TM) and by analysis of RNA-Seq data from patients with mCRPC (SU2C- PCF Dream Team). We next quantify AR-V expression in matching EpCAM-positive vs EpCAM-negative CTCs, as EpCAM-based CTC enrichment is commonly used. We identify lower AR-V prevalence in the EpCAM-positive fraction, suggesting that EpCAM-based CTC enrichment likely underestimates AR-V prevalence. Lastly, using single CTC analysis we identify enrichment for AR-v567es in patients with neuroendocrine prostate cancer (NEPC) indicating that AR-v567es may be involved in lineage plasticity, which warrants further mechanistic interrogation.Quantitation of androgen receptor variant (AR-V) expression in circulating tumor cells (CTCs) from patients with metastatic castration-resistant prostate cancer (mCRPC) has great potential for treatment customization. However, the absence of a uniform CTC isolation platform and consensus on an analytical assay has prevented the incorporation of these measurements in routine clinical practice. Here, we present a single-CTC sensitive digital droplet PCR (ddPCR) assay for the quantitation of the two most common AR-Vs, AR-V7, and AR-v567es, using antigen agnostic CTC enrichment. In a cohort of 29 mCRPC patients, we identify AR-V7 in 66% and AR-v567es in 52% of patients. These results are corroborated using another gene expression platform  expression levels in single circulating tumor cells (CTCs) from prostate cancer patients. They show that current methods for isolating CTCs tend to underestimate the prevalence of AR-V and that a specific variant, AR-v567es, could be potentially used as a biomarker for an aggressive subtype of prostate cancer. Hence, AR-pathway directed therapy is the first line of treatment for patients with advanced prostate cancer4. If not received in the front-line setting, upon progression to the castration-resistant state (CRPC), second-generation potent androgen receptor pathway inhibitors (ARPi) such as abiraterone, enzalutamide, and apalutamide, targeting androgen bio-synthesis and AR-ligand binding, respectively, are routinely used to further inhibit AR-driven cancer progression.Prostate cancer is the most commonly diagnosed cancer in men and the second leading cause of male cancer death in the United States. Active androgen receptor (AR) signaling is central to prostate cancer development and progression6. Two of the most investigated AR splice variants are AR-V7 and AR-v567es, which are transcriptionally active in the absence of androgens. Clinically, AR-V7 mRNA expression, measured in circulating tumor cells (CTCs) of patients with mCRPC, has been associated with enzalutamide and abiraterone resistance7.Nevertheless, most of the tumors eventually develop ARPi resistance due in some patients to aberrant activation of AR signaling including expression of AR splice variants (AR-Vs) which lack the ligand-binding domain and are constitutively active in the nucleus8.While the clinical impact of AR-V7 expression has been actively being investigated, the role of AR-v567es in treatment response and disease progression is not well elucidated. An earlier study reported that AR-v567es was expressed in 23% of mCRPC bone metastases and associated with a high nuclear AR immunostaining score, and shorter overall survival9 implicating both variants in taxane resistance. As both variants are co-expressed in patient samples where bulk tumor or the entire CTC fraction is used, the relative impact of each variant alone on disease progression and treatment response has not been yet determined. Furthermore, although AR-V7 mRNA and protein detection assays have been CLIA approved10, testing for AR-V7 has yet to be fully incorporated in routine clinical decision making, due to the lack of consensus on a unified CTC enrichment method and analytical assay. Currently, both the FDA-cleared CTC capture (CellSearch®) and the CLIA approved CTC enrichment (AdnaTest) platforms, rely on epithelial cell adhesion molecule (EpCAM)-based capture12. However, as EpCAM can be downregulated during epithelial-to-mesenchymal transition (EMT), a biological process that precedes metastatic dissemination, EpCAM-based CTC enrichment may not capture the heterogeneous pool of CTCs13.In addition, our recent results from a prospective multi-institutional clinical trial of patients with mCRPC receiving taxane chemotherapy, showed that AR-V7 and AR-v567es expression in patient CTCs, was associated with lower biochemical response rates and shorter progression free survial (PFS)TM and RNA-Seq, each performed in independent, larger cohorts of patients. Single CTC analyses from patients with mCRPC and neuroendocrine prostate cancer (NEPC) revealed significant enrichment in AR-v567es expression in NEPC, which was confirmed in NEPC organoids, heretofore unrecognized.Herein, we report the analytical specificity and sensitivity of the AR-V ddPCR assay and its ability to detect each transcript in single CTCs. The prevalence of AR-FL, AR-V7, and AR-v567es transcripts observed by ddPCR in CTCs from patients with mCRPC, was corroborated by different gene expression platforms, NanoString14 , AR-V7, and AR-v567es in CTCs isolated from the peripheral blood of patients with prostate cancer. To specifically amplify each transcript of interest, we designed primers and probes to span exon to exon junctions in order to avoid co-amplification of other variants with high sequence similarity, such as the sequence similarity observed between AR-V7 and AR-V914 Fig. . The AR-14 Fig. . In addi14 Fig. .The analytical sensitivity of the assay for each transcript is demonstrated in Fig. Next, we evaluated the performance of the AR-V ddPCR assay in CTCs and matching PBMCs isolated from six patients with mCRPC as well as in PBMCs isolated from 10 male healthy donors. Fig. . We obseTo determine the prevalence of each AR transcript in mCRPC, we next performed the AR-V ddPCR assay in CTCs isolated from 29 patients with mCRPC, following CD45 negative depletion. The clinical characteristics of this patient cohort are shown in Table TM, in three different large cohorts of patients. First we mined the RNA-Seq data available from the TCGA15 consisting of 505 primary prostate cancer samples and from the SU2C cohort consisting of 98 mCRPC patient samples17. After obtaining the raw data from RNA-Seq or NanoStringTM, we determined the expression of each transcript, by counting mapped reads across exon 7 and exon 8 junction for AR-FL; across exon 3 and cryptic exon 3 junction for AR-V7; and across exon 4 and exon 8 junction for AR-v567es  expression for CTC enrichment11. The prevalence of AR-V7 mRNA expression in mCRPC reported by the AdnaTest is lower than what we observed in both the mCRPC patient CTCs and in the SU2C cohort. This discrepancy could be partially explained by the reported EpCAM downregulation during metastatic progression20, which would limit CTC capture to the EpCAMpos-only subtypes.The advantage of using CTCs is that they provide a non-invasive source of tumor cells enabling real-time interrogation in longitudinal fashion that can be used in clinical decision making. For AR-V7, an antibody-based protein expression platform, namely the Oncotype DX AR-V7 Nucleus Detect® test is CLIA approvedTM micromanipulator. We subjected these two pools of EpCAMpos and EpCAMneg CTCs to AR-V ddPCR  EpCAMneg CTCs and only in 6/10 (60%) EpcAMpos CTCs while ARv567es was expressed in 6/10 (60%) EpCAMneg CTCs and only in 2/10 the EpcAMpos CTCs . Taken together these results indicate that CTC subpopulations have distinct AR-V expression patterns, and that antigen agnostic CTC enrichment might provide information on a more comprehensive pool of CTCs which would be missed by antigen-specific enrichment methods.To address this concern, we enriched CTCs by CD45 negative depletion from the peripheral blood of 10 patients with mCRPC and further isolated pools of CTCs based on EpCAM expression using the CellCelectorPCR Fig. . We obseTCs Fig. . Taken tpos vs EpCAMneg CTCs and given that intra-tumoral heterogeneity is implicated in disease progression and treatment resistance, we investigated the intra-patient heterogeneity analyzing AR-V expression in single CTCs. We isolated a total of 159 single CTCs from three patients with mCRPC and subjected them to AR-V ddPCR. Each transcript alone was assessed in single CTCs from each patient. AR-FL was detected in 14/53 CTCs (26%), AR-V7 in 7/53 (13%) and AR-v567es in 6/53 (11%). While these results suggest that AR-FL is the most predominant transcript overall, when we analyzed the data on a per patient-basis we observed inter- and intra-patient heterogeneity in the proportion of single CTCs expressing each transcript, in agreement with a previous report on single CTC RNA-Seq from prostate cancer patients21. For example, patient A had 11 CTCs expressing AR-FL, 4 CTCs expressing AR-V7, and 2 CTCs expressing AR-v567es; making AR-FL the predominant transcript. In contrast, patient B had 1 CTC expressing AR-FL, 0 CTCs expressing AR-V7, and 3 CTCs expressing AR-v567es, which was the predominant transcript in this patient. Patient C had 2 CTCs expressing AR-FL, 3 CTCs expressing AR-V7, and 1 CTC positive for AR-v567es making AR-V7 the predominant transcript . To expand on this interesting observation, we quantified AR-V expression in organoids derived from patients with NEPC, as previously described22. This analysis identified AR-v567es as the predominant transcript, expressed in three of the four NEPC organoids (75%) , AR-V7 in 0/17 (0%) and AR-v567es in 10/17 (59%) Fig. . Compari5%) Fig. .18. Currently, two of the most investigated AR splice variants are AR-V7 and AR-v567es, which are transcriptionally active in the absence of androgens. However, their clinical significance and impact on treatment decision have yet to be established. In addition, AR-V detection rates in patients with mCRPC vary widely, as do the assays used, the patient cohorts, and the source of tumor material encompassing tumor biopsies, whole blood, exosomes, and CTCs.Active AR signaling is central to prostate cancer development and progression; aberrant activation of AR signaling including expression of AR splice variants (AR-V) mediates clinical resistance to androgen deprivation therapy and to second-generation potent ARPis such as abiraterone and enzalutamide9. Currently, there are several assays developed to detect AR-V7 mRNA by qRT-PCR26 or ddPCR30, using liquid biopsies such as CTCs, ctRNA, exosomes, or whole blood. Interestingly, AR-V7 mRNA detection has shown higher sensitivity when CTCs are used as input, compared to other types of liquid biopsy31. In our AR-V assay, we chose to quantify AR-FL and AR-V expression using the ddPCR platform because it has been shown to have increased precision and sensitivity in detecting low template copies33 in a complex biological background without the need for normalization, calibrator, or external references36.AR-V7 has been the most extensively investigated due to its reported association with ARPi and taxane resistance30 to 95%29, even among the reports using ddPCR. In our study using CTCs from patients with mCRPC we detected AR-V7 expression in 66% of patients. This result is consistent with our earlier study where AR-V7 expression was determined in a different cohort of patients with mCRPC, using the same assay but a different CTC isolation platform (PSMA+ selection)9. In agreement with these results, two other studies using ddPCR for AR-V7 quantitation in CTCs from patients with mCRPC reported 50% and 53% AR-V7 detection rates37. With regards to the other ddPCR-based assays, the liquid biopsy input was either whole blood or exosomal RNA isolated from peripheral blood, and as such they cannot be directly compared to ours30.Interestingly, there is a wide range of AR-V7 detection rates, from 1814. Some of the other assays, developed before the discovery of AR-V9, have primers and probes showing the partial overlap between AR-V7 and AR-V937. The extent to which potential co-amplification of AR-V9 contributes to the discrepancy in AR-V7 detection rates remains to be determined.Notably, the primers used in our assay were designed to specifically detect AR-V7 avoiding co-amplification of the structurally similar AR-V9, which share a common nucleotide sequence in the 3′ terminal cryptic exon26. Hornberg et al. were the first to assess the expression of AR-v567es in bone metastases of mCRPC patients and reported AR-v567es expression in 23% of the cases. Liu et al. used whole blood from patients with mCRPC and reported an AR-v567es detection rate of 32%. Our assay is the first to use ddPCR with high specificity for AR-v567es detection in CTCs. We found AR-v567es expression in 52% of patients with mCRPC, albeit the expression level was much lower than that of AR-V7 or AR-FL  and mCRPC (SU2C- PCF Dream Team). Our results showed that AR-V7 was expressed in 29% of patients with primary prostate cancer (TCGA) and in 78% of patients with mCRPC (SU2C- PCF Dream Team) Fig. . These rles Fig. . Similarpos and EpCAMneg mCRPC CTCs showed a higher prevalence of both AR-V7 and AR-v567es in the EpCAMneg CTC subpopulation  supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin (penicillin/streptomycin) in a 5% CO2 incubator at 37 °C. VCaP cells were cultured in DMEM (Corning) with 10% FBS and penicillin/streptomycin in a 5% CO2 incubator at 37 °C.22Rv1 (Cat # CRL-2505) and VCaP (Cat # CRL-2876) cells were obtained from ATCC. ATCC authenticates human cancer cell lines using short tandem repeat analysis. CWR-R1-D567 cells were kindly gifted to us from Dr. Scott Dehm (University of Minnesota)pEGFP-C1-AR-FL, pEGFP-C1-AR-V7, and pEGFP-C2-AR-567es plasmid DNA were used to exogenously express each transcript as positive controls for the development of the multiplex ddPCR assay. Six nanograms of each plasmid was transfected in AR null HEK293T cells using FuGENE® 6 Transfection Reagent according to the manufacturer’s instructions . Post 24 h transfection total RNA was isolated using RNeasy Mini Kit , cDNA was generated from 1 μg of total RNA using ProtoScript First Strand cDNA Synthesis  and cDNA samples were used for evaluating the specificity of the methodology.Each primer set was designed to recognize unique and distinguishable regions of AR-FL and AR variants using Primer3Plus based on the direction of the ddPCR Application Guide Bulletin 6407 (Bio-Rad). AR-FL assay recognizes unique junction between exon 7 and 8, AR-v7 assay recognizes the junction of exon 3 and cryptic exon 3, and AR-v567es assay recognizes the junction between exon 4 and exon 8. The primers were designed such at least one primer from a pair spans on the exon-exon junction to avoid unspecific amplification from other variants and synthesize from genomic DNA. The specificity of the primer design was assessed by a Nucleotide BLAST (blastn) search against the up-to-date version of the human genome reference (hg38) database and IGV (Integrative Genomics Viewer). The primers and probes were designed and purchased from Bio-Rad shown in Table 42. After PCR, droplets that contained a template will have a fluorescent signal (positive droplets) that distinguishes them from the droplets without a template (negative droplets). All positive droplets were used to determine each transcript’s expression values30.Droplet Digital PCR (ddPCR) is a method of absolute nucleic acid quantification based on the partitioning of a qPCR reaction sample into tens of thousands of nano-reactions (droplets) of defined volumeAR-FL, AR-V7, and AR-v567es transcript quantifications were carried out on a QX200 Droplet Digital PCR (ddPCR) system with automated droplet generation (Bio-Rad Laboratories). Reactions were carried out in ddPCR Plates 96-Well, Semi-Skirted (by Eppendorf). Each well contained 5.5 μl of ddPCR Supermix for Probes, 2.2 μl Reverse Transcriptase and 1.1 μl of 300 mM DTT , 1.1 μl of target-specific primers, and 11 μl of sample RNA, for a total volume of 22 μl. Immediately after droplet generation, 96-well plates containing droplet-partitioned samples were heat-sealed and PCR was carried out on a C1000 Touch Thermal Cycler (Bio-Rad) using the following cycling protocol: reverse transcription at 42 C for 60 min, enzyme activation at 95 °C for 10 min followed by 40 cycles of 95 °C for 30 s (for denaturation) and 60 °C for 60 s , followed by a final 10 min incubation at 98 °C for enzyme deactivation. Included on each plate were the following positive and negative “plate controls”: no template control (NTC), HEK 293 cells transfected with either AR-FL, AR-V7, or AR-v567es. Purified nuclease-free water was used as the negative, NTC. Transcript copy numbers for the HEK 293 cells transfected with each AR transcript or NTC can be found in Table To assess the intra-assay variability (repeatability) and inter-assay (reproducibility) of our ddPCR assay, five experimental replicates were performed with 10 ng of RNA extracted from HEK293T cells either non-transfected or transfected with plasmids encoding each respective transcript, AR-FL, ARV7, and AR-v567es. The intra-assay variability was carried out by using the same batch of RNA, pre-mix from the One Step kit, and cartridges, with samples from each repeat randomly positioned on the 96-well PCR plate. The inter-assay variability was evaluated by analyzing the same batch of RNA processed on 5 different days, with 5 different kit reagents. The standard deviation (SD) in copies/μl was calculated for each transcript. The intra- and inter-assay coefficient of variance (CV) was calculated by standard deviation/mean.+ cells , according to the manufacturer’s instructions. We also isolated matched total peripheral blood mononuclear cells (PBMCs) from 1 ml of each corresponding blood sample, by Ficoll-PaqueTM PLUS  density gradient centrifugation. Total RNA was extracted from the enriched CTCs and matching PBMCs using the RNAeasy Plus Micro kit (Qiagen) as per the manufacturer’s instructions. Samples were then processed for ddPCR analysis as described above.Up to 20 mL of peripheral blood were collected in EDTA tubes (BD Vacutainer) from patients with mCRPC, NEPC, and healthy male subjects under Weill Cornell Institutional Review Board (IRB) approval. Each blood sample was processed within 24 h of blood draw. CTCs were enriched from the peripheral blood by depletion of CD4522. RNA was extracted from each of the NEPC organoids, and the same RNA input was used for each transcript. In addition, each sample was normalized to a previously validated reference gene, GUSB as an internal loading control in the HEX channel.The generation and characterization of tumor organoids derived from needle biopsies of metastatic lesions from four NEPC patients was previously described at Puca et. al.pos/CD45neg cells were isolated as single or as pools of up to 50 cells by the robotic arm of the instrument and processed for ddPCR. To identify EpCAMneg/CD45neg cells as bona-fide CTCs, visual inspection was performed based on established criteria including larger size, greater nuclear to cytoplasmic ratio, as well as distinct nuclear morphology43. For single VCaP, 22Rv1 or CWR-R1-D567 analysis, cells were plated on glass-bottom microwell dishes, and individual cells were marked under bright field before microcapillary mediated mechanical suction  was utilized to automatically image, enumerate and select CTC based on immune phenotype. Briefly, after CD45 depletion, the enriched CTCs were stained live for the following cell surface antigens: EpCAM  followed by anti-rabbit Alexa 568 and CD45 . Cells were subsequently plated on glass-bottom microwell dishes (Mat Tek Corporation) for automated scan and live imaging. CTCs were visualized under the microscope and EpCAMion Fig. . Effectiion Fig. . Single For the Cancer Genome Atlas (TCGA) and Stand Up to Cancer (SU2C) datasets, we obtained raw sequencing read data from 505 and 98 patient samples respectively, trimmed them using Trimmomatic to eliminate low quality reads and aligned to human reference genome (version hg38) using STAR. AR-FL expression was determined based on mapped reads across the junction between exon 7 and exon 8 of the AR gene. AR-V7 expression was determined based on mapped reads across the junction between exon 3 and cryptic exon 3. AR-v567es expression was determined based on mapped reads across the junction between exon 4 and exon 8.TM data obtained from patients at our Institution with benign prostate tissue (n = 49), primary prostate cancer (n = 89), and CRPC (n = 39). The clinical characteristics of this patient cohort have been previously published in Table 39. The targeted gene panel developed using the NanoStringTM nCounter was previously described in ref. 39.We assessed AR- FL, AR-V7, and ARv567es mRNA expression by analyzing NanoStringP ≤ 0.05.Statistical analysis was conducted using Prism 8 (GraphPad). Specific statistical tests and parameters are indicated in figure legends. Significance was set at Further information on research design is available in the Transparent Peer Review FileSupplementary InformationDescription of Supplementary FilesSupplementary Data 1Reporting Summary


The caries preventive effect of Colgate Duraphat® and GC Tooth Mousse Plus® has been widely studied, but the remineralisation potential of initial occlusal caries using these two remineralisation materials remains unclear.  This study is aimed at evaluating and comparing the remineralisation of early enamel caries on the occlusal surface of permanent posterior teeth using ICDAS II caries scoring system and DIAGNOdent Pen (DDPen) after remineralisation with Colgate Duraphat® and GC Tooth Mousse Plus®. N = 120) with incipient occlusal caries were included in this study. The occlusal surface of each tooth was scored using DDPen and ICDAS II scoring before remineralisation. Then, remineralisation of the teeth of the experimental group was carried out using either CPP-ACP-F or fluoride varnish. After the remineralisation procedures, the occlusal surface of each tooth was again scored using DDPen and ICDAS II scoring. The teeth were then fixed in dental stone blocks and sectioned longitudinally for histological examination using a stereomicroscope. Statistical analysis was performed to calculate the sensitivity and specificity of DDPen and ICDAS II to detect remineralisation and compare with the gold standard histological examination.  Extracted posterior teeth  revealed a higher association of DDPen score (.738) as compared to ICDAS-II scores (.430).  The study concluded that both ICDAS II and DDPen could detect remineralisation of early enamel occlusal caries. DDPen was more sensitive than ICDAS-II to detect remineralisation compared with the Downers histological scores. Dental caries is a gradually progressive disease that has been identified as the most commonly occurring oral disease/chronic infection , 2. The Several studies have reported that Duraphat® is highly effective in remineralising enamel from other mineralisation products –9. FurthICDAS II supplies a consistent method of lesion detection and evaluation, leading to the diagnosis of caries . ICDAS INarrative and systematic reviews have shown that DDpen is more sensitive to the occlusal aspect of posterior teeth than traditional diagnostic methods, but the specificity is inferior to clinical visual examination –25. DDPeDifferent cut-off values for DDPen have been described in the literature; however, no study has evaluated pre-and post-remineralisation relevance of cut-off values. Moreover, no previous studies have been carried out performing simultaneous comparison between the three assessment tools  on tooth surface remineralisation. Hence, this study is aimed at investigating the potential of ICDAS II and DDPen in detecting remineralisation of the early carious lesion after applying topical fluoride gel (Colgate Duraphat®) and casein phosphopeptide-amorphous calcium phosphate fluoride (Tooth Mouse Plus) at the occlusal surface of extracted human permanent posterior teeth. This study also compared the diagnostic cut-off values of DDPen corresponding with the histological examination after the remineralisation process.Ethical approval for the current study was obtained from the Human Research Ethics Committee of Universiti Sains Malaysia (USM) (USM/JEPeM/18100516). Extracted permanent maxillary and mandibular posterior teeth for orthodontic, periodontal, or other reasons from adult patients that visited the outpatient dental clinics of the School of Dental Sciences, USM, were selected for this study.A total of 120 freshly extracted teeth were included in this study. Before caries assessment, the occlusal surfaces were cleaned using a toothbrush with pumice slurry and water before randomly assigning numbers from 1 to 120. Unrestored mandibular or maxillary posterior teeth without any carious lesion or incipient carious lesion limited to the enamel, denoted as code 01, 02, and 03 according to the ICDAS II scoring system, were included. The teeth samples were examined under 10x magnification individually. Teeth were excluded from the study if there was the presence of dental fluorosis, tetracycline staining or any sources of staining, hypoplasia, and dentinal exposure.Before the remineralisation process, two trained and calibrated examiners (T.Y.N and M.S.H) performed the visual assessment of each sample using ICDAS II. Visual examination was using a dental operating light, a WHO probe, and a 3-way syringe to score each tooth according to the ICDAS II scoring system. Sharp explorers were not used during the visual diagnosis. TYN and MSH scored the teeth individually with no discussion regarding the scores to ensure blinding. Interexaminer reproducibility was assessed using kappa analysis. The third examiner (M.F.R) used the DDPen to score each sample afterwards. A triple air syringe was used for five seconds to dry out the enamel surface before using the DDPen. Before scoring, the device was calibrated according to manufacturer's instructions. The probe tip was positioned on the occlusal surface of the tooth and rotated around its vertical axis until the highest value was found. ICDAS II scores and DDPen readings were repeated for two weeks for all samples to ascertain and evaluate intraexaminer reproducibility.For the control group, the extracted teeth samples were washed with deionised water and then placed in artificial saliva at 37°C. No material was used for remineralisation of the control group. For the CPP-ACP with fluoride (GC Tooth Mousse Plus®) group, the paste was applied once daily (every 24 h) for 21 days on the occlusal surfaces with a microbrush tip applicator left in place for three minutes which simulated at-home use of this cream. The specimens were then rinsed with deionised water and gently dried with an air syringe. The samples were then immersed in artificial saliva and incubated at 37°C . This prAfter the remineralisation process, MFR repeats the scoring using the DDPen post-remineralisation for each sample. To ensure blinding, the examiner did not have access to previous scores of ICDAS or DDPen, and the same procedure for performing the post-remineralisation ICDAS score was followed. The teeth were then fixed in dental stone blocks and sectioned longitudinally using a diamond-coated bandsaw (Exakt system) for histological examination using a stereomicroscope with 10x magnification. Parallel cutting of the samples was performed by using tweezer prongs. The parallelism of the block is of utmost importance to ensure accurate cutting direction. The teeth sections fixed in the blocks were then polished by using slurry pumice. The polished sections were then again examined at 10x magnification under the stereomicroscope. Each section was photographed using a digital camera. This gold standard histological examination was compared with the ICDAS II scoring and DDPen examination for validity assessment.The descriptive statistical analysis was performed using SPSS software version 24 (IBM SPSS Statistics). Inter- and intraexaminer reproducibility was assessed by calculating the unweighted kappa coefficient . WilcoxoKappa statistics revealed almost perfect reproducibility of TYN (0.954), MSH (0.919), and MFR (0.885), whereas good interexaminer agreement (0.673) was observed .  Table Az value of DDPen score (0-13) at D1 (0.972) was higher than ICDAS-II code 0 at D1 (0.811), showing the greater accuracy of the method. Rank correlations (Spearman's coefficient) with histology D1 were .692 for ICDAS-II code 0 and .950 for DDPen score (0-13). Az value of DDPen score (14-20) at D2 (0.894) was higher than ICDAS-II codes 1 and 2 at D2 (0.667), showing the greater accuracy of the method. Rank correlations (Spearman's coefficient) with histology D2 were .769 for ICDAS-II codes 1 and 2 and .854 for DDPen score (14-20). Az value of DDPen score (>21) at D3 (0.838) was higher than ICDAS-II code 3 at D3 (0.721), showing the greater accuracy of the method. Rank correlations (Spearman's coefficient) with histology D3 were .601 for ICDAS-II code 3 and .774 for DDPen score (>21). The current study is aimed at evaluating the sensitivity and specificity of ICDAS II and DDPen to detect the remineralisation of early enamel caries lesion in extracted human permanent posterior teeth and compare with the gold standard histological examination to identify the most suitable cut-off values of DDPen for identification of remineralised carious lesions.The current study utilised ICDAS-II and DDPen scoring, which are designed to enable the clinicians to diagnose the disease at the chairside, making the results of the current study more relatable to a clinical setup. The current study did not find a significant difference in the remineralisation potential of GC Tooth Mousse Plus® and Duraphat®, following previous literature . However∗) with Downer's histological scores for the corresponding study samples, agree with the cut-off value suggested by Lussi et al. [The cut-off values for DDPen, which had the highest and a significant correlation , D2 (0.81), and D3 (0.93), whereas another clinical study by Heinrich-Weltzien et al. [Az values for D2 (0.90) and D3 (0.83). The current study reported the highest Az value of DDPen score (0-13) at D1 (0.972) followed by DDPen score (14-20) at D2 (0.894) and then DDPen score (>21) at D3 (0.838). The difference in the performance of DDPen can be attributed to the determination of cut-off values or the number of cases reported in each study. Our study included the most significant number of cases having carious lesions at the D1 level, which could better represent higher accuracy in that category.An advantage of the area under the ROC curve analysis is that it reflects the diagnostic performance more comprehensively than the sensitivity and specificity, determined by only one cut-off point. It also provides an overall validity of the methods , 43. Dinn et al.  reportedIranzo-Cortés et al.  conducteOn the other hand, a higher specificity is desirable to ensure minimal false-positive cases, which was found true in the case of ICDAS II. A possible explanation of this finding is the subjective nature of ICDAS criteria, which provides clinicians with a specific demarcation of disease progression. Similarly, Akgul et al.  performeThe spearman rank correlation coefficient was calculated for different lesion depths based on Downer's histological classification. The rank score of DDPen ranged from 0.774 to 0.950 compared to the ICDAS-II rank score ranging from 0.601 to 0.769. However, these results revealed the potential of ICDAS-II and DDPen to detect remineralisation. Another notable finding is that the most significant difficulty in diagnosing carious lesions was observed in deeper carious lesions with the lowest accuracy. The results revealed that DDPen could be utilised as a valuable clinical tool to detect remineralisation and monitor the progress of remineralisation of a particular lesion.Az value did not present a statistically significant difference, meaning that the diagnosis and decision-making were not influenced in the three study groups. In comparison, our study focused on carious lesions and found that DDPen was significantly (rank correlation = 0.738) more effective in diagnosing cavitated carious lesions when compared with ICDAS-II (rank correlation = 0.430), which warrants the use of DDPen in cavitated lesions as an adjunct. The higher accuracy of DDPen and electronic caries monitor has also been previously reported [Alomari et al.  carried reported . A possireported  also fouA change in either pH or the presence of free radical ions can create a desired anticariogenic environment, as previously noted in the literature , 49. AnoThe ICDAS-II scoring system and DDPen scoring were able to detect remineralisation. A cut-off value of 0-13 denotes sound tooth, 14-20 denotes enamel caries, and >21 denotes dentine caries. The DDPen scoring had a higher-ranked correlation with Downer's histological scoring as compared to ICDAS-II scoring. The area under the ROC curve revealed that DDPen scoring demonstrated higher sensitivity while ICDAS-II scoring demonstrated greater specificity to detect remineralisation.


IGF2, Gli1, Myc and SOX9), inhibiting cell viability, growth, proliferation, migration and invasion. Significant apoptosis activation was detected in Lnc-THOR-silenced/-KO NSCLC cells. Conversely, ectopic overexpression of Lnc-THOR upregulated IGF2BP1 mRNA targets and enhanced NSCLC cell proliferation, migration and invasion. RNA-immunoprecipitation and RNA pull-down assay results confirmed the direct binding between Lnc-THOR and IGF2BP1 protein in NSCLC cells. Lnc-THOR silencing and overexpression were ineffective in IGF2BP1-KO NSCLC cells. Forced IGF2BP1 overexpression failed to rescue Lnc-THOR-KO NSCLC cells. In vivo, intratumoral injection of Lnc-THOR shRNA adeno-associated virus potently inhibited A549 xenograft tumor growth in nude mice. At last we show that Lnc-THOR is overexpressed in multiple NSCLC tissues and established/primary NSCLC cells. Collectively, these results highlighted the ability of Lnc-THOR in promoting NSCLC cell growth by associating with IGF2BP1, suggesting that Lnc-THOR represents a promising therapeutic target of NSCLC.Long non-coding RNA (LncRNA) THOR (Lnc-THOR) is expressed in testis and multiple human malignancies. Lnc-THOR association with IGF2BP1 (IGF2 mRNA-binding protein 1) is essential for stabilization and transcription of IGF2BP1 targeted mRNAs. We tested its expression and potential functions in non-small cell lung cancer (NSCLC). In primary NSCLC cells and established cell lines, Lnc-THOR shRNA or CRISPR/Cas9-mediated knockout (KO) downregulated IGF2BP1 target mRNAs ( Lung cancer is one major cause of human mortalities around the world , 2. Overvia sponging miR-193a, thereby promoting NSCLC cell growth (Long non-coding RNAs (LncRNAs) are a large family of conserved single strand RNA molecules over 200-nt long –10. LncRGli1 (glioma-associated oncogene homolog 1), Myc, IGF2 and SOX9 (Insulin-like growth factor 2 (IGF2) mRNA-binding protein 1 (IGF2BP1) belongs to the IGF2BP RNA-binding family proteins , requireand SOX9 . Recent and SOX9 . It is eand SOX9 . Lnc-THOand SOX9 , essentiand SOX9 , 25. Lncand SOX9 –31. We hAll the methods applied in this study were carried out according to the ethics guidelines of Zhengzhou University.The cleaved caspase antibody sampler kit (#9929), IGF2BP1, (#8482) and β-Tubulin (#2146) were purchased from Cell Signaling Technologies . Cell culture reagents were obtained from Hyclone Co. . Puromycin, polybrene and other chemicals were purchased from Sigma-Aldrich .Established NSCLC cell lines, A549 and H1299, were from Dr. Li at Wenzhou Medical University . Cells wA set of 10 pairs of NSCLC tumor tissues and the corresponding adjacent normal lung tissues (over two cm away from the boundary of tumor tissue) were obtained from primary NSCLC patients with tumor resection. The patients were administrated at the First affiliated Hospital of Zhengzhou University and received no preoperative treatments. Human testis tissues were from a patient with post-traumatic orchiectomy. The written informed consent was obtained each patient. Human tissues were stored in liquid nitrogen immediately after resection. The protocols of using human tissues were approved by the Ethics Committee of Zhengzhou University, in according to the principles of Declaration of Helsinki.GAPDH was tested as the reference gene, with 2−ΔΔCt method applied for data quantification. The mRNA primers were from Dr. Pan at Shanghai Jiao Tong University . The retrieved RNA was reversely transcribed into cDNA by an SuperScript™ II Reverse Transcriptase Kit (Invitrogen). qRT-PCR was performed by using SYBR Premix Ex Taq™  under the ABI Prism 7900 Fast Real-Time PCR system . iversity .Equal amounts of total protein lysates (40 μg per lane) were resolved by sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) electrophoresis and transferred to polyvinylidene difluoride (PVDF) blots. The blots were blocked and immuno-blotted with the primary antibodies overnight, following by incubation with the corresponding secondary antibodies. An enhanced chemiluminescence (ECL) detection kit  was applied to detect targeted protein bands. The ImageJ software from NIH was utilized for data quantification.5 cells/well into six-well plates (in polybrene containing complete medium) and were infected with the lentivirus (MOI=20). After 24h, puromycin (2.5 μg/mL) was added to select stable cells for 3-4 passages. Lnc-THOR knockdown in stable cells was verified by qRT-PCR assays. Control cells were infected lentiviral particles with scramble control shRNA (“shC”).The lentiviral particles, encoding shRNAs against non-overlapping sequence of Lnc-THOR (“sh-S1/sh-S2”), were provided by Dr. Pan at Shanghai Jiao Tong University . NSCLC c5 cells per well) and were transfected with a LentiCas9-puro construct (Genechem). Stable Cas9 NSCLC cells were established after puromycin selection. Cells were then transfected with a pSpCas9(BB)-2A (PX458) plasmid encoding sgRNA against Lnc-THOR  and wer5 cells per well) and were infected with the adenovirus for 48h. Stable cells were established by puromycin selection and IGF2BP1 overexpression verified by Western blotting and qRT-PCR assays.The recombinant adenovirus encoding IGF2BP1-expressing pSUPER-puro construct was from Dr. Zhao at Soochow University . NSCLC c6 cells per mouse subcutaneously (s.c.)]. Nude mice were monitored every day, xenograft tumor weights and volumes were measured with a sliding caliper, and tumor volumes calculated using the formula (L×W2)/2. When the tumor volume was close to 100 mm3 (“Day-0”), mice were subject to intratumoral injection of Lnc-THOR shRNA (“sh-S1”) adeno-associated virus (AAV) or the scramble control shRNA (“shC”) AAV. All mice were sacrificed at the end of the experiments and the tumors were harvested. All animal studies were performed according to the standards of IACUC of Zhengzhou University, with the protocols approved by the Ethics Committee of Zhengzhou University.The nude mice, half male half female, aged 5-6 weeks, 18.5-19.0g in weights, were randomly divided into two groups, and were inoculated with A549 cells [at 6 ×10P < 0.05 was considered to indicate a significant difference. In vitro experiments were repeated at least three times, with similar results obtained.All values were presented as mean ± standard deviation (SD). Statistical comparisons were performed by Student’s t-test (Excel 2007) between two groups or one-way ANOVA plus a Scheffe’ and Tukey Test (SPSS 23.0) for multiple comparisons. via puromycin, stable cells were established. Alternatively, a Cas9-Lnc-THOR-KO construct [also from Dr. Pan , were tested. The lentiviral particles encoding Lnc-THOR shRNA (“sh-S1”) were added to the NSCLC cells. We next analyzed the potential effect of Lnc-THOR depletion on NSCLC cell apoptosis , 34. As Further experimental results found that Lnc-THOR shRNA or KO induced mitochondrial depolarization in pCan-1 cells, evidenced by accumulation of JC-1 green monomer (In pCan-2 and pCan-3 primary cells as well as in established cell lines (A549 and H1299), Lnc-THOR silencing by sh-S1 shRNA see Figure 1IGF2, Gli1, Myc and SOX9, were significantly increased after Lnc-THOR overexpression , mice were randomly assigned into two groups (with 10 mice per group). The xenograft-bearing mice were subject to intratumoral injection of Lnc-THOR shRNA (“sh-S1”) AAV or the scramble control shRNA (“shC”) AAV. The virus was injected daily for seven consecutive days. As shown, A549 xenografts with Lnc-THOR shRNA AAV injection grew significantly slower than control tumors with shC AAV injection (3 per day) was calculated. The results further confirmed that Lnc-THOR shRNA virus injection potently inhibited A549 xenograft growth in mice  and Henan Medical Science and technology research plan (LHGJ20190218). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Treatment results achieved after fulfilling an inpatient psychosomatic rehabilitation are often not permanent. Additional participation in outpatient rehabilitation aftercare may reduce the risk of recurrent disorders and support a successful reentry to working life. A therapy diary should accompany the aftercare and bring about the self-reflection process of psychosomatic rehabilitates, which could reduce recurrent disease progressions and support the recovery process as a whole. The study focuses on the evaluation of the effectiveness and implementation potentialities of a therapy diary in outpatient rehabilitation aftercare.In a qualitative study, seven therapists for outpatient rehabilitation aftercare in Central Germany and eleven outpatient psychosomatic rehabilitation patients were interrogated using partially standardized, guideline-based expert interviews.The data evaluation is based on the Qualitative Content Analysis according to Mayring.The results show that an accompanying use of a therapy diary during the outpatient rehabilitation aftercare enables an intense commitment through own thoughts and feelings. By writing down thoughts, emotions, dysfunctional behaviors in problematic situations, great successes are experienced. Through this initiated self-reflection process, the rehabilitant gains a better knowledge of one’s behavior in dealing with oneself and the environment and thereby, whenever necessary, learns to create new ways of acting.The voluntary use of the therapy diary in the outpatient rehabilitation aftercare could assist the therapy process and henceforward the recovery of the rehabilitants, and also increase the prospect of successful occupational rehabilitation. According to the Federal Health Monitoring System, the granting of reduced earning capacity pensions, about a third of which can be associated with psychiatric disorders, has almost doubled in the last decade , 2. WhenN = 18).The presented study was conducted as side research of a randomized intervention survey. Information on sample and method can be found separately.n = 7) for outpatient rehabilitation aftercare were able to take part in the investigation. All of them had at least two years’ professional experience in the outpatient rehabilitation environment.To form the expert group “therapists”, a full survey of all aftercare facilities in Central Germany  was sought. A total of 37 therapists for outpatient rehabilitation aftercare received a mailing call to participate in the study. Eventually, seven therapists (n = 11) took part in the study. All of them actively attended outpatient rehabilitation aftercare and used the therapy diary. Table To compose the expert group “rehabilitation patients” for the study, a total of 81 outpatient psychosomatic rehabilitation patients were recruited to participate. Merely eleven outpatient psychosomatic rehabilitation patients ; which possible opportunities and difficulties they see through the use of the intervention diary in the outpatient rehabilitation aftercare regarding the quality of the outpatient rehabilitation aftercare, the compliance of the participants and the effect on the dropout rate (reduction through self-reflection by intervention diary use); and nonetheless, how the therapy diary can be efficiently implemented in the outpatient rehabilitation aftercare environment.Based on Qualitative Content Analysis according to Mayring , the int1) Assessment of the effectiveness of the therapy diary in the treatment process in outpatient rehabilitation aftercare.a) Perspective of the therapists:“Yes, I also think that it can be beneficial for the participants to see progress more clearly and above all to reflect on it, to overthink what was said and done in the group. And possibly also better prepare for the group sessions when it comes to addressing issues. [ … ] The size is so beneficial. If it’s bigger, it’s going to be harder to always take it with you or have it by hand.” (quote from therapist 01).b) Perspective of the rehabilitation patients:Overall, the therapy diary was appraised by the surveyed therapists as a tool that supported the treatment process, which can also positively affect the recovery process. The open questions encourage the rehabilitation patients to state what they have learned from previous meetings. Furthermore, this allows reflecting self-critically on whether and in what way this can or has been transferred to everyday life. This way, potential difficulties or concerns can be written down and picked up on directly in the following session, as required, to find a solution with the help of the other group members. Patients who are outgoing and have already particularly ‘reflected’ could benefit from this method.“It’s really what I wanted. Help for me, tools in hand and a wide range of things you can do to stop falling into deep holes.” (quote from rehabilitation patient 08).When asked about the effect of the therapy diary, some of the rehabilitation patients answered that they found the use of the therapy diary helpful when reflecting on and reworking the sessions. Also, when preparing and formulating their discussion requests and goals. However, these were patients who kept their own ‘aftercare diary’ independently of the therapy diary.“But I thought it was good to put it all in writing. Yes, it made me stronger, so I truly liked it a lot. To express your feelings once in writing, to search for the words for how I would say it. I still have it all here, like I said, I look at it from time to time.” (quote from rehabilitation patient 07).Rehabilitation patients who have regularly completed, or are expected to finish aftercare, evaluated the subjective benefit of participation in aftercare as overall (very) high. The aftercare met expectations and encouraged the pursuit of individual goals in everyday life conditions.a) Perspective of the therapists:“So if there are additional individual sessions, then it should be clear that they will be financed. For sure, an intermediary session would be very useful for some. [...] Well, it would have to be something where it's obvious that if there's a necessity, we can do it. It doesn't have to be regularly, but if things get difficult, it is just inevitable.” (quote from therapist 04).b) Perspective of the rehabilitation patients:As a suggestion for optimization, the introduction of a ‘sub-goals’ page at the beginning, in the middle, and at the end of the therapy diary was recommended. Giving the possibility to compare achieved and previously set goals allows enhancing the reflection process. However, this should not solely be done by the patient, but rather in a two-way dialogue with the therapist. Nevertheless, this may lead to a higher expanse of the session and the appropriate reimbursement.“Perhaps it would be useful, in addition to the introductory and final discussions, to have the chance to achieve an intermediate step together with the group therapists in a practical manner, one-to-one, to evaluate in that session what the expectations have been and which expectations I do have in real terms.” (quote from rehabilitation patient 01).In general, an additional intermediate discussion with the therapist in a one-to-one setting is also something that rehabilitation patients increasingly want. This should take place after about half of the appointments in order to reflect on previous progression and the patient’s own development and therefore enable them to enter the second phase of aftercare in a more conscious and targeted manner. The scheme of this intermediate discussion could be based on the records in the therapy diary.a) Perspective of the therapists:“If they come to such an intermediate conclusion, I think that’s OK, then it must be in there: which problems have I implemented or addressed so far? [ … ] which changes have occurred and how have I approached them?” (quote from therapist 02).b) Perspective of the rehabilitation patients:In the therapists’ opinion, the therapy diary could improve the overall quality of outpatient rehabilitation aftercare as it is a tool that the patient can apply to assist in encouraging him or her even when uncertainties arise in everyday life. In particular and as required, the patient could read back through what progress he or she has made since the beginning of the aftercare. Successfully coping strategies were recorded “in black and white” (quote from therapist 01), which could then be reexamined at a later similar time and applied again in a similar situation.“And above all, [...] so that you just stick with it and do not simply go back home to the everyday's life rat race neglecting everything. Not only making it last 4 to 6 weeks, and then you switch back to your old habits, but you should always remember your personal goals.” (quote from rehabilitation patient 02).2) Implementation of the therapy diary in the outpatient rehabilitation aftercare setting.For the participants, one repeatedly mentioned motivation to participate in the aftercare was the desire for therapeutic support after the end of rehabilitation. Aftercare is intended to stabilize the achieved state of health or to prevent a recurrence. To avoid shifting back into old lifestyle and behavior patterns, newly acquired strategies should be strengthened and implemented in everyday life situations. We are aware that the required therapy is not concluded with the end of rehabilitation, but furthermore accompanies the process of reintegration into working life. The rehabilitation patients are willing to follow their personal therapy goals and want to “stick with them”. To underpin this, the therapy diary could be a perfect tool.a) Perspective of the therapists:“There it is, model learning. Is someone else’s dilemma interesting, or more even, does it reflect my problem? This means that the patient furtherly recognizes that he or she is not alone with that problem, there are many more people struggling. And if you approve, that serves as an icebreaker. Oh well, I am not alone in this, I may as well let it out. That is also a taboo, isn’t it?” (quote from therapist 02)b) Perspective of the rehabilitation patients:An evaluation in or with the group would likewise be useful if the outpatient rehabilitation aftercare is taking place in a closed group environment. To support the self-reflection process, the rehabilitation patient needs to receive “external” feedback. Hereabouts, the social support factor from the group would once again come into play, increasing the commitment of the rehabilitation patients. By regularly reporting the partial successes both through the feedback from the group and the therapist, the drop-out rate could be reduced as a result.“It is like being in any crisis area. If you see suffering somewhere else, you will start to perceive some earlier observations quite differently, although you might have seen things more narrowly earlier. [ … ] I have realized it could be worse.” (quote from rehabilitation patient 03).For many of the interviewed rehabilitation patients, it was essential to exchange experiences with other group members, for instance, on topics that have been recorded in the diary. It is also seen as a chance, to being able to learn from the companions’ experiences and to receive positive or even critical feedback, specific tips, and advice regarding their issues. In dialogue with other group members, the rehabilitation patients felt understood, accepted, and bolstered, and in some cases even experienced relativization of their predicaments.a) Perspective of the therapists:“It is the kind of suggestion you get, and I would be in favor of it if it is voluntary. Of course, if you desire to do it, you can do it. But if you do not want to, you do not have to. Then, I think it can be a good thing.” (quote from therapist 03).The therapists consider the use of the therapy diary at the beginning of the respective sessions as very beneficial. It enables an immediate start with the rehabilitation patients’ relevant or urgent topics and, if required, to identify common topics and to prioritize them accordingly. Nonetheless, this requires the motivation of the participants.“Well, I would first try to use the diary actively, so that the participants bring it into the group settings where they use it as a basis for what they say in the initial discussion round. Then they can work from there. Of course, this only makes sense if the majority of the participants actually do it. And if it cannot be done that way, if I had it available, I would at least offer it.” (quote from therapist 01).b) Perspective of the rehabilitation patients:Regarding the usage possibilities of the therapy diary within outpatient rehabilitation aftercare, the interviewed therapists highlighted that it was simple to work with, and that the clarity and range of the questions were good. The rehabilitation patients also reported that they valued the use of the diary as overall straightforward and considered the questions to be clearly and easily structured. The therapists, however, stressed the voluntary character of the diary and the absence of penalties for non-use as extremely important. The intervention diary should therefore not be an obligatory part of the aftercare, as there are rehabilitation patients who are less open to this method or who have limited access to it. Some rehabilitation patients may feel overburdened by “schooling” and, as a result, their motivation to participate could be jeopardized.“So the very first few times, you sit down straight after the therapy session and do it right away, that’s OK. But if you come home at 8:30 pm, you do not have enough energy to do it. And when would you do it then? Maybe at the weekend. And then you will have to go back through it all, and if I’m quite honest, you actually do not want to.” (quote from rehabilitation patient 06).From the rehabilitation patients’ point of view, using the therapy diary as an introduction to the current session is also considered to be very beneficial. Patients can write notes in advance of what they would like to address in the next session, a question, or a concern that may have remained outstanding since the last sitting. The notes from the previous session should be written as soon as possible. This way, it requires way less effort.“If I did not write it straight away in the evening, I would do it first thing the next morning because then I still had everything that was discussed in my mind. So it was no trouble for me at all. You can write it down in no time.” (quote from rehabilitation patient 05).The rehabilitation patients highlighted furthermore, that managing the therapy diary routinely can be an additional burden if there is too little time for it in everyday life.The center of this qualitative research, to answer the following questions, has been the experience from outpatient psychosomatic rehabilitation patients, as well as from therapists working in outpatient rehabilitation aftercare: 1) How do you rate the effectiveness of a therapy diary? 2) How can the therapy diary be efficiently implemented in the outpatient rehabilitation aftercare environment? To answer these questions, we drew upon the expertise of 7 therapists for outpatient rehabilitation aftercare and 11 outpatient rehabilitation patients.The therapists considered the therapy diary intervention tool to be suitable for practical use, not only because it was simple to manage and very clear, but also because it widely helped the therapy process. By writing down thoughts, emotions, and dysfunctional behavior in difficult situations but also the experienced triumphs, the therapy diary allows the patient to scrutinize his or her thoughts and feelings. This initiated self-reflection process enables the patient to gain a better perception of his or her behavior while dealing with himself or herself and the environment. Besides, it permits the patient to reflect and, wherever necessary, to create innovative ways of acting . This waAs therapists and rehabilitation patients state, using the therapy diary would be suitable for both: as an introduction to the individual group sessions, as well as a support for the therapy process. It allows rehabilitation patients to think through and to write down their concerns, difficulties, and issues in advance. In the latter case, the therapy diary would serve as documenting progress where accomplishments and failures are recorded and reflected upon. Even more, functional behaviors could be extrapolated, established, and advanced. The therapy diary triggers self-reflection and therefore supports this process. It encourages the patients to better understand their thoughts, emotions, and behavior and enables them to take appropriate action . To implIn this study, therapists in outpatient rehabilitation aftercare with previously tested knowledge in care practice were interviewed. That experience suited practical application and the additional prerequisites of formal training. They also had a wealth of experience with psychosomatic rehabilitation patients who actively worked with the therapy diary. This made it possible to obtain the expertise of proven and effective aftercare experiences that support the recovery process. Furthermore, it allowed achieving a substantiated evaluation of the effectiveness of the therapy diary as well as possible implications. As a matter of fact, due to the number of participants (18), this qualitative study is not a representative sample. Nevertheless, the criterion of theoretical saturation was clearly met. It must also be disclosed that the interviews were particularly attended by rehabilitation patients with an overall motivation to keep a therapy diary. Among these patients, the acquiescence was generally high, which meant that they were more focused on the benefits of the therapy diary. In a further analysis and to determine (in) congruent results, it would be useful to compare the results of the therapist’s and rehabilitation patients’ interviews with the experiences of other workers in the healthcare system who operate in the structure of outpatient rehabilitation aftercare groups. This could create a possibility to define further differentiated implementation options and applying recommendations, which then methodically lead to generalizable results in a quantitative study.Occupational rehabilitation is not only focusing on symptom reduction. Occupational rehabilitation as well means training, coping and activity and can also be done despite of recurring or lasting symptoms. Also the therapy diary is focusing on what patients have learned, which is also a coping-orientation rather than a symptom-orientation. For rehabilitation patients, the voluntary use of the therapy diary in outpatient rehabilitation aftercare could bolster the course of the therapy, the recovery, and also the process of reintegration into working life. Especially if the notes serve as a type of documentation of the therapy advancement: for practical behavior tips and coping tactics which result in direct feedback. This feedback could take place in a two-way dialogue with the therapist, but fundamentally also in a group setting. Purposely, the additional factor of social support from the group could increase the patients’ commitment. By regularly recording the respective partial victories during the course of the therapy, the already achieved progress could be advanced. Besides, the chances of successful reintegration into working life could also be increased by train the patients to cope with their allday life and potentially reoccurring symptoms as well. The topic, whether patients who use the therapy diary have more sustainable outcomes than patients who do not use a therapy diary, should be examined in a forthcoming analysis.


Regardless of the established cytotoxicity of lyso-sulfatide and the anti-inflammatory effects of FAAH inhibition seen in mouse models of several neurological diseases, genetic inactivation of FAAH did not mitigate, but rather exacerbated the disease phenotype of MLD mice. This unexpected finding was reflected by worsening of rotarod performance, increase of anxiety-related exploratory activity, aggravation of peripheral neuropathy, and reduced life expectancy. Thus, we conclude that FAAH has a protective function in MLD and may represent a novel therapeutic target for treatment of this fatal condition.An inherited deficiency of arylsulfatase A (ASA) causes the lysosomal storage disease metachromatic leukodystrophy (MLD) characterized by massive intralysosomal storage of the acidic glycosphingolipid sulfatide and progressive demyelination. Lyso-sulfatide, which differs from sulfatide by the lack of the N-linked fatty acid, also accumulates in MLD and is considered a key driver of pathology although its concentrations are far below sulfatide levels. However, the metabolic origin of lyso-sulfatide is unknown. We show here that ASA-deficient murine macrophages and microglial cells express an endo-N-deacylase that cleaves the N-linked fatty acid from sulfatide. An ASA-deficient astrocytoma cell line devoid of this activity was used to identify the enzyme by overexpressing 13 deacylases with potentially matching substrate specificities. Hydrolysis of sulfatide was detected only in cells overexpressing the enzyme fatty acid amide hydrolase (FAAH). A cell-free assay with recombinant FAAH confirmed the novel role of this enzyme in sulfatide hydrolysis. Consistent with the  The sac linkage . They arvia calcium-mediated process collapse , an endoplasmic reticulum-localized amidase renowned for its function in the endocannabinoid metabolism.The main challenge in identifying such an endo-N-deacylase is that it might accept and convert sulfatide only as a secondary substrate and with a low catalytic efficiency ksporters  or intersporters . Here weA and B). Compared with the classical BSA complexation method, uptake of NBD-sulfatide was three- to four-fold increased. We designate this novel loading method as translipofection.Cultured cells take up fluorescently or radioactively labeled GSLs from the medium and target them to lysosomes for degradation. Such cell loading assays are an established means to diagnose GSL storage diseases and to analyze the intracellular degradation pathways of GSLs. To increase the sensitivity of sulfatide loading assays, we exploited the negative charge of its sulfate group and tested cationic cell transfection reagents on their ability to promote cellular uptake. Complexation of NBD-labeled sulfatide with FuGENE HD proved to be more efficient with regard to internalization efficacy and cell viability than other cationic transfection reagents and previously published methods of complexation with BSA or packaging in synthetic lipoprotein particles , A and Btranslipofected and analyzed by life-cell fluorescence microscopy. For this purpose we used two astrocytoma cell lines derived from wild-type mouse brain (11+/+C1) and ASA-knockout mouse brain (17−/−A1), respectively  was synthesized . This redistribution can be explained by intralysosomal degradation of sulfatide to ceramide, its cleavage by ASAH1, export of the liberated BODIPY 500/510-labeled fatty acid to the ER, and reincorporation into complex phospho- and sphingolipids passing the Golgi apparatus to various cellular membranes. Due to the lack of ASA, BODIPY-labeled sulfatide cannot be hydrolyzed in 17−/−A1 cells causing accumulation of the green fluorescence in the endosomal/lysosomal compartment . Notably, also in this condition BODIPY 500/510-labeled sulfatide (or sulfatide-derived lipids) eventually escaped from lysosomal entrapment and reached the Golgi apparatus. Compared with wild-type cells, this redistribution was, however, substantially retarded and Golgi localization of BODIPY 500/510 could be seen 4 h after translipofection for the first time. Similar kinetics of redistribution were obtained for NBD-labeled sulfatide (not shown).To evaluate the dissemination of fluorescently labeled sulfatide and its breakdown products on a subcellular level, wild-type and ASA-deficient cells were ectively . To circthesized . Cells wtranslipofected cells were separated by thin layer chromatography (TLC) and the chromatograms were scanned with a FLA-5100 fluorescence scanner showing a limit of detection around 10 fmol NBD per band. In a first experiment, wild-type human fibroblasts were translipofected. After 6 h, sulfatide (undegraded), galactosylceramide, ceramide, and free fatty acids could be identified by coseparation of NBD-labelled standards (A). Liberated NBD-labeled fatty acids were also reincorporated into composite lipids representing sphingolipids and phosphoglycerolipids as suggested by their Rf values and differential resistance to mild alkaline hydrolysis (not shown). Interestingly, signals were much more intense in lipid extracts from the medium than in cellular extracts indicating efficient export of NBD-containing metabolites probably via ATP-binding cassette transporters . Thus, translipofection of NBD sulfatide allows to measure also a low residual turnover of sulfatide.In a next experiment cells from a juvenile MLD patient with the disease-causing mutation p.P426L   were traC, arrow) indicating sulfatide degradation. In contrast, no fatty acid signal was discernible for 17−/−A1 cells. This reflects a complete blockade of the sulfatide catabolism in these ASA-deficient astrocytoma cells.Next, we compared the sulfatide catabolism of the two murine astrocytoma cell lines 11+/+C1 and 17−/−A1. ASA-expressing 11+/+C1 cells were able to liberate the NBD-labeled fatty acid C, arrow translipofected primary bone-marrow-derived macrophages and brain microglial cells isolated from ASA knockout mice. Surprisingly and in contrast to 17−/−A1 cells, both cell types released significant amounts of the NBD-labeled fatty acid . Furthermore, NBD-galactosylceramide representing a prominent catabolic intermediate of the lysosomal degradative pathway in wild-type cells (A–C) was undetectable. Taken together, these observations pointed toward an ASA-independent pathway of sulfatide degradation in macrophages and microglial cells involving direct deacylation (A).To investigate the sulfatide catabolism of ASA-deficient macrophages and microglial cells, we pe cells , A–C wascylation A.Figure https://www.brenda-enzymes.org/) by applying one of the following three criteria. (i) lysosomal localization (according to Ref (http://genevisible.com). Enzymes with complex substrate specificity requirements that could not be brought into compliance with the molecular structure of sulfatide were arbitrarily omitted. This led to the elimination of, e.g., cathepsins with endopeptidase activity , two bacterial endo-N-deacylases accepting complex GSLs as substrates. BlastP (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to compare the active sites with human nonredundant protein sequences and revealed weak similarities with dipeptidase I  and cathepsin S , respectively  or other linear amides (E.C. 3.5.1) were selected from the BRENDA database (g to Ref ) and higvity see . (ii) Novity see . (iii) Evity see  or Pseudectively . Figure C), they could be utilized to evaluate alternate sulfatide-hydrolyzing pathways using transgenic expression of ectopic deacylases. For this purpose, 17−/−A1 cells were transiently transfected with the candidate enzymes (B), translipofected with NBD sulfatide, and the release of NBD fatty acid to the medium was analyzed by high-performance liquid chromatography (HPLC). As a control, 17−/−A1 cells were metabolically corrected by transfection of wild-type human ASA. Such ASA-expressing control cells released the NBD-fatty acid eluting from the HPLC column as a single peak with a retention time of ∼16 min (A). Confirming the TLC data, no such peak could be seen for uncorrected 17−/−A1 cells (B). When the sensitivity of the fluorescence detector was increased, a faint signal was, however, detectable also for mock-transfected 17−/−A1 cells . When the 13 candidate enzymes were individually expressed in 17−/−A1 cells, none of them but one increased the signal for the NBD fatty acid compared with control cells transfected with empty vector. The exception was FAAH that increased the peak area by a factor of 2.01 ± 0.42 (mean ± SD) in five independent transfection experiments. Overexpression of ASAH1, on the contrary, had no effect (factor 1.12 ± 0.38). Liberation of the NBD fatty acid by FAAH-transfected cells was confirmed by TLC (E). We then generated 17−/− A1 cells stably transfected with either FAAH, or as a control, with ASAH1. Expression levels of single clones were analyzed with a fluorescence-based assay measuring deacylation of AMCAA (for FAAH) and western blotting (for ASAH1), respectively. The clone with the highest FAAH activity, called FAAH-25, released 2.40-fold more NBD fatty acid than control cells on average (D), whereas the clone with the highest ASAH1 expression showed no increase . Increased release of NBD fatty acid by FAAH-25 cells could be independently demonstrated by TLC (F).Because ASA-deficient 17−/−A1 astrocytoma cells are not capable to degrade NBD sulfatide C, they c enzymes B, transl ∼16 min A. ConfirA1 cells B. When td by TLC E. We the average D, wheread by TLC F.Figure in vitro assay. As a positive control, we used a bacterial SCDase that is known to hydrolyze the N-acyl linkage between fatty acids and sphingosine bases in various GSLs. Both, recombinant SCDase and, to a lower extent, also recombinant FAAH released significant amounts of NBD-labeled fatty acid from NBD sulfatide (G). Importantly, the competitive FAAH-inhibitor URB597 reduced liberation of the fatty acid in the FAAH-catalyzed reaction. The residual turnover of sulfatide in the presence of competitive amounts of inhibitor might be explained by the limited thermostability of URB597 possibly leading to a progressive decline of its inhibitory activity over the applied incubation time of 24 h.To provide further evidence for a possible role of FAAH in the hydrolysis of sulfatide, NBD-labeled sulfatide was reacted with recombinant human FAAH in a cell-free ulfatide G. Importin vitro. To investigate if FAAH is also involved in the catabolism of (unlabeled) sulfatide in vivo, we constructed mice with a combined deficiency of ASA and FAAH. Furthermore, the mice were transgenic for the cerebroside sulfotransferase catalyzing the last step in the biosynthesis of sulfatide , confirming this prediction and thus an in vivo function of FAAH in the sulfatide catabolism. FAAH deficiency did not affect sulfatide levels (B) being approximately two orders of magnitude higher than lyso-sulfatide levels (not shown).The described experiments suggested that FAAH deacylates NBD sulfatide ulfatide . Due to by 18.4% A, confire levels B being aC). Compared with the control mice, mean weights were, however, significantly increased to 15.1 mg in MLD mice (expressing FAAH) confirming the previous data for the phrenic nerve . FAAH deficiency, however, increased mortality of MLD mice significantly and ten of 20 mice died between ages 8 and 16 months. Thus, lack of FAAH diminishes the reduced life expectancy of MLD mice further. None of the mice developed hindlimb paralysis in the observation period.MLD mice develop a paralysis of hindlimbs around age 16 months and their life expectancy is reduced to approximately 16 to 20 months . Due to Fig. 5A–H). As expected, significant behavioral changes were seen, however, in MLD mice compared with wild-type controls (compare the first with the third bar). Thus, the swimming velocity of MLD mice was severely reduced (A) and rotarod tests revealed deficits in motor coordination and balance (B). Furthermore, open field tests revealed trends for less frequent changes between phases of activity and inactivity (C and D) with a simultaneous increase of migration distances (E), more frequent transitions between center and border zones of the cage (F), and a trend to spent less time in the corners and more in the center (G and H). These alterations in the open field behavior were not significant, but in accordance with the previously detected increased anxiety-related exploratory activity of MLD mice . Whereas FAAH deficiency has no impact on the low swimming velocity of MLD mice (A), it further worsened the weak rotarod performance and the time it took the mouse to fall off the rod was reduced by an additional ∼30% (B). Additional effects of FAAH deficiency became apparent also in the open fields tests. Here, FAAH-deficient MLD mice switched ∼2.5-fold more often between phases of activity and inactivity, which led to a formal normalization of this parameter (C and D). Notably, the shift to shorter walks and shorter breaks did not affect the total distances covered by the mice within the observation period (E). It did, however, correlate with a tendency for an increased mobility between the different zones of the cage (F) and the time spent in the corners was further reduced (G and H).Important conclusions can be drawn from the comparison of FAAH-expressing MLD mice and FAAH-deficient MLD mice . Thus, FAAH deficiency increased AEA concentrations and ASA deficiency had no additional effect on the elevated levels.FAAH is the principal enzyme that degrades the neurotransmitter anandamide (AEA) . To evalFig. 7B). Importantly, the absolute levels of this chemokine were indistinguishable between FAAH-expressing and FAAH-deficient MLD mice. Also for the other seven cytokines identical levels were measured in FAAH-expressing and FAAH-deficient MLD mice (not shown). In contrast to MIP-1α, none of them was significantly upregulated in any of the two groups of MLD mice compared with non-MLD controls. Thus, FAAH deficiency did not enhance the early stage of neuroinflammation seen in the present study.To detect possible effects of FAAH deficiency on neuroinflammation typical of MLD-mice approaching 2 years of age , we evalFig. 73H- or 14C-labeled sulfatide (data not shown). To further elevate the sensitivity of the assay, we increased the cellular uptake of NBD sulfatide by preincubation with FuGENE HD, a cationic cell transfection reagent. Cellular uptake was increased by a factor of 3 to 4 supposedly due to the incorporation of the anionic sulfatide into complexes with positive surface charges that can bind to the negatively charged cell surfaces. The high sensitivity of our nonradioactive loading assay allowed us to identify an ASA-independent sulfatide-hydrolyzing activity of macrophages and microglial cells. This activity was demonstrated by the release of the fatty acid from sulfatide in the complete absence of lysosomal ASA activity. No such reaction was detectable in ASA-deficient astrocytoma cells. Of note, more than 25 years ago evidence for an ASA-independent pathway of sulfatide degradation has already been provided for immortalized B-cells from a late infantile MLD patient  sulfatide was verified by crossing ASA knockout mice with FAAH knockout mice with the resulting double-deficient mice showing significantly reduced levels of lyso-sulfatide. Still, sizable ∼80% of the lyso-sulfatide level seen in FAAH-expressing MLD mice persisted, indicating that FAAH-independent pathways contribute to the bulk of lyso-sulfatide production. Additional experiments are required to find out if other sulfatide-hydrolyzing endo-N-deacylases exist and/or if de novo synthesis by sulfation of psychosine via the galactose-3-sulfotransferase-1 plays a major role in lyso-sulfatide formation in vivo fvia activator proteins (saposins) that are indispensable for lysosomal degradation  and cloned in frame to generate myc-tagged proteins. Exceptions were ASAH1, NAAA, and FAAH, which were expressed as untagged proteins to avoid interference with proteolytic processing and/or hydrolytic activity suggested by published data. The plasmid pcDNA3-ASA-myc harboring the myc-tagged full-length human ASA cDNA was cloned accordingly. The transmembrane domain of the exoenzyme DPEP1 was deleted to obtain a soluble enzyme variant. All cDNAs were of human origin except NAAA, for which only a murine full-length clone was available.E.coli stab cultures were purchased from Invitrogen and ImaGenes-SourceBioscience, respectively . Plasmid17−/−A1 astrocytoma cells were transiently transfected with Turbofect (Thermo Fisher Scientific) following the instructions of the manufacturer. To generate stably transfected cells, 500 μg/ml G418 disulfate  was added to the medium for 10 to 14 days and growing single clones were isolated by aspiration with a pasteur pipette. Transgene expression of transfected cells was verified by immunoblotting using anti-myc antibodies and, for ASAH1, NAAA, and FAAH, protein-specific antibodies, respectively. The following primary antibodies and dilutions were used: (i) mouse monoclonal α-c-myc antibody 9e10 ; (ii) goat polyclonal α-FAAH V-17 antiserum ; (iii) goat polyclonal α-ASAHL (NAAA) G-13 antiserum ; (iv) rabbit polyclonal α-ASAH1 antiserum . The following secondary antibodies were used: (i) peroxidase-conjugated bovine α-goat antibodies ; (ii) peroxidase-conjugated goat α-rabbit antibodies .translipofection, 5 nmol NBD sulfatide was dissolved in 1.5 μl methanol and mixed with 1.5 μl FuGENE HD transfection reagent (Promega) prior to loading. In pretests also TurboFect, ExGen 500, and Lipofectamine 2000 transfection reagent were used instead of FuGENE . Loading by complexation with bovine serum albumin (BSA) and synthetic low-density lipoproteins (sLDL) was as described  and dried under a nitrogen stream. The dry lipids were dissolved in 200 μl chloroform/methanol 1:1 (v/v) for separation by thin-layer chromatography (TLC) and in 250 μl methanol/H2O bidest/85% phosphoric acid 550:450:1.5 (v/v/v) for separation by HPLC, respectively.The GSL metabolism of cultured cells was analyzed using the sulfatide fluorescent analogue N-[12-[amino]dodecanoyl]-cerebroside 3-sulfate . In this compound the NBD fluorophore is linked to the terminal (omega) carbon atom of the N-linked fatty acid. In general, 5 nmol NBD sulfatide was added to subconfluent cells in a 35 mm dish. For escribed , 38. Aft2O bidest/85% phosphoric acid in different volume fractions: (A) 550:450:1.5, (B) 750:250:1.5, (C) 1000:0:1.5. For the separation of lipids, the column was run with a constant flow rate of 0.5 ml/min at room temperature. The gradient cycle was 50 min in length with a 5 min isocratic segment with solvent (A), a 15 min linear gradient to solvent (B), a 15 min isocratic segment with solvent (C), and a 15 min isocratic segment with solvent (A). Lyso-sulfatide and sulfatide levels of mouse brain were quantified by mass spectrometry on a Xevo TO-S triple quadrupole mass spectrometer (Waters GmbH)  with an automatic TLC sampler 4 (CAMAG). Separation was with chloroform/methanol/acetic acid 190:9:1 (v/v/v) as solvent system. NBD sulfatide (Matreya), NBD galactosylceramide (Avanti Polar Lipids), and NBD dodecanoic acid (Invitrogen-Thermo Fisher Scientific) were coseparated as standards. Metabolic products harboring an NBD-labeled fatty acid were identified with a FLA-5100 fluorescence scanner (Fujifilm Life Science) at an excitation and emission wave length of 473 and 537 nm, respectively. For HPLC, a Bio-Tek HPLC system (Kontron AG) equipped with a Zorbax C18-column  and an FP-2020 fluorescence detector (Jasco) was used. The wavelength for excitation was 465 nm and for emission 535 nm. Mobile phases consisted of methanol/Hrs GmbH)  and TLC,rs GmbH) .AEA levels of total brain were analyzed by LC–mass spectrometry (MS) essentially as described  using deg and 4 °C. Protein concentrations of the supernatants were determined using the Bio-Rad Dc assay (Bio-Rad). Cytokine levels were measured with ABTS-ELISA kits according to the manufacturer's recommendations (Peprotech). For the ELISAs appropriate volumes of the supernatant supplemented with 0.05% Tween-20 were used. Homogenates of 3 to 5 mice per group and eight dilutions of each cytokine standard were measured in duplicates. The resulting calibration curves were used to determine the cytokine levels that were subsequently normalized on the corresponding protein concentration of the homogenate.Brains were dissected, cut midsagitally, and one hemisphere (about 200 mg wet weight) was homogenized in 2 ml ice-cold phosphate-buffered saline (PBS) pH 7.4 supplemented with Halt protease inhibitor cocktail (Thermo Fisher Scientific) and 5 mM EDTA using an Ultra-Turrax T25 homogenizer (Janke & Kunkel). Homogenates were centrifuged for 5 min at 10,000translipofection as described before. The labeled sulfatide was synthesized by standard methods from lyso-sulfatide and BODIPY 500/510 C12-fatty acid (Thermo Fisher Scientific) (Cells were loaded with the Golgi marker Invitrogen BODIPY TR ceramide (Thermo Fisher Scientific) for 30 min according to the recommendations of the manufacturer. Then, the cells were washed and BODIPY 500/510-labeled sulfatide was loaded by entific) . FluoresE and G).Recombinant human FAAH expressed in Sf21 insect cells was from Cayman Chemicals. The activity of FAAH was measured with 7-amino-4-methyl coumarin arachidonoyl amide  that is cleaved to arachidonic acid and highly fluorescent 7-amino-4-methyl coumarin . To testad libitum access to food and water. Mice were kept according to the German “Tierschutzgesetz” and the European Union directive 2010/63/EU on the protection of animals used for scientific purposes. Breeding, handling, and investigation of the mice were approved by the local authorities . To evaluate the degree of hypertrophic neuropathy, sciatic nerves of full length were dissected from female mice (Mice with a deficiency of murine ASA (mASA−/−) being double transgenic (tg) for cerebroside sulfotransferase (PLP-CST) and the inactive human ASA (hASA) mutant hASAc69s represent an aggravated mouse model of MLD . For reaale mice , the twoMouse behavior was tested at an age of 12 months. All 63 mice analyzed (13–18 mice per group) were born in a small time interval of 16 days. They were trained and tested one by one in a fixed order on identical days. Motor coordination was evaluated using a rotarod apparatus  at an accelerated speed mode with a ramp duration of 300 s and a minimum and maximum speed of 4 and 40 rpm/min, respectively. Mice were accustomed to the task on four consecutive days at a constant speed of 4 rpm for 2 min. On each of the following 4 days the mice were tested at the accelerating speed mode once. The time spent on the rotarod  was recorded. The swimming velocity was measured in a self-constructed opaque swimming pool with the dimensions 150 × 10 × 15 cm (length × width × height). The water temperature and depth were 26 °C and 10 cm, respectively. An accessible platform of 10 × 10 cm was placed at one end of the lane and allowed the mice to get out of the water. The mice were first trained on four consecutive days to swim increasing distances to the platform following a light gradient from bright to dark . On the following 3 days, mice were put into a 10 × 10 cm starting area opposite to the platform and filmed with a video camera from above. The time needed to cover the last 100 cm to the platform was extracted from the recordings and the swimming velocity (m/s) was calculated. Motor activity, anxiety-related responses, and exploratory activities were analyzed by open field tests using a dimly lit arena measuring 30 × 30 cm (30 cm high) divided into a wall zone (7 cm wide), corner zones (7 × 7 cm), and a center zone (16 × 16 cm). Mice were placed into the center of the arena and tracked for 30 min using a computer-operated EthoVision camera system and the EthoVision XT9 software (Noldus Information Technology). Various exploratory activities and anxiety-related responses were extracted from the tracks including the distance moved, the mean velocity, the duration and frequency of activity/inactivity intervals, and the times spent in the wall-, corner-, and center zones. Due to significant gender-specific differences and a higher variability of the behavior of male mice in the open field tests, only female mice were evaluated.matzner@uni-bonn.de) upon reasonable request, respectively.The authors confirm that the data supporting the findings of this study are available within the article, its supporting materials, and from the corresponding author, Ulrich Matzner (Institute of Biochemistry and Molecular Biology, Medical Faculty, University of Bonn, Email: This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Prostate cancer (PCa) is a top-incidence malignancy, and the second most common cause of death amongst American men and the fifth leading cause of cancer death in men around the world. Androgen receptor (AR), the key transcription factor, is critical for the progression of PCa by regulating a series of target genes by androgen stimulation. A number of co-regulators of AR, including co-activators or co-repressors, have been implicated in AR-mediated gene transcription and PCa progression. Epigenetic regulators, by modifying chromatin integrity and accessibility for transcription regulation without altering DNA sequences, influence the transcriptional activity of AR and further regulate the gene expression of AR target genes in determining cell fate, PCa progression and therapeutic response. In this review, we summarized the structural interaction of AR and epigenetic regulators including histone or DNA methylation, histone acetylation or non-coding RNA, and functional synergy in PCa progression. Importantly, epigenetic regulators have been validated as diagnostic markers and therapeutic targets. A series of epigenetic target drugs have been developed, and have demonstrated the potential to treat PCa alone or in combination with antiandrogens. Prostate cancer (PCa) is the second most commonly diagnosed cancer type and the fifth leading cause of cancer death in men around the world . PerturbEpigenetics refers to biological processes that regulate gene expression and function without altering DNA sequences . EpigeneThese epigenetic modifications enhance the establishment of a context-specific transcriptional profile, and aberrations ultimately cause genomic instability. Aberrations of epigenetic regulators such as gene amplification or mutations have been frequently found in PCa , and plaIn this review, we summarized the known epigenetic regulators, including readers, writers, and erasers working together with AR in PCa progression, and discussed their potential as epigenetic diagnostic and therapeutic targets, as well as future translational applications for PCa.Androgen is mainly produced by the conversion of cholesterol in the testes, muscle, adrenal glands, ovaries, skin, adipose tissue and endometrium . AndrogeAR, also named NR3C4, is an important member of the nuclear receptor superfamily. Its full-length gene transcript contains eight exons, which synthesize three major functional protein domains with individual functions . The N-tAR co-regulators, recruited by the individual domains of AR protein, modify the epigenetic conditions around the ARE binding motifs in the chromatin, thereby affecting the transcriptional activity of AR. Compelling evidence has highlighted the central roles of chromatin structure and histone posttranslational modifications (PTMs) in determining the spectrum of genes regulated by AR and other transcription factors . MultiplThe crystal structures of AR-FL, and the domains of hinge/DBD/LBD have been resolved , but thaThe NTD of AR protein, a relatively long and flexible domain, accounts for more than 60% of AR protein, but the crystal structure of the NTD of AR protein has not been resolved yet. Within the NTD is activation function 1 (AF-1), which is essential for AR transactivational activity . Co-actiThe DBD/hinge domain of AR protein is the most conserved region and is composed of two zinc fingers based on the crystal structure of the DBD  Shaffer. Its funThe hinge region is important for the subcellular distribution of nuclear receptors . NuclearThe ligand-dependent functional binding domain (AF2) in glucocorticoid receptor (GR) or estrogen receptor (ER) shows strong transcriptional activity . HoweverThe absence of LBD domain in splice variant AR forms (AR-Vs) constitutively sustains active AR signaling after anti-androgen drug treatment .AR-V7, a truncated AR splicing variant 7 without LBD, is highly active in CRPC, and promotes abiraterone and enzalutamide resistance . Its mRNde novo DNA methyltransferases, mainly initiate the DNA methylation  mediate the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC) . And theHistone modifications play vital roles in cancer progression . MethyltHistone acetylation is generally associated with active transcription, regulated by HATs and HDACs  Mathis .Tip60, a histone acetyltransferase abnormally overexpressing in CRPC tissues, directly induces the acetylation of H2A, H3 and H4 through the HAT region at the C-terminal, and directly induces AR acetylation through the activation of the intrinsic acetyltransferases . HistoneNon-coding RNAs (ncRNAs) refer to RNAs that are not translated into proteins. According to their length, ncRNAs are divided into two categories: small ncRNAs are transcripts with a length of 18–200 nt, and long ncRNAs are those more than 200 nt. NcRNAs participate in epigenetic regulation directly or indirectly .LncRNAs primarily interact with mRNA, DNA, protein, and miRNA and consequently regulate gene expression through epigenetic mechanisms. LncRNAs repress gene transcription in collaboration with chromatin remodeling complexes or histone modifying enzymes. The polycomb repressive complexes PRC1 and PRC2 as the most common protein partners of lncRNAs, result in chromatin compaction. PRC2 is mainly composed of SUZ12, EED and EZH2. Among them, histone methyltransferase EZH2, responsible for H3K27 methylation, leads to gene silencing . MultiplLncRNAs have been involved in transcription activation. PRNCR1 and PCGEM1 bind successively to AR and enhance AR-mediated gene transcription in a ligand-dependent or independent manner . Linc006miRNAs, by binding to the 3′ untranslated regions (UTR) of mRNA, repress gene expression. Aberrant miRNAs may be the outcome of epigenetic dysregulation, including CpG islands hypermethylation of promoter . The metin vivo and in vitro by downregulating ROCK1 and ROCK2  by decreasing ZEB1 and ZEB2 . miR-135nd ROCK2 .The interaction between AR signaling and miRNAs contributes to the progression of PCa. AR binds to miR-21 promoter and increases its expression . The uprConversely, miRNAs repress AR gene expression. miR-31 directly binds to the mRNA of AR or other cell cycle regulatory genes and inhibit the proliferation of PCa cells . In addiMYC, which results in the degradation of MYC mRNA through RISC complex  mRNA, resulting in the translation inhibition and RNA degradation. SHP is a common suppressor of AR, which inhibits AR transcriptional activity. miR-141 is commonly elevated in PCa, thus indirectly promotes AR transcriptional activity . In addi complex . Table 3Liquid liquid phase separation (LLPS) is a biological phenomenon, which means that components with similar properties form droplets and condense in cells. The intracellular components form a reaction chamber through phase separation, so as to establish an environment in which multiple biological reactions occur at the same time . NcRNAs in vitro, and SPOP strongly promotes the formation of droplets  has been approved by the United States FDA for detecting the levels of PCA3 in the urine of men over 50 years of age. In most prostate tumors, the level of PCA3 is 60–100 times higher than in adjacent non-tumor tissues, and is not detected in other types of tumors. Compared with serum PSA, the combination of urine PCA3 and fusion gene TMPRSS2-ERG significantly improved the specificity of PCa diagnosis and reduced unnecessary prostate biopsy .PCA3 is an ideal diagnostic marker, but is not a prognostic marker  3-dezaneplanocin-A (DZNeP) showed significant antitumor efficacy by inhibiting the activity of multiple histone methyltransferases including EZH2 . LSD1, aHDAC molecules regulate gene expression by altering chromatin structure and regulating the functions of non-histone proteins . SeveralAdditionally, natural compounds like anacardic acid, from cashew nut shell liquid, and a polyisoprenylated benzophenone derivative from the fruit rind of garcinia indica, known as garcinol, inhibit the HAT activity of both p300 and PCAF . Small mThe androgen/AR signaling pathway is one of the most important drivers for PCa initiation and progression, as well as the transformation to CRPC. Therefore, the anti-androgen/AR drugs have become the first line therapy for PCa.With a deeper understanding of PCa biology and epigenetics, additional epigenetic regulators critical for the development of PCa have been identified. Epigenetic regulators, individually or in combination with AR, promote PCa progression and have the potential to become novel PCa diagnostic markers and therapeutic targets. Although the development of epigenetic drugs is still in its infancy, undoubtedly, they will become increasingly important therapeutic tools for PCa, particularly for CRPC and neuroendocrine prostate cancer (NEPC) when used alone or in combination with anti-androgen/AR drugs.