my opinion or my interpretation…therefore that gave it more weight as far as he M062However, some participants preferred to use m2Hear alone, as they did not think it would be suitable or relevant for communication partners that do not have a hearing loss, such as M069, who said, “Well, I didn’t think it was particularly important, you know, really. They were…appropriate to me, but not to anybody else.”Although m2Hear facilitated self-management of hearing loss, there were occasions when it was considered necessary to seek help and advice from an audiologist. Changes in the overall hearing level, for instance, would prompt participants to arrange an audiological reassessment; “If my hearing changes I would ring audiology,” remarked M060. In addition, participants reported that they would arrange an appointment if their hearing aid was faulty or causing discomfort:I suppose if I was having specific problems with my hearing aids and thought there was something malfunctioning or something like that, then I would ring up the [audiology department] and speak to somebody.M062M069 also commented that they felt it was permissible to contact audiology if they experienced difficulties they were unable to resolve themselves using m2Hear: “I think if something goes wrong, you need advice about that type of thing, then…it’s nice to know that you’ve got somebody to talk to.” On this basis, m2Hear is a useful “tool” (M062) that can be used to supplement the provision of face-to-face appointments with an audiologist.Whether participants opted to access m2Hear from a handheld device  was based on immediacy and convenience. For example, M066 said, “I had a smartphone…that's what I use all the time…It's just easier to pick up and use.” Similarly, M069 commented:I’ve got a laptop, but I don’t use it very often. The tablet is much more convenient...it’s easier to handle. It’s smaller, and also, to be able to access the Internet, it’s almost immediate. Whereas, the laptop isn’t.Accessing m2Hear from smartphone technologies was advantageous because of portability: “It’s transferrable isn’t it, between devices…it’s portable, everywhere, any device. It makes it interesting” (M067). Furthermore, m2Hear could be used in multiple listening situations whenever desired. For example, comparing m2Hear with written information, M066 commented, “It's more convenient to use, wherever you are. You can just get your phone out, whereas you might not have the booklet with you.” Nevertheless, some participants preferred to use a laptop or desktop computer to access m2Hear, citing that a larger screen size was necessary to optimize visual acuity: “My eyesight is going as well. So, whilst the phone, I’ve got an iPhone, is great, I’m finding if I need to look at something that I feel I get a bigger picture” (M061). Taken together, while the device used to access m2Hear varied across participants, this was often based on personal preference and convenience. Nevertheless, smartphone technologies have improved accessibility.All participants commented that m2Hear was simple and straightforward to use, “It wasn’t loads of diving off into other areas and that, it was…nice and simple. There wasn’t anything complicated” (M067). It was also well-organized and structured, ensuring that information could be easily located: M062 said, “It’s very easy to get to the section you need to look at or you want to look at…you can see quite clearly which elements you need to go to. That was good.” In relation, the questions accompanying each individual mRLO facilitated navigation:They seemed relevant to the [mRLO] you'd just watched. I think the questions really weren't difficult questions. I presume the questions were designed in mind of, “Have you got the hang of what the [mRLO] was on about?” I think, yes, the questions tease that answer out quite well.M064Participants also reported that the organization of m2Hear enabled greater personalization:It means that you haven’t got to start right from the beginning again…I could just go straight to the one that I want. That’s all I want to go to, and it doesn’t hold you back, it just makes it easier to access it.M067For these reasons, participants reported that m2Hear was preferable to written information: M065 summarized, “In the written information you’ve got to do a whole lot of searching. [m2Hear] leads you by the nose, to be quite honest, and everything is logical about it, and it just takes a click.”Participants commented that the mRLO content was comprehensive, useful, and relevant, which they believed facilitated their knowledge of hearing aids and communication. For example, M060 said, “I can’t think of any topics that were actually left out...It’s got everything in there that I needed to know, and it’s there for me to look back on if I forget.” However, some participants stated that they would have liked additional information to have been included, such as the physiology of the ear and hearing loss, “I’d like a bit more technical depth to it...I’d like a little bit more depth about how the hearing system works” (M063). In addition to the mRLOs, optional quizzes and interactivities were also perceived to improve participants’ learning and understanding. For example, remarking on one of the activities, M060 said:It gives you a bit of food for thought, so you think about it again. It’s no good learning something by rote, as it were. It’s understanding it. So next time you come across that you’ve got an understanding.However, some stated that the quizzes were not beneficial, as they understood the content of the mRLO:In my case, I think I pretty much understood everything on the [mRLOs]. The quizzes for me were slightly irrelevant in terms of my understanding, my learning. I didn’t need them.M060Therefore, although some participants expressed ambivalence, most stated that they used m2Hear because they believed that both the mRLOs and interactive components would improve their knowledge of hearing aids and communication, resulting in more successful outcomes.Several participants reported that they regularly referred back to m2Hear as a means of support and reassurance that they were handling their hearing aids correctly:I think it's a kind of reassurance thing...You watch it and you think, “Well, yes, I am.” I think it's just useful as a reminder, having forgotten something or so on. Yes, I think it's a useful tool, isn't it? Yes, it's a good backup, I think.M064Subsequently, m2Hear improved both self-efficacy for hearing aids and coping with hearing loss:I can’t reinforce how useful I feel that [m2Hear] is. As I say, I've gone back to recap on different things...It’s just really given me the confidence...I feel that I can cope with any situation with my hearing aids now.M060In addition, m2Hear reduced feelings of loneliness and despair, with M060 saying:I think when I was looking at it I was not only soaking in the information and stuff, I was thinking, well, this is something that’s relevant to a lot of people...I'm not on my own, and I've got something to help me.Therefore, using m2Hear facilitated an optimistic outlook, empowering participants to self-manage their hearing loss.The most commonly cited reason participants used m2Hear was to improve their hearing aid handling and communication skills. m2Hear helped participants to manage their expectations and encouraged them to persevere using their hearing aids:You expect to be able to put [hearing aids] on like you put glasses on. I did think it would work in the same way. I thought it would be instant and it’s not. [m2Hear] is a useful reminder...this isn’t going to be straightforward and you’re going to have to work at it, but the benefits will be worthwhile.M062Moreover, m2Hear facilitated hearing aid use, especially when experiencing communication difficulties that might otherwise result in social withdrawal. For example, M067 commented that the mRLOs:made a difference, they helped me. I mean I wouldn’t have said to people, “I can’t hear you,” I probably would have just switched off, and sort of just not bothered probably. It did help me; it gave me a bit of a boot.As such, m2Hear had a positive impact on hearing aid use and adherence as well as communication during the 10-week evaluation.In relation to the themes identified, differences between the barriers and facilitators that impacted the use of m2Hear compared with those that impacted the use of C2Hear were also reported, whereby m2Hear was consistently viewed more positively.m2Hear was considered more appropriate for any level of digital literacy compared with C2Hear when accessed via YouTube. For example, M075 stated, “[m2Hear] is presented so well that I think most people, no matter how poor their understanding is, they’d still get on well with that.” In comparison, the C2Hear YouTube channel was viewed as more difficult to use if an individual had poor digital literacy skills. When using YouTube, M078 remarked:I was terrified in case I’d make a mistake and I ruin my computer, you see. Because I’m not very computer-savvy. So, I just looked atm2Hear. Conversely, participants commented that the C2Hear RLOs were lengthy in duration: “I lost interest in the [C2Hear RLOs]. I found them a bit distracting because sometimes they told you a bit too much” (M076). Nevertheless, the longer RLOs might be preferable for individuals who like to access all the information in one sitting:A key difference reported between m2Hear and C2Hear was the difference in length of the mRLOs. The shorter mRLOs were preferred, as participants felt that they could retain the information more easily. For example, M075 commented, “You can assimilate the knowledge more easily and you retain it perhaps better.” A further advantage of the shorter mRLOs was that it was easier to locate personally relevant information: “m2Hear was covering a topic which was concise and to the point, so you could jump straight to the information that you wanted” (M074)Sometimes the length of the [RLOs] could be nice. When you have got all the information, it’s not so bad to watch it all from beginning to end.M076m2Hear was more accessible than C2Hear, as it was more convenient to use with smartphone technologies. For example, M074 remarked:[m2Hear] was smarter on my iPhone to use, the interface was more workable to me on the iPhone, whereas the C2Hear...because it was on YouTube, it kept jumping up with other clips of other hearing research which was frustrating.A further benefit of m2Hear being easily accessed via smartphone technologies was that it could be used whenever and wherever required:It’s easier just to use [m2Hear] when you’re out and about and you just want to sit down and have a look at it and just go through it, just to remember things more, rather than wait till I get home and just look on the laptop.M076Conversely, participants reported that their use of C2Hear was restricted to when they had more time available due to the length of the RLOs: M076 said, “I used to do [C2Hear] when I’d got more time, so I could watch all of them, not dip in and out of it quite so much”. Taken together, convenience and portability improved the accessibility of m2Hear compared with C2Hear.An important difference between m2Hear and C2Hear was that m2Hear offered the opportunity for greater personalization. In addition, it was easier to find personally relevant information in m2Hear compared with C2Hear: “[m2Hear] is so easy, and it’s so user-friendly, and it’s so clear” (M075). C2Hear was less personalized and difficult to navigate. For example, M071 remarked, “In C2Hear you’ve got to go back, find various bits, and not quite start again, but it’s much more difficult to go back.” Nevertheless, C2Hear would have been acceptable if they had not been able to compare it to m2Hear: M071 stated, “If you hadn’t shown me [m2Hear], I’d probably have been perfectly happy with [C2Hear].” Therefore, although participants were satisfied with C2Hear, they preferred m2Hear overall..Participants consistently commented that m2Hear was more interactive than C2Hear, which reinforced the knowledge gained from each mRLO and would appeal to different learning preferences. For example, M076 remarked, “It’s a different way of learning, a different way of putting information across.” In comparison, C2Hear was considered less interactive, with some participants failing to notice that there was the opportunity to take quizzes at the end of each RLO: “I didn’t spot [the quizzes] on [C2Hear]. Probably because…the [RLO] went on too long” (M076)Compared with C2Hear, m2Hear improved confidence and enabled greater independent use of hearing aids. M074 commented:[m2Hear] increases my confidence for looking after my hearing aids myself without needing to go and get help...It gives me reassurance that I’m doing the right thing and that I’m not going to break them.Participants also reported that the m2Hear interactivities provided further reassurance: “They made you think and made you realise what there was and how easy it was to get the information out” (M075). Thus, in comparison to C2Hear, m2Hear improved self-efficacy for hearing aids and communication, facilitating greater self-management of hearing loss.The functionality of mHealth technologies has been shown to enable greater individualization and interactivity in multiple chronic health care domains, which has the potential to improve self-management ,24. In tWith regard to capability, digital literacy skills were identified as important for the usability and adherence of m2Hear. For m2Hear, participants commented that any level of skill would be sufficient in this area, given that it was relatively straightforward to use and navigate, whereas C2Hear accessed via YouTube required a high level of digital literacy. It is likely that the ease of use of m2Hear is attributable to the iterative, user-centered, and participatory design approach that was employed during the development of m2Hear ,26. SuchA further theme related to capability was that m2Hear was reused throughout the 10-week study period because it provided useful reminders that expanded upon the information provided by the audiologist during the hearing aid fitting appointment. This was further facilitated by the concise duration of the mRLOs, which enabled participants to easily locate and revisit the desired information with ease. In support of these findings, existing research in the area of multimedia learning recommends dividing content into shorter learning segments to reduce cognitive load (or memory capacity), improving knowledge acquisition and long-term retention . On thisOne of the most pertinent social factors identified in this study was whether participants shared m2Hear with their family and friends . Although some participants felt it necessary to share m2Hear with others to improve mutual communication, others did not, citing that their hearing loss was not a concern for others who did not experience hearing difficulties. This latter finding should be addressed, given that frequent communication partners play a pivotal role in hearing loss management and communication . Furthercommunication tactics C2Hear RLO into an mRLO suitable for communication partners [others, such as family members and the general public. Interactive components have also been incorporated, including simulated hearing losses in the presence and absence of background noise. This mRLO for communication partners and the general public is available on the web [Meeting the informational needs of communication partners has been shown to be highly beneficial ,47-49. Fpartners . Specifi the web . The qua the web . We foun the web .Another theme related to opportunity included environmental factors that promoted greater use of m2Hear such as convenience and portability. These findings reflect a key benefit of mHealth technologies, as they can be used when comfort and convenience are paramount . FurtherParticipants consistently reported that they were motivated to use m2Hear because it improved their knowledge of specific topics relating to hearing aids and communication. Supplemental interactivities further improved perceived learning and understanding, presumably through active engagement with learning materials ,55. Partcompetent users of digital technologies, which arguably limits the generalizability of the study findings to individuals with lower levels of self-perceived competency. Future studies could address this limitation by enhancing the representativeness of the sample in terms of digital literacy skills as well as other demographic and clinical characteristics such as age, gender, and hearing loss severity.There are several caveats to the design of this study that could be addressed in future research. For example, a purposive sampling strategy was employed, whereby participants were recruited based on prespecified inclusion and exclusion criteria, such as familiarity with smartphone technologies. This was necessary to ensure that participants would be able to use and access m2Hear throughout the home-based evaluation. As a result, it is perhaps unsurprising that most participants self-reported as A further consideration is that this study used a formalized, deductive (or theory-driven) thematic analysis approach, whereby themes were underpinned by the COM-B model and TDF. The application of theories and models from health psychology in audiological rehabilitation research continues to rise ,64-68. HAs we have argued from the outset, a key advantage of mHealth interventions is that they enable the individual to tailor the information they need as well as increase user interaction, resulting in a more patient-centered approach. Patient-centered care is widely accepted as a fundamental practice that supports an individual to be an active participant in the management of their health . CriticaThis qualitative study provides an in-depth assessment of an mHealth educational intervention used by first-time hearing aid users in their everyday lives. Underpinned by a contemporary model of health behavior change, the COM-B model, we identified key factors that influenced intervention use. Specifically, m2Hear was viewed as a concise and comprehensive resource that is simple and straightforward to use and enables greater individualization and independence. In addition, m2Hear provides greater support and reassurance, improves confidence, and empowers users to self-manage hearing loss. On this basis, this study suggests that m2Hear can be used to supplement existing aural rehabilitation practices to support successful self-management in first-time hearing aid users. Furthermore, this study demonstrates the utility of employing theoretical and ecologically valid approaches in the development of mHealth educational resources to meet the individual needs of the end user.


This Special Issue is focused on breakthrough developments in the field of assistive and rehabilitation robotics. The selected contributions include current scientific progress from biomedical signal processing and cover applications to myoelectric prostheses, lower-limb and upper-limb exoskeletons and assistive robotics. In recent years, the use of robotics to help motor-disabled people has experienced a significant growth, mostly based on the development and improvement of biosensor technology and the increasing interest in solving accessibility and rehabilitation limitations in a more natural and effective way. For this purpose, biomedical signal processing has been combined with robotic technology, such as exoskeletons or assistive robotic arms or hands. However, efforts are still needed to make these technologies affordable and useful for end users, as current biomedical devices are still mostly present in rehabilitation centers, hospitals and research facilities. This Special Issue covers several of the recent advances in robotic devices applied to motor rehabilitation and assistance.The Special Issue has collected eight outstanding papers covering different aspects of assistance robotics and biosensors. The selected contributions cover several main topics related to assistance robotics, from the control of myoelectric prostheses to the rehabilitation and assistance of the lower and upper limbs. What follows is a brief summary of the scope and main contributions of each of these papers, provided as a teaser for the interested reader.Upper-limb transradial amputation is cause of severe disability, and a key technology to solve this issue is the use of prosthetic devices for motor substitution. A particular way of controlling these devices is through electromyographic (EMG) signals. In , a low-cOther issues that affect classification accuracy are evaluated in ,3. An adThe rehabilitation of the lower limb has been addressed using a variety of devices, from exoskeletons to walkers. Lower-limb exoskeletons are mainly used in gait rehabilitation. HYBRID is an ambulatory robotic gait trainer with movement induction and partial weight support . This deIn , the autIn upper-limb rehabilitation, the physiological interaction of the patient with the robotic device is critical. In , two dif


AR amplification and expression of AR splice variants, demonstrating that AR remains a key therapeutic target in advanced disease. For the first time we show that IKBKE drives AR signalling in advanced PC. Significant inhibition of AR regulated gene expression was observed upon siRNA-mediated IKBKE depletion or pharmacological inhibition due to inhibited AR gene expression in multiple cell line models including a LNCaP derivative cell line resistant to the anti-androgen, enzalutamide (LNCaP-EnzR). Phenotypically, this resulted in significant inhibition of proliferation, migration and colony forming ability suggesting that targeting IKBKE could circumvent resistance to AR targeting therapies. Indeed, pharmacological inhibition in the CWR22Rv1 xenograft mouse model reduced tumour size and enhanced survival. Critically, this was validated in patient-derived explants where enzymatic inactivation of IKBKE reduced cell proliferation and AR expression. Mechanistically, we provide evidence that IKBKE regulates AR levels via Hippo pathway inhibition to reduce c-MYC levels at cis-regulatory elements within the AR gene. Thus, IKBKE is a therapeutic target in advanced PC suggesting repurposing of clinically tested IKBKE inhibitors could be beneficial to castrate resistant PC patients.Resistance to androgen receptor (AR) targeting therapeutics in prostate cancer (PC) is a significant clinical problem. Mechanisms by which this is accomplished include  AR gene amplification and through AR splice variants (The androgen receptor (AR) is a key molecule in the development and progression of prostate cancer (PC) and as such is a critical therapeutic target. Current androgen-deprivation therapy (ADT) is initially effective at reducing AR signalling and PC progression, but most patients inevitably become resistant to these treatments via multiple mechanisms including variants . TherefoAR activity is regulated by numerous post-translational modifications (PTM) which suggests that targeting AR modifying enzymes which enhance AR activity may provide therapeutic benefit when direct AR targeting therapies have failed; particularly as a number of these coregulatory proteins are themselves often dysregulated in PC . The besIKBKE  is a non-canonical I-kappa-B kinase which can be activated by numerous stimuli including TNFα and IL1. It plays a role in numerous signalling pathways, for example it has been shown to phosphorylate CYLD, which in turn activates the NF-κB pathway via deubiquitination of several NF-κB regulator proteins . IKBKE cde novo synthesis in models of PC. Targeting IKBKE with small molecule inhibitors in both PC cell line xenografts and patient ex vivo explant models resulted in reduced tumour volume, inhibition of proliferation and reduced AR expression. Collectively, our data suggest that IKBKE is a viable therapeutic target for the treatment of PC. Interestingly, pharmacological inhibitors of IKBKE are used in treatment of asthma, allergic rhinitis and aphthous ulcers , PSA , IKBKE , α-tubulin , LATS2 , YAP , c-MYC , TMPRSS2 , PARP1/2 , FKBP5 , GFP  Ki67 .All compounds were purchased in powder form and resuspended in DMSO to a concentration of 10 mM unless otherwise stated. CAY10576 (Santa Cruz Biotechnology), MRT67307 (Stratech Scientific), BX795 (Cambridge BioScience), CYT387 (Adooq Biosciences and Cambridge BioScience), Ruxolitinib (Cambridge BioScience) and R1881 (Sigma) were stored at −80°C for no more than 6 months. Enzalutamide (Selleckchem) was prepared at a concentration of 30 mM and stored at −80°C for no more than 6 months. MG-132 (Sigma) was purchased as a 10 mM stock in DMSO and stored at −80°C.l-glutamine (Sigma) at 37°C in 5% CO2 atmosphere. LNCaP-AI , supplemented with 1 nM R1881. VCaP cells were maintained in High Glucose DMEM-6171 media (Sigma) containing 10% FCS and 2 mM l-glutamine.VCaP, CWR22Rv1 , PC3 andLNCaP-AI  were mail-glutamine for 72 h, prior to the addition of 1 nM R1881 for indicated lengths of time.For androgen stimulation assays, cells were grown in RPMI-1640 media (Gibco) supplemented with 10% dextran coated charcoal stripped serum FCS and 2 mM Cell line authentication was performed by short tandem repeat profiling  and mycoplasma testing was performed routinely using MycoAlert kit .Cell lines were reverse transfected with individual siRNAs (25 nM) using Lipofectamine RNAiMAX (Invitrogen) according to manufacturer's instructions (for siRNA target sequences see LNCaP cells were cultured as previously described and treated with DMSO or IKBKE inhibitors CAY10576 (5 μM) or CYT387 (5 μM) for 16 h. For knockdown experiments, cells were reverse transfected as detailed above and cells collected after 72 h knockdown. Nuclear-cytoplasmic fractionation was performed using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific) according to the manufacturer's protocol and analysed by western blotting.LNCaP and LNCaP-Enz cells were reverse transfected with 25 nM N/S or IKBKE siRNA and incubated for 72 h. Cells were then reseeded and incubated for 21 days to allow colony formation. Cells were then fixed and stained with crystal violet. Colonies were counted and the surviving fraction calculated.LNCaP cells were either reverse transfected with 25 nM N/S or IKBKE siRNA and incubated for 72 h or pre-treated with CAY10576 for 24 h prior to seeding into migration chambers in basal media and inserted into wells containing serum supplemented media as a chemoattractant. Cells were incubated for 16 h, then inner chambers scrubbed to remove cells which did not migrate through the pores of the membrane. Cells which had migrated to the other side of the membrane were then fixed in ice cold methanol and stained with haematoxylin. Cells were then imaged using Aperio CS2 Digital Pathology scanner (Leica) and counted.Western analysis was performed as previously described .15 (Promega) and 0.4 mM dNTPs (Bioline) per reaction. Quantitative reverse transcriptase–polymerase chain reaction (qPCR) was performed in triplicate for genes of interest using Platinum® SYBR® green qPCR SuperMix-UDG with ROX (Invitrogen) in 384-well clear optical reaction plates using the QuantStudio 7 Flex real-time PCR system (Applied Biosystems) according to the manufacturer's protocol. Data were normalized against the house keeping gene hypoxanthine phosphoribosyltransferase 1 (hprt1). Primers sequences are described in RNA was isolated using TRIzol (Invitrogen) according to the manufacturer's protocol. RNA was quantified using Nanodrop spectrophotometer, and 200 ng of RNA was reverse transcribed using 1× Moloney Murine Leukemia Virus (MMLV) buffer (Promega), 60 U MMLV reverse transcriptase, 0.5 μM oligo d(T)et al. (Chromatin immunoprecipitation (ChIP) assays were performed in LNCaP and LNCaP-AR-GFP cells reverse transfected with either 25 nM N/S or IKBKE siRNA for 72 h, according to the protocol described by Schmidt et al. . Brieflyet al.  purifiedCell growth was assessed using the IncuCyte ZOOM live cell imager (Essen) according to the manufacturer's recommendations. In each case, cells were treated and grown in their respective normal growth media, unless otherwise stated. For knockdown experiments, cells were reverse transfected with 25 nM siRNA using RNAiMax according to the manufacturer's protocol. For compound assays, cells were seeded and allowed to adhere for 24 h, prior to the addition of compound. Images were taken every 6 h for at least three doubling times. The IncuCyte ZOOM 2015A software package was trained to identify cells from each cell line and measure the % confluence of each well. Cell confluence was normalized for each well at the 0 time point and the relative change in cell confluence was calculated for each time point thereafter. Data were presented as the mean relative cell confluence at each time point from at least three independent experiments.LNCaP cells were reverse transfected with 25 nM siRNA using RNAiMax according to the manufacturer's protocol and grown for 96 h. Cells were collected in 100 μl citrate buffer . DNA staining and lysis buffer (20 μg/ml propidium iodide (Sigma), 0.5% NP-40 , 250 μg/ml RNaseA (Qiagen) 0.5 mM ethylenediaminetetraacetic acid (EDTA), in PBS) was prepared and 400 μl added to the cell suspension and incubated for 40 mins in the dark at 4°C. Using FACScan together with CellQuest software (Beckton Dickenson) 10 000 events were collected and analysed using Cyflogic software (CyFlo Ltd). The percentage of cells in each cell cycle phase was determined and data presented as the mean % of cells from three independent experiments +/− SEM. Student's t-test was used to determine statistical significance between the experimental conditions.6 per mouse) were implanted subcutaneously in 0.2 ml Matrigel into CD1 nude mice . Once palpable tumours were detected at an average volume of 100 mm3, CYT387 was administered via daily oral gavage at a dose of 100 mg/kg or vehicle control. Tumour volume was measured using callipers according to the equation a2 × b/2, . Median relative tumour volume (RTV) was calculated, where RTV1 = tumour volume on day 1 of treatment for each mouse. Time to RTV4, defined as the time it takes for tumours to reach four times the initial volume, was calculated as described previously  and performed according to ethical guidelines set out by Workman et al. under a UK home office licence in accordance with the Animals (Scientific Procedures) Act 1986  or channel transurethral resection of the prostate (chTURP).Ex vivo explant cultures were established as described by Centenera et al.  in a 24-well plate containing 500 μl RPMI-1640 HEPES modification medium (Sigma-Aldrich) with 10% foetal calf serum and 1% L-Glutamine supplemented with 1× antibiotic anti-mycotic solution (Sigma-Aldrich), 0.01 mg/ml hydrocortisone (Sigma-Aldrich), 0.01 mg/ml bovine insulin solution (Sigma-Aldrich) and synthetic androgen 1 nM R1881. Treated explants were cultured in ex vivo medium containing CYT387 (10 μM). Control explants were treated with a matched volume of DMSO. Explants were cultured at 37°C with 5% CO2 for 48 or 72 h before being formalin-fixed and paraffin embedded. Patient information detailed in a et al. . CollectFour μm-thick sections of formalin-fixed paraffin-embedded (FFPE) explant tissue were stained for the proliferative marker Ki67. Automated immunohistochemistry (IHC) was carried out on a Discovery XT staining module (Roche). Antigen was retrieved by incubating slides in Cell Conditioning Solution (Roche). Endogenous peroxidase activity was reduced by incubating slides in Inhibitor CM (Roche). Slides were incubated in Ki67 primary antibody  or Anit-AR primary antibody  for 1 h at 37°C followed by HRP-conjugated Discovery OmniMaP HRP secondary antibody (Roche) for 30 min. A ChromoMap DAB kit (Roche) was used for detection. Slides were counterstained in hematoxylin and mounted in DPX. Stained slides were imaged using an Aperio CS2 Digital Pathology scanner (Leica). Regions of interest  were manually annotated and the percentage of Ki67 positive cells was determined using Aperio Image Analysis software (Leica) in a minimum of four representative 40× magnification regions containing a total of at least 1000 nuclei. Positive control tissue for Ki67 is shown in t-test was used for data sets containing two experimental arms and one-way ANOVA with Greenhouse-Geisser correction and Dunnett's multiple comparison test was performed where there were three or more experimental arms to the experiment. Two-way ANOVA with Dunnett's multiple comparison test was performed on data with two variables. Tests were undertaken in GraphPad Prism 6 software.The numbers of independent experimental repeats are detailed within figure legends. Where data is expressed as mean ± SEM of three or more independent experiments, statistical analysis was performed to indicate significance. Specifically, Student's The NHS Health Research Authority NRES Committee North East, Newcastle and North Tyneside, has provided approval for human tissue studies in the laboratory of Dr. McCracken (REC reference: 15/NE/0400).in vivo experiments were approved by the local animal welfare ethics review body (AWERB) and the Home Office in accordance with the Animals (Scientific Procedures) Act 1989.The PSA) and transmembrane serine protease 2 (TMPRSS2) in the presence or absence of exogenous androgen (R1881) stimulation  was performed, in which each kinase in the human genome was individually depleted in androgen sensitive LNCaP-7B7  cells inn Figure , B. Addin Figure . Consists Figure . Due to n Figure . Taken tAs IKBKE modulates the expression of genes which can be driven by both full length AR and AR-Vs we questioned whether IKBKE could alter AR levels as a mechanism underlying these observations. Firstly, we examined the effects of IKBKE knockdown using both a pool of three siRNAs and individual siRNAs in LNCaP cells growing in serum containing media. After 72 h, western blotting confirmed IKBKE knockdown, and PSA expression was inhibited but further demonstrated that total AR protein levels were found to be robustly reduced Figure . LNCaP-APSA enhancer (P < 0.001)  Figure . TogetheP < 0.05) with the PSA promoter-enhancer region comparable with control IgG background levels  compared to prostate adenocarcinoma  and normal prostate  . FurtherPSA and AR mRNA levels , controls the stability and nuclear translocation of the transcriptional co-activator Yes-associated protein (YAP) . The Hipoduction ,35. TherYAP mRNA levels increased 5-fold (±SEM 1.64) and 4 fold (±SEM 0.96) with two independent siRNA sequences, whilst c-MYC mRNA was significantly reduced by >50% in response to significant IKBKE knockdown , confirming that IKBKE is required for normal PC cell growth   Figure  which wa) Figure . Furtherd Figure ,C. Colond Figure . A simild Figure  and CWR2d Figure  cells. Md Figure  indicatid Figure . The datin vivo was tested using the Phase III, dual IKBKE/JAK2 antagonist, CYT387, on CWR22Rv1 tumour xenograft growth. This inhibitor was favoured over CAY10576 as it has superior chemical properties for in vivo studies. As this inhibitor also has JAK2 activity we first compared the growth of CWR22Rv1 cells in vitro, in the presence of either CYT387 or a specific JAK2 inhibitor, Ruxolitinib. This showed that JAK2 inhibition does not inhibit the growth of CWR22Rv1 cells (3), CYT387 was administered via daily oral gavage at a dose of 100 mg/kg. Compared with a vehicle control, CYT387 treatment at this dose effectively inhibited tumour growth (Figure The efficacy of IKBKE pharmacological antagonism v1 cells  hence anh Figure  and signh Figure , with noh Figure . We nexth Figure . Furtherh Figure . Taken tThe AR remains a key therapeutic target in advanced, anti-androgen resistant disease. As an alternative means to reduce AR activity when anti-androgens have failed, co-regulator proteins which control the activity of the AR can be targeted. Indeed, many of these co-regulators are dysregulated in advanced disease . IdeallyHere, we demonstrate that IKBKE plays a role in the production of AR mRNA and as such results in enhanced AR regulated gene expression. As IKBKE mediated AR regulation occurs at the level of transcription, all AR mRNA species are affected including those which result in the production of constitutively active splice variant forms for which there are currently no targeted therapeutics available. The kinase activity of IKBKE appears to be important in regulating AR mRNA production therefore, we hypothesize that inhibition of IKBKE may be therapeutically beneficial to CRPC patients who frequently retain expression of functional AR and AR-Vs. Indeed, we show that knockdown and inhibition of IKBKE can reduce proliferation, migratory ability and tumour volume supporting our hypothesis that IKBKE inhibition is a viable clinical strategy. Furthermore, it has been reported that high cytoplasmic IKBKE expression associates with the development of bone metastases and castration resistance  consisteCurrently, two compounds are available in the clinic that target IKBKE, each displaying minimal side effects; Amlexanox and CYT387. Amlexanox, an IKBKE/TBK1 inhibitor developed to treat ulcers, allergic rhinitis and asthma, results in a G0/G1 arrest in glioblastoma cells and induction of apoptosis . We alsoIn order to determine the mechanism by which cytosolic IKBKE positively regulates AR mRNA we investigated transcription factors that have been shown to regulate the expression of AR and which functionally interact with IKBKE. Firstly, we ruled out NF-κB inactivation as p65 is cytoplasmic in LNCaP derivative cell lines. Secondly, c-MYC was investigated as a mediator between IKBKE and AR expression. Interestingly, we observed a decrease in c-MYC protein levels in response to IKBKE knockdown or pharmacological inhibition which resulted in decreased c-MYC binding to the AR gene. We confirmed that c-MYC down-regulation was at the transcriptional level and not due to increased c-MYC turnover. This contrasts with observations in pancreatic ductal adenocarcinoma cell lines where IKBKE mediated phosphorylation of Akt and subsequent inhibition of GSK3β regulates the nuclear retention and stabilization of c-MYC without impacting on c-MYC mRNA . HoweverNumerous studies describe c-MYC as transcriptionally regulated by YAP, a component of the Hippo pathway ,43,44. Ade novo protein synthesis. Upon knockdown of IKBKE in LNCaP cells engineered to constitutively over express a GFP-tagged AR driven by a CMV promoter, no alterations in AR-GFP protein levels were observed, however PSA levels were robustly depleted. Furthermore, AR-GFP recruitment to the PSA enhancer was significantly reduced. One potential mechanism whereby IKBKE regulates AR protein activity may be via IKBKE-mediated AKT phosphorylation at Thr308/ Ser473 sites. Activated pAKT can directly phosphorylate AR to promote activation (Whilst IKBKE regulation of AR mRNA production appears to be the primary mechanism of control, we have evidence that IKBKE affects AR transcriptional activity independently of tivation  or indirtivation . The IKBtivation ,47. FurtWith links established between IKBKE gene amplification and AR independent neuroendocrine PCs and our data regarding IKBKE knockdown and PC3 cellular proliferation we acknowledge that there is a potential for AR independent effects. Indeed, IKBKE expression in PC3 cells is much higher than in LNCaP cells. Interestingly, upon knockdown of c-MYC in PC3 cells a robust inhibition of cellular proliferation is observed which would explain why IKBKE knockdown, which decreases c-MYC levels, has this effect. Whilst this requires further investigation, there is also scope to investigate the utility of IKBKE targeting therapeutics in highly aggressive AR negative tumours.In summary, this study demonstrates that IKBKE plays a role in AR signalling. Small molecule inhibitors of IKBKE kinase function diminishes AR signalling by simultaneous downregulation of AR mRNA and transcriptional activity. This downregulation is a result of IKBKE crosstalk with the Hippo pathway resulting in LATS2 upregulation, which in turn downregulates YAP by phosphorylation-mediated nuclear exclusion and degradation, to deplete its transcriptional target c-MYC which is important in the production of AR mRNA Figure . The inhgkaa271_Supplemental_FileClick here for additional data file.


KLF5, in turn, reduced AR transcriptional activity and inhibited androgen-induced cell proliferation and tumor growth in vitro and in vivo. Mechanistically, KLF5 occupied the promoter of AR, and silencing KLF5 repressed AR transcription. In addition, KLF5 and AR physically interacted with each other to regulate the expression of multiple genes  to promote cell proliferation. These findings indicate that, while transcriptionally upregulated by AR signaling, KLF5 also regulates the expression and transcriptional activity of AR in androgen-sensitive prostate cancer cells. The KLF5-AR interaction could provide a therapeutic opportunity for the treatment of prostate cancer.Androgen/androgen receptor (AR) signaling drives both the normal prostate development and prostatic carcinogenesis, and patients with advanced prostate cancer often develop resistance to androgen deprivation therapy. The transcription factor Krüppel-like factor 5 (KLF5) also regulates both normal and cancerous development of the prostate. In this study, we tested whether and how KLF5 plays a role in the function of AR signaling in prostate cancer cells. We found that KLF5 is upregulated by androgen depending on AR in LNCaP and C4-2B cells. Silencing  PTEN [TMPRSS2 and ERG [MYC, and inactivation of P53 and RB [AR, and functional alterations leading to androgen independence [Prostate cancer (PCa) is prevalent among older men; and is one of the common causes of cancer-related death in men. While genetic and epigenetic alterations of multiple genes, including loss of PTEN ,2,3, fus and ERG ,5, ampli3 and RB ,7,8, and3 and RB ,10. AR ipendence ,12,13.AR is a member of the nuclear steroid receptor superfamily that is predominantly activated by testosterone and di-hydrotestosterone ,15. AR sCCND1 and MYC [Klf5 promotes Pten loss-induced prostate tumors, and the Klf5-/-/Pten-/- tumors also have increased basal to luminal differentiation [KLF5 is a basic transcription factor that belongs to the Krüppel-like factor (KLF) family. It regulates a variety of biological processes including cell proliferation, apoptosis, angiogenesis, stemness and the epithelial-mesenchymal transition (EMT) ,18. KLF5 and MYC ,27,28. I and MYC ,30. Andr and MYC , and botntiation . KLF5 in PCa cells [KLF5 inhibited cell proliferation and tumor growth of PCa cells. In addition, as a transcription factor, KLF5 occupied the promoter of AR to promote its transcription; and KLF5 was also required for AR’s transcriptional activity. Furthermore, KLF5 and AR interacted with each other to regulate transcription of AR target genes  to promote cell proliferation and tumor growth. These findings suggest that specific targeting of the AR-KLF5 interaction could be a potential therapeutic strategy for disrupting androgen signaling in PCa treatment.Taken together with the facts that androgen/AR signaling is the driving force in both normal prostate development and regeneration and PCa development, both KLF5 and AR are transcription factors, and androgen appears to induce the expression of Ca cells ,33, we pPSA, TMPRSS2, and FKBP5 at the mRNA level  treated with varying concentrations of R1881, a synthetic androgen, specifically binds to AR with higher affinity than dihydrotesterone (DHT), and R1881-bound AR dimerizes and translocates to the nucleus to interact with coregulators to regulate gene transcription ,35. AndrNA level b. TreatmNA level e,f. PSA, TMPRSS2, and FKBP5) as expected  or enzalutamide (10 µM) and analyzed KLF5 expression. AR silencing by siRNA, which was confirmed by western blotting m, eliminTMPRSS2 and FKBP5 [To determine whether androgen-upregulated KLF5 has a functional role in androgen/AR signaling, we evaluated whether knockdown of KLF5 affects AR’s transcriptional activity in LNCaP cells with KLF5 silencing by siRNA and C4-2B cells with KLF5 silencing by shRNA. Cells were cultured in regular media, which contains hormones as regular FBS was used. Enzalutamide treatment was applied to inhibit AR signaling activity. KLF5 silencing clearly decreased the expression of PSA, a classic transcriptional target of AR , at bothnd FKBP5 ,42, was PSA and MMTV [PSA and MMTV promoters were significantly decreased by KLF5 knockdown, and enzalutamide treatment eliminated both the promoter activities and the effect of KLF5 silencing on promoter activities  with pSG5-AR or FLAG-tagged AR (Flag-AR) with HA-tagged KLF5 (HA-KLF5) into HEK293T cells, and performed IP with anti-Flag antibody. Western blotting detected AR in the KLF5 precipitate a and KLFWe also divided KLF5 into two fragments, one with residues 1–200 and the other with 201–453, and tested which fragment mediates KLF5′s association with AR using the same approaches as in We also performed IP with anti-KLF5 or anti-AR antibody to pull down their respective protein complexes in C4-2B cells. Western blotting detected AR in the KLF5 complex and KLF5 in the AR complex e,f, indiMYC and CCND1, and KLF5 has also been shown to upregulate the same two genes in epithelial cells [MYC and CCND1 in androgen-responsive PCa cells. In LNCaP and C4-2B cells cultured in normal medium, knockdown of KLF5 decreased the expression MYC and cyclin D1 at both protein . Consistent with colony and sphere formation results, KLF5 silencing reduced tumor growth, as indicated by tumor images and tumor weights at excision e,f. EnzaIn the tumor xenografts, IHC staining demonstrated enzalutamide treatment reduced the expression of both KLF5 and AR, which is consistent with in vitro findings g–l, and KLF5 silencing decreased the expression of PSA, a classic transcriptional target gene of AR in the prostate [TMPRSS2 and FKBP5, two other AR target genes [PSA and MMTV [MYC and CCND1 [KLF5 silencing   and immune responses. For example, pro-inflammatory TNFα and lipopolysaccharide (LPS) induce KLF5 expression, and TNFα depends on KLF5 to induce MCP-1 . In addiKLF5 locus often undergoes hemizygous deletion [KLF5 expression because the gene is haploinsufficient [During late stages of tumor progression, AR becomes activated even when androgen levels are low, causing CRPC ,10. Furtdeletion ,55, whicfficient . As KLF52. During experiments, cells recovered from a liquid nitrogen freezer were used within two months (<20 passages) with no noticeable morphological changes. All cell lines were authenticated by STR profiling before experiments were started. For all experiments involving R1881  treatments, the medium was replaced with phenol red-free medium containing 10% charcoal-stripped FBS. Human PCa cell line LNCaP was purchased from American Type Cell Culture  and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum . The C4-2B cell line, originally derived from a bone metastasis of a LNCaP clone in mice , was kinKLF5, which was developed and validated in a previous study with various PCa cell lines [KLF5 is stably knocked down. For RNAi with siRNAs, siRNA  oligos against human KLF5 and AR were synthesized by Sagon Biotech  and transfected into cells using the Lipofectamine RNAiMAX reagent  according to the manufacturer’s protocol.For RNA interference (RNAi) with shRNA, C4-2B cells were infected with lentiviruses expressing an shRNA specifically targeting human ll lines , to estaCultured cells were lysed in lysis buffer . Cell lysates were subjected to polyacrylamide gel electrophoresis (PAGE), and proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane. Membranes were soaked in 5% nonfat milk or 5% BSA solution for one hour to block nonspecific binding of proteins, and then incubated with primary antibodies overnight at 4 °C. On the following day, membranes were incubated with secondary antibodies for 2 h at room temperature, and WesternBright ECL  was used with the luminescent image analyzer  to capture images. Uncropped scans can be found in Antibodies used in western blotting were: KLF5 , AR , PSA , MYC , cyclin D1 , FLAG , HA , GAPDH .GAPDH gene served as an internal control. The comparative 2-△△Ct method was used to calculate gene expression levels. Each sample was analyzed in triplicate. Primer sequences for real-time qPCR are listed in Total RNA was extracted from cells using the Trizol reagent (Invitrogen) and 2 μg total RNA reverse-transcribed using the PrimeScript™ RT reagent Kit with gDNA Eraser . Real-time qPCR was performed with the SYBR Green MasterMix reagent (Takara) using the Mastercycler Realplex real time PCR system . Human Mammalian expression plasmids for Flag-KLF5, HA-KLF5, pSG5-AR, Flag-AR, and luciferase reporter plasmids for pGL3-KLF5, pGL3-PSA, and pGL3-MMTV were generated in our laboratory. For promoter-luciferase plasmids, pGL3-AR, pGL3-AR1, and pGL3-AR2, primers were respectively designed with the entire 2-Kb immediate AR promoter sequence (AR), the upper 1.5-Kb (AR1) and the lower 0.5-Kb (AR2). PCR and cloning of PCR products were used to generate luciferase reporter plasmids for AR, AR1, and AR2 in pGL3-Basic vector following standard procedures. Primer sequences for gene cloning are listed in LNCaP and C4-2B cells were transiently transfected with pGL3-Basic, pGL3-KLF5, or pGL3-PSA, or pGL3-MMTV and pRL-TK  as an internal control. After 48 h of transfection and enzalutamide  treatment, cells were lysed in 5 × lysis buffer (Promega) for 30 min and luciferase activity was measured using a luminometer . Firefly luciferase activity was normalized to Renilla luciferase activities in each reaction. Experiments were performed in triplicate.One thousand C4-2B cells/well or 2000 LNCaP cells/well were seeded in 6-well plates and cultured in the presence of RPMI-1640 medium containing 10% FBS with DMSO or 10 μM enzalutamide . The plates were incubated for 2 (C4-2B) or 3 (LNCaP) weeks, after which cultures were fixed with 4% paraformaldehyde for 30 min and stained by 0.05% crystal violet  for 1 h at room temperature and then photographed. In a single experiment, assays were conducted in triplicate and then as three independent experiments.In 8-well chamber slides , 30 µL growth factor reduced BD Matrigel  was added per well. Slides were placed in a cell culture incubator for at least 15 min to solidify the Matrigel. Next, 2000 C4-2B cells/well or 4000 LNCaP cells/well were seeded in RPMI-1640 medium containing 10% FBS with DMSO or 10 μM enzalutamide (Beyotime Biotechnology) and 2% Matrigel. Media were replenished every 3 days. Chamber slides were incubated for 2 (C4-2B) or 3 (LNCaP) weeks, and then photographed. Image J program  was used to measure the diameter of each sphere. Spheres with a diameter larger than 80 μm were counted.For exogenous immunoprecipitation, HEK293T cells were cotransfected with pcDNA3.0-Flag or Flag-KLF5 in the same vector with pSG5-AR plasmid using the Lipofectamine 2000 Transfection Reagent (Invitrogen) according to the manufacturer’s protocol. After 48 h, the cells were collected and resuspended in cell lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 2 mM EDTA, 0.8% NP-40, 0.2% Triton X-100, and 3% glycerol. EDTA-free Protease Inhibitor Cocktail  and PMSF (Sangon Biotech) were added to the cell lysis buffer. Cell lysates were incubated with anti-FLAG-agarose beads at 4 °C for 2 h (Sigma). Beads were washed and eluted and supernatants analyzed by western blotting. For endogenous immunoprecipitation, C4-2B cells or mouse prostate were collected and resuspended in lysis buffer and then rabbit or mouse normal IgG, KLF5 antibody (self-made) or AR antibody  added to the lysate, followed by overnight incubation at 4 °C. Lysates coupled with antibody were incubated with magnetic Dynabeads (Invitrogen) for 2 h. Beads were washed extensively and eluted and supernatants analyzed by western blotting as above.7) cells in 100 µL PBS-Matrigel (1:1)  were implanted via subcutaneous injection into the flanks of mice. Cells were left to grow for one week. Mice were randomly divided into control or enzalutamide (10 mg/kg)  [C4-2B (2×10Y-70002)  groups, 2O2 for 10 min, tissue sections were blocked with 10% normal goat serum, incubated with primary antibodies at 4 °C overnight, and then with EnVision PolymerHRP secondary antibodies  at room temperature for 30 min. After chromogenic reaction using DAB (MXB Biotechnologies), nuclei were stained with hematoxylin (MXB Biotechnologies). Finally, tissue sections were dehydrated and mounted.IHC staining was performed to detect protein expression of KLF5 , AR , Ki67 , cyclin D1  and MYC  in tumor xenografts. Formalin-fixed paraffin embedded tissues were sectioned at 4 μm, deparaffinized in xylene, rehydrated in graded ethanol (100–75%) and repaired antigen by boiling the slides in a citrate buffer  for 3 min using a pressure cooker. After treatment with 3% HC4-2B cells were grown for 3 days in RPMI-1640 medium supplemented with 10% FBS. ChIP was performed using the Simple ChIP Enzymatic Chromatin IP Kit  according to the manufacturer’s instructions. Firstly, cells were cross-linked with 1% formaldehyde for 10 min and quenched with glycine at room temperature. Samples were collected and digested using micrococcal nuclease for 20 min at 37 °C; reactions were stopped by the addition of 0.5 M EDTA and incubated in ChIP buffer with protease inhibitors on ice for 10 min. After sonication, chromatin extracts were immunoprecipitated using anti-AR , anti-KLF5  or anti-IgG antibody. ChIP products were detected by regular PCR (with input loading quantity of one fourth of IgG or AR) or real-time qPCR (each gene in triplicate). Sequences of PCR primers are described in p < 0.05 was considered statistically significant.All in vitro experiments were repeated at least three times. All numerical results are expressed as mean ± SD. Two group comparisons were compared using Student’s t test by SPSS 21 . A value of In summary, we demonstrated that KLF5 is crucial for androgen/AR signaling to activate the transcription of specific genes, including some that mediate cell proliferation, and to promote cell proliferation and tumor growth in PCa cells. Mechanistically, androgen promotes the expression of KLF5 via AR, KLF5 in turn promotes the transcription of AR by binding to AR promoter, and KLF5 and AR coordinate to transactivate the target genes of AR. These findings not only suggest that KLF5 is a crucial factor for the function of AR in PCa cells, they will also further the understanding of how AR signaling is sustained in CRPC. In addition, they will likely facilitate the development of therapeutic strategies for the treatment of CRPC.