The anatomical and physiological heterogeneity of strokes and persons with stroke, along with the complexity of normal upper extremity movement make the possibility that any single treatment approach will become the definitive solution for all persons with upper extremity hemiparesis due to stroke unlikely. This situation and the non-inferiority level outcomes identified by many studies of virtual rehabilitation are considered by some to indicate that it is time to consider other treatment modalities. Our group, among others, has endeavored to build on the initial positive outcomes in studies of virtual rehabilitation by identifying patient populations, treatment settings and training schedules that will best leverage virtual rehabilitation's strengths. We feel that data generated by our lab and others suggest that (1) persons with stroke may adapt to virtual rehabilitation of hand function differently based on their level of impairment and stage of recovery and (2) that less expensive, more accessible home based equipment seems to be an effective alternative to clinic based treatment that justifies continued optimism and study. Virtual reality (VR) is an approach to human computer interface that utilizes multisensory feedback designed to foster a sense of immersion or agency in a simulated task or activity. Virtual environments, designed for the purposes of upper extremity rehabilitation in persons with stroke have been studied for more than 15 years. Unique aspects related to the control of sensory information make it an ideal method for presenting tasks in a manner consistent with the principles of use dependent neuroplasticity, affording scientists an opportunity to gain insight into the tenets of neuroplasticity and apply them to develop more effective rehabilitation interventions . VirtualUse of virtual environments to distort the relationship between actual participant movement and simulated movement can be leveraged in different ways to drive neuroplasticity in sensorimotor circuits at any impairment level. In severely impaired individuals with trace or absent hand movement, VR can be used to provide a modified form of mirror visual feedback training in which the unaffected limb is used to control a virtual avatar, visually representing movement of the affected limb. Mirror visual feedback training has been shown to enhance excitability in the ipsilateral  hemisphere to moving hand via facilitation of compensoatory parieto-frontal networks –8. VirtuWhile the theoretical advantages of virtually simulated rehabilitation are many, the adoption of this technology in clinical settings has been slow. Initially, the cost of custom-made virtual rehabilitation systems was the most important initial barrier to adoption of this approach in clinical settings. This barrier hasbeen overcome by leveraging advancements made by the consumer electronics industries into lower cost rehabilitation technology. The other major hurdle that has been effectively overcome by technology advances is cyber-sickness which was experienced by early users of virtual environments. With these barriers addressed more domain specific limitations are being addressed. For example, the major technological limitation slowing development of the virtual rehabilitation of dexterity in persons with stroke (the focus of this paper) is the fidelity of lower cost motion capture systems .Rehabilitation of the hemiparetic hand caused by stroke has been one of the challenges that VR rehabilitation research has endeavored to overcome for a substantial portion of the field's existence. Early studies examined the ability of persons with chronic stroke to train safely and productively using this approach and comparisons between this approach, traditional interventions and repetitive task practice were conducted. Similar to the other labs in the growing field , 15, ourThe early work in this area was followed by extensive work across the field. Large systematic reviews and meta-analyses support our assertion that virtual interventions elicit upper extremity function gains as measured by clinical test batteries that are comparable to, or better than traditionally presented interventions in persons with chronic stroke , 28. DemIt is important to note that while consistent, measurable and statistically significant, the effect size of the gains demonstrated in studies of any rehabilitation intervention, virtual or otherwise were small and tended not to result in returns to full, pre cerebrovascular accident (CVA) levels of function or participation –33. The p = 0.049, UC group r = −0.31, p = 0.61). This may be due to the fact that the VR group, which received additional hand focused therapy in the very early recovery period, may have integrated expansion of the FDI motor map into better hand function. A companion study of 17 individuals who all received an additional 8 h hand focused VR/robotic training initiated within 3 months post lesion demonstrated a similar expansion of FDI area, and similar correlations between expansions in ipsilesional FDI map area and improvements in WMFT score   mapping in subsets of subjects participating in the pilot study we describe above. The first study compared 7 moderately impaired individuals who received an additional 8 h of VR/robotic intervention within 1 month post-stroke, to 6 similarily impaired individuals who did not receive additional hand focused rehabilitation . In both= 0.017) .This correlation between lesioned hemisphere motor map expansion and hand function improvements following intensive hand training, but not usual care during the early recovery period, has been identified in studies by other labs , 44. Twop = 0.042) in Jebsen Test of Hand Function change as the result of a 2 week training intervention   or presence of attention or neglect still fail to predict accurate motor recovery in a high percent of stroke survivors , 48. Rohrvention . Rohafza < 0.05) . The lim < 0.05) . TMS-bas < 0.05) . Therefo < 0.05) . While M < 0.05) , CMC dur < 0.05) . MeasureOne adavantage afforded by virtual environments is the opportunity to manipulate sensory information . These mTMS mapping of a sub-set of patients from this more impaired group showed a different pattern of adaptation than the pattern identified in our less impaired subjects. In the more impaired subjects, extensive damage along the lesioned corticospinal tract made it impossible to elicit lesioned hemisphere motor evoked potentials at the impaired FDI, causing us to focus attention on the relationship between the contralesional motor cortex and the impaired UE. This sub-study showed an increase in the contralesional FDI map representation from pre to post training followed by a decrease from post to 1 month. The increase from pre to post intervention motor map area was associated with pre to 6 month increases in the UEFMA and maximum pinch force scores . We idenp < 0.001)] ] . They al 0.001)] . These o 0.001)] –70. DireOver the years our group has been a collaboration between biomedical engineers, physical therapists and neuroscientists, all sharing an interest in motor learning and neuroplasticity. Our approach is unique in the participation of all of these disciplines in the earliest stages of intervention technology design. Our main lab, which is housed in an engineering building, is equipped with lab grade kinematic measurement equipment, EEG and TMS equipment, several 3-D printers and a fittings shop. More recently, we have added satellite labs on the campuses of two rehabilitation hospitals. The balance of this paper will present unpublished and synthesized findings from previously published and unpublished studies that all examined virtual rehabilitation simulations in an attempt to present three contrasts: (1) adaptations made by persons with severe impairments Upper Extremity Fugyl Meyer Assessment (UEFMA) <20 and those with moderate to mild impairments (UEFMA 21–60) (2) long term adaptations made by subjects in the early subacute phase of recovery; and finally; (3) adaptations made by patients performing directly supervised interventions using costly facility based equipment with those of subjects using inexpensive equipment, in their homes, independently. Synthesis of these findings provide an overview of the progression of technological interventions for the hemiplegic hand and arm post stroke based on non-immersive virtual rehabilitation and, importantly, may provide a window into the most promising future pathways.Our group has continued its examination of early rehabilitation in more impaired persons. Three additional subjects from our new study of early hand rehabilitation have completed the study through the 6 month data collection point and we have collected a total of 10 subjects with 6 month follow up that were less impaired as well. n = 10) during the 1 month post-stroke can be beneficial when compared to usual care (UC) (n = 11) at 1 month after the end of training, but this advantage seems to disappear at 6 months post-stroke   outcomes with our older work examining therapist supervised lab-based repetitive task practice (RTP) intervention and robot assisted virtual rehabilitation (RAVR), all in persons with chronic stroke offers some insight as well as some considerations for future study. In our 2015 paper comparing a robotically facilitated virtual rehabilitation intervention, to a therapist supervised circuit training intervention of 12 table-top repetitive task practice activities, both groups of subjects demonstrated a mean improvement of 2 points on the UEFMA. Both groups demonstrated inconsistent change patterns with some subjects improving, some staying the same and others regressing . This paThe training schedules for these two interventions differed. The two lab based interventions were delivered in a 2 week period with 8, 3 h sessions. Our home based subjects averaged less therapy, closer to 18 total h, but the intervention occurred over 12 weeks. It is possible that a training schedule that distributes training time across a greater time period may be more conducive to motor improvements than a concentrated schedule with a larger volume of training. More rigorous testing will be required to determine if one approach to treatment or treatment schedule was definitively better. This said, the possibility that subjects using inexpensive equipment, in their homes, independently, might make similar to or better gains, than patients performing directly supervised interventions using costly equipment will have important implications for patient access to treatment and the cost of health care delivery. Additional further study will examine the feasibility of adding movement based priming to our home based interventions. Proof of concept testing of a low cost, admittance controlled finger training robot, that will utilize the same platform that presents our home based VR intervention is in progress .The anatomical and physiological heterogeneity of strokes and persons with stroke, along with the complexity of normal upper extremity movement make the possibility that any single treatment approach will become the definitive solution for all persons with upper extremity hemiparesis due to stroke unlikely. This situation and the non-inferiority level outcomes identified by many studies of virtual rehabilitation are considered by some to indicate that it is time to consider other treatment modalities. Data generated by our lab and others suggesting that (1) persons with stroke may adapt to virtual rehabilitation of hand function differently based on their level of impairment and stage of recovery and (2) that less expensive, more accessible home based equipment seems to be an effective alternative to clinic based treatment that justifies continued optimism and study.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Internal Review Board, Rutgers The State University of New Jersey. The patients/participants provided their written informed consent to participate in this study.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.GF, QQ, AMo, AC, AMe, and SA have applied for a patent for the Home Virtual Rehabilitation System. QQ, AMo, and AC have interests in NeuroTech3R, a company working toward bringing the Home Virtual Rehabilitation System to market. The remaining 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.


The expanded use of second-generation antiandrogens revolutionized the treatment landscape of progressed prostate cancer. However, resistances to these novel drugs are already the next obstacle to be solved. Various previous studies depicted an involvement of the enzyme AKR1C3 in the process of castration resistance as well as in the resistance to 2nd generation antiandrogens like enzalutamide. In our study, we examined the potential of natural AKR1C3 inhibitors in various prostate cancer cell lines and a three-dimensional co-culture spheroid model consisting of cancer cells and cancer-associated fibroblasts (CAFs) mimicking enzalutamide resistant prostate cancer. One of our compounds, named MF-15, expressed strong antineoplastic effects especially in cell culture models with significant enzalutamide resistance. Furthermore, MF-15 exhibited a strong effect on androgen receptor (AR) signaling, including significant inhibition of AR activity, downregulation of androgen-regulated genes, lower prostate specific antigen (PSA) production, and decreased AR and AKR1C3 expression, indicating a bi-functional effect. Even more important, we demonstrated a persisting inhibition of AR activity in the presence of AR-V7 and further showed that MF-15 non-competitively binds within the DNA binding domain of the AR. The data suggest MF-15 as useful drug to overcome enzalutamide resistance. Because of the strong relevance of androgens in all stages of prostate cancer (PCa), androgen deprivation therapy (ADT) is a mainstay in the treatment of metastatic PCa. However, progression to castration-resistant prostate cancer (CRPC) frequently occurs after 18 to 36 months on average . The proA number of different enzymes are involved in the biosynthesis of intratumoral androgens like CYP17A1 and AKR1C3. Various studies including recently published findings by our group associated increased expression of AKR1C3 with CRPC ,20,21 asMelodorum fruticosum, in PCa with an emphasis on their efficacy in overwhelming enzalutamide resistance. To this end, we used 22Rv1 cells, which are enzalutamide resistant and express AR-V7 [The aim of our study was to investigate the anti-neoplastic effects of three natural AKR1C3 inhibitors, which originated from the plant ss AR-V7  and an ess AR-V7 . In addiss AR-V7 . In addiMelodorum fruticosum (MF-11 and MF-14) or synthetically derived from MF-14 (MF-15), proved to be the most effective in an AKR1C3 inhibition assay as described by Schuster et al. [In a previous study, Mayr et al. described the general suitability of various natural chalcones as potential AKR1C3 inhibitors . In thisr et al. . BrieflyTo validate the anti-neoplastic effects of the compounds, we incubated 22Rv1 prostate cancer cells with increasing concentrations of MF-11, MF-14 and MF-15 over 5 days. As shown in To further investigate cell line-dependent effects, we validated MF-15 in two other AR-positive prostate cancer cell lines. As shown in Previous findings by our group have demonstrated the usefulness of 3D tumor spheroids as relevant tissue-mimicking in vitro tools for drug testing . TherefoWe next investigated MF-15 in co-culture spheroid assays. In a previous study, we demonstrated that prostate cancer cells become enzalutamide-resistant upon spheroid co-culture with CAFs, associated with increased production of pro-inflammatory cytokines and elevation of steroid biosynthesis with significant upregulation of AKR1C3 in particular . CorrespPrevious AKR1C3 inhibitors were shown to exert dual inhibitory effects on AKR1C3 and AR ,24. To eTo further investigate this, we next examined if MF-15 affected AR expression. As shown in We next evaluated the influence of MF-15 in AR activity using a luciferase reporter gene assay. We used AR-negative PC-3 cells for these experiments to avoid potential confounding effects of endogenous AR and transiently transfected them with full-length AR (AR-FL). As shown in Since AR-V7 transcript levels usually occur at substantially lower expression levels than AR-FL in vivo (42), we next transfected PC-3 cells with AR-FL and AR-V7 at two different ratios (95:5 and 80:20), thereby mimicking a more likely patient situation. As summarized in Based on the fact that MF-15 strongly inhibits AR-V7, we hypothesized that the inhibitor most likely does not bind to the LBD of the AR as the anti-androgen enzalutamide does. To further elucidate the potential binding sites of MF-15 within the AR protein, we performed molecular docking studies on known binding sites aside from the AR-LDB. We first focused on the AR-DBD, where two binding sites, the P-box and the D-box amino acid motifs, have been previously described in the literature. The P-box is involved in binding to DNA, while the D-box mediates dimerization ,36. As dx-axis compared to the mock control, suggesting non-competitive inhibition of AR transcriptional activity. These data suggest that MF-15 acts differently from enzalutamide, which is known to be a competitive AR antagonist [To further investigate the binding mode of MF-15 to the AR, we measured reporter gene activity after incubation of PC-3 cells transiently transfected with AR-FL with increasing concentrations of R1881 in the absence or presence of 10 µM MF-15. As shown in tagonist ,42. To ftagonist ,34,36. AResistances to potent anti-androgens like enzalutamide and abiraterone acetate are becoming a growing problem in the treatment of advanced prostate cancer. Therefore, the development of new drugs, which would help to overcome these resistances, is largely demanded. AKR1C3 has gained increasing attraction as a novel target based on studies demonstrating its impact on prostate tumor progression, mCRPC and development of resistances to enzalutamide and abiraterone acetate ,44,45,46Several potent AKR1C3 inhibitors, including steroidal, non-steroidal and natural compounds, have been developed so far [In vitro cell culture experiments revealed that 10 µM MF-15 significantly inhibited various prostate cancer cell lines, including enzalutamide resistant DuCaP EnzaR and 22Rv1 cells, though with varying response rates. Western blot analysis, however, revealed substantial levels of AKR1C3 in all three cell lines, thereby suggesting that a low response rate to MF-15 can hardly be explained by faint AKR1C3 expression. In addition, we showed here that MF-15 also effectively prohibited 3D spheroid growth. Most importantly, MF-15 was also highly effective in inhibiting spheroid growth of DuCaP/CAF and DuCaP EnzaR co-culture spheroids, which were previously shown to exhibit increased AKR1C3 expression associated with enzalutamide resistance and a strong pro-inflammatory phenotype . In compAnother interesting aspect of some previously described AKR1C3 inhibitors is their bifunctional activity on AKR1C3 and the AR. The group of Verma et al. for instance reported on the antineoplastic activity of an AKR1C3 inhibitor called KV-37, which reduced prostate tumor cell growth in vitro and in vivo associated with a reduction of PSA expression and reduced AR transactivation . In our Molecular docking simulations on known binding sites of the AR-DBD were conducted to propose a mechanism of action for MF-15 on the AR. The best results were found directly in the AR-DBD. These findings were further corroborated by reporter gene assays using the glucocorticoid receptor, which exhibits high similarity with the AR in its DBD ,34,36. MAn AKR1C3 inhibitor like MF-15 with dual-inhibitory function on AKR1C3 and AR as well as on truncated AR variants holds the advantage of simultaneously inhibiting intratumoral androgen biosynthesis and the AR signaling cascade, thereby covering the action of antiandrogens such as enzalutamide and abiraterone acetate. Nevertheless, it should be noted that the growth-inhibitory effects of MF-15 were not restricted to AR-positive prostate cancer cells, since also AR-negative PC-3 cells were affected by MF-15 in our study, exerting a similar effect as the chemotherapeutic drug docetaxel. Anti-proliferative effects of ARK1C3 inhibitors in PC-3 cells have previously been shown by several other groups ,59. A poMelodorum fruticosum have been shown to also possess significant anti-inflammatory properties, inhibiting microsomal prostaglandin E synthase-1 (mPGES-1), which represents a key enzyme in the synthesis of pro-inflammation prostaglandins [Another advantage of an AKR1C3 inhibitor like MF-15 would be that a co-administration of prednisone, as it is common to prevent side effects of abiraterone acetate, would not be necessary ,26. MF-1glandins . Based oglandins ,62. Moreglandins . In summFrom the clinical point of view, AKR1C3 inhibitors could be used in patients with enzalutamide resistance as a new additional treatment option, especially in cases where the patients do not meet the criteria for undergoing another mCRPC treatment like chemotherapy. However, clinical trials are warranted to proof the findings of this study.Melodorum fruticosum were collected and extracted with CH2Cl2 as described in Engels et al. (61). The crude extracts (23 g) were subjected to silica gel column chromatography  and eluted with a gradient from petrol ether (100–0%), to ethyl acetate (0–100%), and methanol (0–100%), and collected time-dependently. The 170 obtained fractions were inspected with thin-layer chromatography using MF-14 as authentic standards and pooled to 10 comprehensive fractions (A-J). Fraction I (689 mg) was then separated over Sephadex® LH-20  using CH2Cl2:acetone  as a mobile phase to yield nine comprehensive fractions (I-a—I-i). Finally, 132.8 mg of fraction I-h were separated over Sephadex® LH-20  using methanol as a mobile phase to yield 35.1 mg MF-11 and 53.5 mg MF-14. MF-15 was obtained through catalytic hydration of MF-14. To this end, 23.8 mg MF-14 were dried and dissolved in methanol before the addition of Pd/C  and H2 at room temperature under stirring. The reaction was monitored with thin-layer chromatography and stopped after 15 min by filtration. MF-15 was ultimately purified over Sephadex® LH-20 with methanol as mobile phase and its identity and purity confirmed by 1H NMR by comparison literature (60). NMR spectra were acquired on a Bruker Advance II 600 NMR spectrometer . Finally, 18.5 mg MF-15 were obtained with a purity of >95%.The leaves of Docking simulations were carried out on a crystal structure of the AR-DBD  , using GTM (Gibco) and 1% penicillin and streptomycin (Lonza). DuCaP cells were a gift from Prof. J. Schalken  and routinely cultured in RPMI with 10% FCS [2. AR-V7 status of the used cell lines: 22Rv1: AR-V7 positive [22Rv1 cells and PC-3 cells were obtained from the American Type Culture Collection  and cultured in RPMI  with 10% fetal calf serum , 1% GlutaMAX 10% FCS . Enzalut 10% FCS . Immorta 10% FCS  and were 10% FCS . All celpositive , DuCaP: positive , DuCaP Epositive , PC-3: Apositive .Enzalutamide (MedChemExpress), R1881 , the AKR1C3 inhibitor (AKRi) (3-(4-trifluoromethyl)phenylamino) benzoic acid, Calbiochem), indomethacin (Sigma-Aldrich), dexamethasone , docetaxel (MedChemExpress) and mifepristone  were dissolved in dimethyl sulfoxide (DMSO). All solvents used for the isolation were purchased from VWR International .® Aqueous one solution cell proliferation assay (Promega). Briefly, 10 µL of reagent were added to 100 µL of cell culture medium, and absorbance was measured at 490 nm on a Cytation™ 5 Cell Imaging Multi-Mode Reader (BioTek). In each individual experiment, changes in cell viability were expressed as percentage of mock control.Cell viability was determined via CellTiter 962. To obtain co-culture spheroids, DuCaP tumor epithelial cells were seeded together with CAFs at a ratio of 1:1 as previously described [® S3 LiveCell Analysis System.3D spheroids were established as described previously . Brieflyescribed . Spheroi−ΔΔCT [Total RNA was isolated from cells with ExtractMe total RNA isolation Kit  and quantified with the NanoDrop ND-2000c (Thermo Scientific). RNA was transcribed into cDNA by reverse transcription using SuperScript III reverse transcriptase (Invitrogen). qPCR was performed with TaqMan™ Assays (Thermo Fisher Scientific) for the quantification of FKBP5 (Hs01561006_m1), AKR1C3 Hs00366267_m1), and hydroxymethylbilane synthase , which was used as the endogenous control. Primer and probe sequences of AR-FL, AR-V7 and PSA were as follows: AR-FL , AR-V7 , PSA . qPCR was carried out with ABI Prism 7500 Fast RT-PCR System (Applied Biosystems) cycler. Fold change in gene expression was determined using the mathematical model ratio 2267_m1, aCells were lysed using RIPA buffer supplemented with 0.5 mM PMSF, 2 µg/mL leupeptin, 2 µg/mL aprotinin, 2.5 mM NaF and 1% Triton X-100 by shaking at 4 °C for 1 h. Fifty micrograms of protein were separated using 4–12% NuPAGE Bis-Tris protein gels  and transferred onto a 0.2 µm nitrocellulose membrane . Blocking of membranes and antibody incubation was performed in StartingBlockTM (PBS) blocking buffer  using the following antibodies: anti-AKR1C3 , anti-androgen receptor , anti-glyceraldehyde-3-phosphate dehydrogenase . Visualization and quantification of protein bands were performed with Image Studio software Version 5.2 (LI-COR Biosciences).To assess reporter gene activity, AR negative PC-3 cells were transiently transfected with a CMV promoter-driven pFlag tagged full-length AR (AR-FL), the AR truncated splice variant 7 (AR-V7) or GR , and a NanoLuc luciferase reporter gene under the control of a promoter element consisting of two consecutive canonical androgen response elements (AREs) and a TATA box (pNL1.1.-ARE2TATA) using ViaFect lipofection reagent (Promega). Cells were then seeded into 96-well plates in RPMI + 2% FCS at 37 °C overnight. Then, 0.2 nM R1881, 2.5 µM enzalutamide or 10 µM MF-15 were added. After 24 h, luciferase reporter gene activity was determined with Nano Glow Dual Assay (Promega) and measuring absorbance on a CYTATION multiplate reader instrument.Our results show that the natural AKR1C3 inhibitor designated MF-15 achieves a significant inhibitory effect in enzymatic assays as well as anti-neoplastic effects in various prostate cancer cell cultures models. Moreover, MF-15 expressed a stronger inhibitory effect than other known AKR1C3 inhibitors under examination like indomethacin. Particularly interesting was the strong inhibitory effect of MF-15 on the AR and on its variant AR-V7 in cell lines, which were mostly insensitive to enzalutamide. Therefore, we are confident that a combination of AKR1C3 and AR inhibitory activity, as displayed by MF-15, is a powerful combination to combat prostate cancer. Especially the activity on AR-V7 could aid in overcoming the problem of enzalutamide resistance.


Frontiers in Oncology by Zhize Wang et al.  or chemotherapy, while Z. Wang included only results from patients treated with novel hormonal therapy (NHT). For these reasons  response after chemotherapy. However, Z. Wang also looked at AR-V7 positivity before and after treatment, while J. Wang furthermore subdivided results by method of detection.Not surprising—though reassuringly, given the overlap between the two meta-analysis—the findings between the two studies were quite similar: AR-V7-positive patients have worse prognosis, a trend that held regardless of treatment and endpoint. The hazard ratios for progression-free survival and overall survival were fairly similar . More difficult to reconcile were the odds ratios for PSA response. Z. Wang concluded that AR-V7-positive patients had a decreased PSA response rate . J. Wang concluded AR-V7-positive patients had lower response rates . Looking at the OR values, this would be a higher odds of response, rather than a lower, for the J. Wang study. It could be that J. Wang calculated the OR for AR-V7 negative, instead of positive, or that they calculated the OR for a non-response, rather than a PSA response. The fix to either is taking the reciprocal, which produces ORs of 0.17 and 0.45, respectively, somewhat in line with those of the Z. Wang study.Both studies argue for a prognostic role of AR-V7, with positive patients faring worse. The major limitation of studies like these is that they are not multivariable in nature. There are several prediction models available for men with progressive or hormone-refractory metastatic prostate cancer , 5. TheyMK wrote this article. RB reviewed and advised on this piece. All authors contributed to the article and approved the submitted version.Code DSC-08001617819PRD.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.


Strongylid nematode infections may negatively affect both animal health and welfare, with deleterious consequences for livestock productivity. Many farmers in recent decades have relied on anthelmintics as the sole strategy of control, but the intensive use of these chemotherapeutics has led to the development of anthelmintic resistance (AR). Knowledge of both the efficacy of anthelmintics and factors promoting AR are essential to effectively control nematode infections, but no information on these topics for goats in the Czech Republic (CR) is available. This survey aimed to determine the occurrence of AR at Czech goat farms and to identify risk factors for the development of AR. A total of 24 herds of dairy goats across the CR were evaluated using in vitro tests for detecting AR, and a questionnaire survey was carried out to evaluate factors associated with AR.Teladorsagia/Trichostrongylus and Haemonchus were the predominant types of resistant larvae. Eight (62%) of the affected herds were evaluated as highly resistant to ivermectin. Eleven (46%) of the herds were resistant to both benzimidazoles and ivermectin. This report is the first on dual AR in the CR. A univariate logistic regression analysis indicated that a high stocking rate and farmer inexperience were significantly associated with ivermectin and benzimidazole resistance, respectively.Resistance against benzimidazoles was confirmed at 18 (75%) farms, and the level of resistance was high in four (22%) of the affected herds based on the egg hatch test. Ivermectin-resistant nematodes were detected in 13 (54%) herds using the larval development test; The results of our survey suggest that AR is widespread amongst herds of dairy goats in the CR, likely due to inappropriate practices of pasture and health management. AR may be an issue for expanding dairy-goat production in the CR in the near future unless both veterinary practitioners and farmers widely adopt strategies to prevent the development of AR.The online version contains supplementary material available at 10.1186/s12917-021-02819-8. Resistance to anthelmintic drugs in gastrointestinal (GI) nematodes of farmed ruminants is widespread and is an important factor limiting ruminant production worldwide , 2. AnthAR is of particular interest in small ruminants, where it has become very common at the vast majority of sheep farms in Europe , 7. GoatAlternative approaches to control GI nematode infections in grazing livestock are available , 23, butData for AR in sheep within the Czech Republic (CR) are limited, and no information about this topic is available for goats. Chroust  recordedWe conducted a survey to gather information on both anthelmintic efficacy and risk factors for developing AR at Czech goat farms where the interest in goat farming is steadily increasing and the demand for goat milk and other dairy products are growing. Our objectives were to: i) evaluate the efficacy of BZs and IVM at dairy-goat farms using in vitro tests, and ii) identify associations between AR and farmer, management, and control factors at the surveyed farms.Both in vitro tests for detecting AR were possible at all goat farms due to sufficient numbers of nematode eggs recovered from the faecal material. The egg hatch test (EHT) was performed in triplicate, but the larval development test (LDT) was only performed in duplicate for all drug concentrations due to a lower number of nematode eggs isolated. The mean proportion of eggs hatching in the control wells of all tissue-culture plates (EHT) was 98 ± 2%, and the mean proportion of infective larvae in the control wells of all microplates (LDT) was 79 ± 8%.50) of thiabendazole (TBZ) ranged from 0.01 to 0.62 μg.ml− 1  of the BZ-resistant herds had a moderate level of resistance based on the hatching categories , and the other herds (22%) were evaluated as highly resistant. IVM resistance was detected in 54% (13 of 24) of the herds based on the selected threshold. Medium lethal dose (LD50) ranged from 0.18 to 184.2 ng.ml− 1 . Infective larvae were recovered from all microplate wells containing 21.6 ng IVM-AG.ml− 1, which were considered to be resistant  varied considerably and a mixture of nematode species was detected in most wells containing 21.6 ng ivermectin aglycone (IVM-AG).ml− 1  of the goat farms using the in vitro tests. A high level of resistance to both drug classes was identified in two (18%) of all dual-resistant herds.Resistance to BZ was detected in 75% 18 of 24) of the herds using the EHT. The medium effective dose (ED8 of 24 oP = 0.04). Pasture rotation was regularly practised at half of the farms, and animals were not drenched at any farm when moved to a new paddock. Kidding occurred mainly from January to March, and goats were turned out on pasture in April or May. Animals were grazed from spring to autumn (October or November) at 17 (71%) farms, and at the remaining herds had year-round access to pasture, depending on the meteorological conditions. All farms had a closed herd turnover, but a new buck was briefly introduced to the herd for mating at most (90%) of the farms.The putative farmer, management, and control factors associated with AR are summarised in Table P = 0.04).The age of farmers ranged between 28 and 65 years, with a mean of 45 years. The majority (71%) of farmers had completed elementary or secondary education, and only eight farmers (33%) were educated in agricultural fields. Experience in dairy-goat farming varied highly from three to 46 years; the farmers had reared animals for an average of 9.5 years. Our results indicated a significant negative association between farming experience and BZ resistance . Only four (17%) farmers declared that treatments were used strictly selectively. This factor was not significantly associated with resistance to BZs (P = 0.14) or IVM (P = 0.77) or with dual resistance (P = 0.92). Fourteen (58%) farmers occasionally sent faecal samples to a service laboratory for examination, and nine of these farmers (64%) drenched animals based on the results of the examinations. Drug efficacy after anthelmintic treatment is not commonly evaluated at Czech goat farms.Anthelmintics were applied twice a year at twelve (50%) farms. All these farms had a predetermined treatment plan; the vast majority (92%) drenched herds in spring when turned out on pasture and thereafter in autumn at housing. Six herds (25%) were treated once annually, and drenching in these herds was scheduled either in spring (33% of herds) or winter during the housing period (67% of herds). The remaining herds (25%) were not drenched regularly every year, and no anthelmintic had been applied for the last five years at one farm (4%). Farmers relied mainly on BZs (83% of farms) and MLs (70% of farms). Nearly all farms treated goats twice annually, rotating between BZs and MLs; oral drenches of BZs were applied by farmers in spring, and injectable IVM was administered by a veterinary practitioner in autumn or winter. The other anthelmintic class (LEV) was used in only four (17%) herds. Unlike other EU countries where the use of injectable IVM is prohibited in dairy animals, this route of IVM administration is allowed in animals during the drying period in the CR. Animal live weight for calculating drug dose was estimated visually at most (91%) of the farms, and the standard dose  of the drug was applied at eight (35%) farms following the manufacturers’ recommendations. More farms had AR in the group receiving the sheep dose (vs. sheep dose × 2), but these associations were not significant  have begun to raise goats in the last decade. Establishing a commercial farm requires specific knowledge and experience, which is a challenge for new farmers . AcquiriOther factors evaluated in our survey (including targeted selective treatment [TST] or underdosing) were not significantly associated with AR. TST is a promising strategy to effectively control nematode infections while maintaining anthelmintic efficacy , 50. AntA more rapid metabolism of xenobiotics is responsible for the faster clearance of anthelmintics from goats than sheep. Drenching goats at the standard dose recommended by the manufacturer (sheep dose) will thus lead to underdosing , 56. InsThis first survey of AR at Czech goat farms found that AR was well established amongst strongylid nematodes. This high prevalence of AR may be a particular issue for dairy-goat production due to the limited number of anthelmintic classes licensed for goats and/or applicable to lactating animals. Anthelmintic treatment will remain the cornerstone for controlling nematode infections in the near future, so measures are required to preserve the efficacy of anthelmintics. We identified high stocking rates and farmer inexperience as two potential important factors driving the development of AR. Control programmes based on understanding parasite biology and local epidemiology of infections should be implemented to prevent the further spread of AR. Stronger knowledge transfer and capacity building in farmer and veterinary communities for sustainable methods of parasite control will be necessary. Further monitoring of AR in goats and other ruminants in the CR is recommended.The goat herds were selected based on known histories of parasitic burdens and on the willingness of the farm owner to participate in the survey. The vast majority of these herds had been investigated in a parasitological survey in 2017 . Owners − 1 were applied to wells of 24-well tissue-culture plates containing an aqueous suspension of nematode eggs (~ 100 eggs per well). All dilutions contained 0.5% dimethyl sulphoxide . Three replicates were evaluated for each TBZ concentration, and three control wells without anthelmintic (0.5% DMSO) were included. The plates were sealed and incubated at 27 °C for 48 h. One drop (~ 10 μl) of Lugol’s solution was then added to each well both to prevent further hatching of eggs and to inactivate developed larvae. The numbers of eggs (those that failed to form larvae and those containing larvae) and L1 larvae in each well were determined under an inverted microscope.The EHT was used for detecting BZ resistance, as recommended by the World Association for the Advancement of Veterinary Parasitology , 62. The− 1 amphotericin B (Sigma-Aldrich) to prevent fungal contamination, 20 μl of culture medium for larval development, and 10 μl of the IVM-AG solution at the appropriate concentration were added to each well of a 96-well microtitrer plate. A total of 12 final concentrations of IVM-AG  were investigated. The culture medium consisted of yeast extract dissolved in physiological saline solution, Earle’s solution, and lyophilised Escherichia coli (see 62 or 64 for details). All reagents were purchased from Sigma-Aldrich. Each sample (including the control) was tested in duplicate. The drug was replaced by 0.5% DMSO in the control wells. The plates were covered and incubated at 25 °C for 7 days. This process was terminated by adding one drop of Lugol’s solution to each well, and the numbers of unhatched eggs and developed larvae (L1–L3 stages) in the wells were counted under an inverted microscope. Infective (L3) larvae recovered from each microplate well were identified based on both their morphological and morphometric features to the genus level as described by van Wyk et al. [Resistance to IVM was assessed using the LDT. A slightly modified method from that previously described by Hubert and Kerboeuf  was applk et al.  and van k et al. .A questionnaire on putative risk factors for AR development was administered in personal interviews with the person responsible for farm management. This questionnaire , the concentration of TBZ required to prevent 50% of the eggs from hatching, was evaluated in the EHT, and a medium lethal dose (LD50), the concentration of IVM-AG required to inhibit 50% of the eggs from developing to the L3 stage, was evaluated in the LDT. ED99 and LD99 were determined concurrently. We followed the recommendations [50 was > 0.2 μg TBZ.ml− 1. The percentage of hatched eggs at 0.3 μg TBZ.ml− 1 was also taken into account in evaluating AR based on the study by Babják et al. [− 1; hatching of 20–50% of the eggs indicated moderate level of AR, and hatching of > 50% of the eggs indicated a high level of AR. An in vitro assessment of IVM resistance requires the definition of discriminating doses for individual nematode species due to the different in vitro sensitivities of the species to IVM. The discriminating dose for in vitro detection of IVM resistance has not yet been clearly established for all species of strongylid nematodes, so we used a concentration of 21.6 ng.ml− 1 as a threshold for IVM resistance in this survey, based on previous results [− 1.A mixed logit model  was applndations , 71 intrk et al. . Only a  results . The ass results ; a high P values ≤0.05 were considered significant. The analyses were performed using R statistical software [The association between farmer characteristics, farm and pasture management, factors of parasite control, and AR status of the herds were evaluated by a univariate logistic regression analysis based on the questionnaire survey. The tests were performed independently for detecting BZ, IVM, and dual resistance (yes/no). The results were expressed as odds ratios (ORs) with the corresponding 95% confidence intervals (CIs). A two-sided Fisher exact test was applied if the model did not converge due to the lack of observations in a particular category. software .Additional file 1. A questionnaire to gather information about farmer characteristics, farm and pasture management, and parasite control measures. Data on risk factors for anthelmintic resistance development.


The Androgen Receptor (AR) is emerging as an important factor in the pathogenesis of breast cancer (BC), which is the most common malignancy among females worldwide. The concordance of more than 70% of AR expression in primary and metastatic breast tumors implies that AR may be a new marker and a potential therapeutic target among AR-positive breast cancer patients. Biological insight into AR-positive breast cancer reveals that AR may cross-talk with several vital signaling pathways, including key molecules and receptors. AR exhibits different behavior depending on the breast cancer subtype. Preliminary clinical research using AR-targeted drugs, which have already been FDA-approved for prostate cancer (PC), has given promising results for AR-positive breast cancer patients. However, since the prognostic and predictive value of AR positivity remains uncertain, it is difficult to identify and stratify patients that would benefit from AR-targeted therapies. Herein, through a review of preclinical studies, clinical studies, and clinical trials, we summarize the biology of AR, its prognostic and predictive value, as well as its therapeutic implications by breast cancer molecular subtype. Breast cancer (BC) is the most common tumor in the female population . It is cInterestingly, it has been noticed that the androgen receptor (AR) is expressed in 70–90% of the BC incidents , and thaThe Androgen Receptor (AR) is a type I nuclear receptor. In the absence of a ligand, it is located in the cytoplasm associated with heat shock proteins (HSP) and other chaperons. Once circulating androgens passively diffuse through the cell membrane, they bind to AR causing conformational changes. Subsequently, AR disassociates from heat shock proteins, gets activated, and forms dimers. These dimers translocate to the nucleus, binding to androgen-responsive elements (AREs) within target genes, causing modulations in DNA transcription. AR dimers can regulate gene transcription positively or negatively, leading to differentiation, proliferation, apoptosis, or angiogenesis  Figure . Non-ERKAR knockout mice showed dysfunctional ovulation and impaired follicular growth [AR-knockout mice showed reduced epithelial cell proliferation and reluctant ductal extension in the breast [AR is expressed in a variety of tissues in both sexes, including the breast. Interestingly, AR plays a significant role in female biology . In partr growth . Althougr growth ,14. Stude breast . It has e breast , revealip = 0.02) [The androgen receptor is expressed in more than 70% of primary breast cancers; usually, its expression is correlated to ERα and PgR. AR prevalence is higher in ERα-positive early breast tumors than ERα-negative ones (74.8% vs. 31.8% of cases respectively) . Patient = 0.02) .p = 0.0005), whereas ERα was higher in non-relapsed ones. The AR:ERα ratio was different among the subgroups (p = 0.0033), indicating the unfavorable prognostic role of AR and AR/ERα in this subset of patients [A retrospective analysis of 42 Ductal Carcinoma in situ of the breast (DCIS) patients treated with surgery followed by radiotherapy showed that AR expression was considerably higher in relapsed tumors  but lower rates of pathological complete response to neoadjuvant chemotherapy [In triple negative breast cancer, AR positivity may represent more than 50% of cases, and its expression levels vary considerably among TNBC molecular subtypes . For thiotherapy . In lumiotherapy . A recenotherapy . Taken tIt seems that the AR:ERα ratio is able predict the response to endocrine therapy . More spOn the other hand, plenty of studies have supported the hypothesis that AR status can predict the efficacy of AR inhibitors . In TNBCAs discussed, AR can trigger or suppress the oncogenic features of breast tumors depending on the bioavailability of estrogens . In thisSo far, although, natural and synthetic steroidal androgens have been used as therapeutic approach for ERα-positive BC, they have induced serious side effects . SelectiAR antagonists are also being investigated in preclinical and clinical studies. Presently-available AR inhibitors are being widely used to treat prostate cancer and are showing encouraging results in several clinical trials in breast cancer. A phase II clinical trial evaluating bicalutamide, a first-generation AR antagonist, in AR-positive/ERα-negative/PgR-negative advanced breast cancers, showed a clinical benefit rate of 19% at 6 months and a median progression-free survival duration of 12 weeks . This waEnzalutamide is a second-generation AR antagonist approved for castration-resistant prostate cancer patients . A phaseAt present, the standard treatment for advanced TNBC is exclusively nonspecific, cytotoxic chemotherapy . AlthougThe development of AR testing on liquid biopsy is an active area of research. In prostate cancer, AR concentration in the serum/plasma or urine has already been correlated with diagnosis, prognosis, and outcome prediction ,43. ReceStudying other prostate cancer research achievements, we can open new horizons in breast cancer research. For instance, the concentration of programmed death-ligand (PD-L1)-positive and PD-L2-positive dendritic cells in circulation may play a role in the selection of BC patients who are resistant to enzalutamide, as has already been shown for PC patients .Moreover, PC research showed that the co-administration of enzalutamide with a therapeutic vaccine targeting the Twist protein, a transcription factor regulating metastasis, achieved overall survival ; howeverThe co-administration of enzalutamide with a therapeutic vaccine targeting the Twist protein in an in vivo prostate cancer model achieved overall survival , openingThe value of AR as a biomarker and therapeutic target in breast cancer remains elusive. The contradictory results are due to the heterogeneity of the disease, as well as to the fact that there is no well-defined cut-off value of AR positivity. The dual role of AR as either a suppressor or inducer of tumor progression enables both androgens and antiandrogens to be used in potential therapeutic regiments. Since clinical trials have shown promising results, research should focus on the identification of novel biomarkers in order to stratify patient subgroups that will benefit the most from AR-targeted therapies.


In recent years, Augmented and Virtual Reality (AR/VR) has matured technically, delivering higher levels of immersion and presence to users, while it has also become a widely available tool to create a new range of applications and experiences. AR/VR technology allows to create scenarios which are much more stimulating and expressive than standard desktop applications, covering a wide variety of areas, namely entertainment, education, art, and health, among others.The fusion of Brain-Computer Interfaces (BCI) with AR/VR can provide additional communication channels, by increasing the bandwidth of the human-AR/VR interaction. This is achieved either explicitly through active BCIs or implicitly using passive BCIs. Active BCIs notably allow users to issue commands to devices or to enter text without physical involvement of any kind, while passive BCIs monitor a user's state  and can be used to proactively adapt the VR/AR interface. See Lotte et al.  for a geBCIs, together with AR/VR, offer the possibility for immersive scenarios through induced illusions of an artificially perceived reality that can be utilized not only in basic BCI research but also in many fields of application: In basic research, AR/VR can be used to adjust intensity, complexity, and realism of stimuli smoothly while maintaining full control over their presentation. In therapeutic applications, AR/VR can create motivating training paradigms that make use of gamification approaches or believable illusions, e.g., of virtual limbs interacting with the environment. In Human-Computer Interaction, AR/VR can be used for rapid prototyping of new interface paradigms in realistic settings. To live up to these expectations, methodological advances are required for BCI interaction and stimulus design, synchronization, or dealing with VR/AR specific artifacts , from 15 participants performing 0-, 1-, and 2-back trials and could show that workload levels could be discriminated from the scalp recordings despite the large level of physical movement occuring during VR usage. Additionally, they showed the feasibility of using functional Near Infrared Spectroscopy (fNIRS) as an alternative modality  into bi-manual control, Vourvopoulos, Škola et al. presented a study with a gamified BCI-VR training including virtual avatar designed with the aim to maintain high levels of attention and motivation. This was achieved using a progressively increasing training, event-driven and not self-paced by providing participants with score points about their progress. Performance levels in terms of classifier performance show higher than chance level (65%) and the strength of Event-Related Desynchronizations (ERDs) during session was positively correlated to the subjective magnitude of the sense of ownership while the perceived ownership of the avatar body was not correlated to the BCI performance nor to the sense of agency.Finally, The presented articles present the large potential of combining AR/VR technology with BCIs to further increase the immersiveness of AR/VR and improve the usability of BCIs for rehabilitation and control. All presented papers use elaborate, task-specific experiment setups with custom AR/VR environments and custom solutions to set up sensors, input devices, and displays [see Putze,  for an oFP created the structure and initial draft. CH, AV, and FP contributed the paper summaries. All authors contributed to the overview of the field.TM is CEO & Research Director of Intheon Labs. The remaining 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.


The androgen receptor (AR) plays a leading role in the control of prostate cancer (PCa) growth. Interestingly, structurally different AR antagonists with distinct mechanisms of antagonism induce cell senescence, a mechanism that inhibits cell cycle progression, and thus seems to be a key cellular response for the treatment of PCa. Surprisingly, while physiological levels of androgens promote growth, supraphysiological androgen levels (SAL) inhibit PCa growth in an AR-dependent manner by inducing cell senescence in cancer cells. Thus, oppositional acting ligands, AR antagonists, and agonists are able to induce cellular senescence in PCa cells, as shown in cell culture model as well as ex vivo in patient tumor samples. This suggests a dual AR-signaling dependent on androgen levels that leads to the paradox of the rational to keep the AR constantly inactivated in order to treat PCa. These observations however opened the option to treat PCa patients with AR antagonists and/or with androgens at supraphysiological levels. The latter is currently used in clinical trials in so-called bipolar androgen therapy (BAT). Notably, cellular senescence is induced by AR antagonists or agonist in both androgen-dependent and castration-resistant PCa (CRPC). Pathway analysis suggests a crosstalk between AR and the non-receptor tyrosine kinase Src-Akt/PKB and the PI3K-mTOR-autophagy signaling in mediating AR-induced cellular senescence in PCa. In this review, we summarize the current knowledge of therapeutic induction and intracellular pathways of AR-mediated cellular senescence. PCa is the most frequently diagnosed and the second leading lethal cancer among men in Western countries where the incidence has a strong correlation with aging . NotablyThe standard PCa hormone therapy includes the inhibition of the AR-signaling by ADT and treatment with AR antagonists . ADT inhThe AR is a nuclear hormone receptor and a ligand-controlled transcription factor belonging to the nuclear receptor superfamily. In the absence of ligand, the AR is primarily localized in the cytoplasm bound in a complex with multiple chaperones (heat shock proteins), which incapacitate AR from entering to the nucleus ,12. UponIn addition to this genomic activity, also a rapid non-genomic signaling of AR is known. Within few minutes, androgen treatment leads to phosphorylation events of signal transduction factors such as MEK1/2, Src, Akt and ERK1/2 as well as other proteins like filamin A through Rac1 GTPase ,21. In CRPC despite resistance to ADT, the AR-signaling is still active likely mediated through adaptive responses that activate AR-signaling by other pathways ,23,24,25Since the CRPC growth depends on AR-signaling, targeting the AR is beneficial also in CRPC . ImportaInterestingly, some PCa cell lines, such as LNCaP, exhibit an opposing response towards androgen concentrations. The growth of the CSPC human LNCaP cell line is promoted by low, physiological androgen levels  whereas paradoxically supraphysiological androgen levels  inhibits PCa growth ,31,32. ITherefore, besides novel AR antagonists, pharmacological SAL are used in clinical trials ,36. The Interestingly, both AR antagonists and androgens at SAL can trigger cellular senescence in PCa in cell culture and ex vivo tumor samples ,37, whicIn general, cellular senescence is originally referred to a stage of irreversible cell cycle arrest and has been proposed as one of the cancer suppression strategies ,39,40. ICIP1 and the p16INK4A-pRb pathways [INK4B, p19INK4D, and p27KIP1, have also been reported to mediate cellular senescence [Two known classical pathways to induce cellular senescence are the p53-p21pathways . Howevernescence ,47. An anescence . Other snescence ,49,50,51nescence ,53. It wnescence .INK4A-pRb tumor suppressor pathway seems to be an important pathway for AR-mediated cellular senescence in PCa [In PCa, the link between tumor suppressor proteins and AR-induced cellular senescence was also described. AR-induced cellular senescence associates with increased localization of PML tumor suppressor to SAHF . Additioe in PCa .Most AR antagonists prevent by competitive binding on one hand the binding of androgens to the AR. On the other hand, AR antagonists change the conformation of the AR and inhibit its activity as a transcription factor . This coIn general, AR antagonists can be structurally divided into steroidal and non-steroidal substances . A cliniInterestingly, other steroidal compounds of the 20-aminosteroidal class act as complete AR antagonists . These sINK4A and p27KIP1 are induced by Bic treatment [Several non-steroidal AR antagonists are widely used in clinics to treat PCa, most of them target the AR ligand-binding domain. The clinically used non-steroidal AR antagonist, Bicalutamide (Bic), sold under the brand name Casodex, is a first-generation antagonist ,68,69,70reatment ,59.INK4A is induced by Enz in both cell lines. Interestingly, a recent study suggests that Enz significantly enhances radiation-induced cellular senescence in LNCaP cells by potentiating radiation-mediated DNA damage [A second-generation AR antagonist, Enz, is clinically used for mCRPC patients and sold under the brand name Xtandi. Enz is an oral AR antagonist targeting the C-terminal LBD ,75,76. TA damage .Another next generation AR antagonist is Apa, sold under the brand name Erleada . It is aINK4A in both LNCaP and C4-2 cells [Due to an intermediate response characterized by slowly rising PSA, and patients who do not respond at all to Enz and Apa treatment , additio-2 cells .Pygeum africanum, which is sold under the trade name Tadenan© to treat prostate adenoma [INK4A and to a hypophosphorylation of the Rb protein, whereas the p53-p21CIP1 pathway is not significantly affected [The benzolic methylester, atraric acid (AA), is the first described natural AR antagonists and non-steroidal compound. It was isolated from bark extracts of the African tree  adenoma ,89,90. A adenoma . Neverth adenoma ,92. Furt adenoma . The treaffected . Another novel chemical platform that provides a leading structure for novel and specific AR antagonists includes halogen-substituted anthranilic acid esters. Specifically, the substitution with a halogen group at the benzene ring, exemplified by the compound C28, strongly inhibits the androgen-induced transactivation, chromatin, and DNA recruitment, as well as cell proliferation. Interestingly, cellular senescence is induced by these AR antagonists . An inhiTaken together, the molecular mechanisms to inactivate AR-mediated transactivation by AR antagonists are distinct among the various compounds. Nevertheless, AR antagonists induce cellular senescence despite the inhibition of AR-mediated transactivation, indicating that they do not completely block all AR functions. These observations suggest that AR antagonist-mediated cellular senescence does not process through a classical genomic AR-signaling pathway.Supraphysiological levels of androgens inhibit the growth of CRPC in vitro . ClinicaINK4A as well as hypophosphorylation of pRb, downregulation of E2F and cyclin D1 [INK4A suggests that SAL-induced cellular senescence is mediated through the p16INK4A-pRb pathway. Moreover, cellular senescence was induced by SAL treatment in PCa samples ex vivo obtained from patients with prostatectomy [Accordingly, the treatment of PCa cells with the natural androgen dihydrotestosterone (DHT) or the synthetic androgen methyltrienolone (R1881) leads to a concentration-dependent proliferation arrest ,31,32,97Notably, AR-negative PCa cells do not show SAL-dependent change of cell proliferation or induction of cellular senescence. Using an inducible system in AR-negative cells, SAL induces cellular senescence, revealing an AR-dependent effect ,32. ThusThe classical AR-signaling, with AR as a ligand-controlled transcription factor, is known as a genomic pathway, which is suggested to occur over several hours . HoweverIt is meanwhile known that by androgen treatment not all AR molecules translocate into the nucleus. Rather a minor fraction remains in the cytoplasm and interacts with lipid rafts containing Akt . Also, oThe activation of Src pathways is often observed in PCa ,105. SrcTherefore, a link between AR-induced cellular senescence and the PI3K/Akt/mTOR has also been suggested. Phosphoinositide 3-kinases (PI3Ks), also called phosphatidylinositol 3-kinases, are aberrantly activated by various genetic and epigenetic alterations in PCa ,109. TheAlso, the Akt family has been shown to drive PCa formation in vivo . A largeInterestingly, Akt substrates play different roles in the AR pathway. Studies showed that the Akt target FOXO3a increases AR expression via direct binding to the AR promoter , while FFor prostate tumorigenesis, it seems that the downstream effector of Akt signaling, the serine/threonine protein kinase mammalian target of rapamycin (mTOR), is essential . mTOR foTaken together, these findings suggest a non-genomic, but not necessarily rapid signaling, of AR-mediated pathways to induce cellular senescence. In line with the non-genomic action, so far many known AR antagonists reduce nuclear localization, which could be mediated by an increase of nuclear export and/or enhance nuclear mobility as a characteristic of reduced chromatin binding. Since these AR antagonists induce cell senescence, it may be hypothesized that cytosolic non-DNA-bound AR mediates cellular senescence, which eventually leads to changes in the transcriptome .The benefit of senescence induction is controversial due to emerging reports on tumor promoter effects of SASP ,44,45. HInterestingly, different senescence inducers can lead to distinct activation/upregulation of pro-survival/anti-apoptotic pathways ,138,139.Inhibition of Akt by highly selective allosteric Akt inhibitor MK2206 sensitized AR antagonist-treated cells to apoptosis, while SAL-treated cells were resistant . ImportaInterestingly, although SAL-mediated cellular senescent PCa cells seem to be resistant for senolytic agent MK2206, they are sensitive to another senolytic agent Ganetespib, a HSP90 inhibitor . The datIn conclusion, both androgens at supraphysiological levels  and AR a


Although Bell's palsy is a common etiology for isolated facial paralysis, it is important clinicians perform a complete neurologic examination to avoid misdiagnosis. Multiple cranial neuropathy is often caused by tumor or infection. Although Bell's palsy is a common etiology for isolated facial paralysis, it is important clinicians perform a complete neurologic examination to avoid misdiagnosis. Multiple cranial neuropathy is often caused by tumor or infection. We present 2 patients misdiagnosed with Bell's palsy and reviewed reported cases. Several exhibited multiple cranial neuropathies with more ominous pathology. This report illustrates the importance of thorough neurologic examination and the need for precise language in clinical practice.Acute facial paralysis is a common neurologic condition whose underlying etiology may have significant morbidity and mortality. With an annual incidence of approximately 15‐30 in 100 000 Bell's palsy is the most common reported cause of acute facial paralysis accounting for 60%‐80% of cases.2This case series and literature review describe two patients who were diagnosed with Bell's palsy and ultimately found to have multiple cranial neuropathies, including their clinical presentation, workup, treatment, and outcomes. An extensive review of the literature published before October 2019 was conducted by searching the PubMed database for reports of patients diagnosed with Bell's palsy that went on to have an underlying identifiable cause for their facial palsy. The search terms “bell's palsy,” “bell's palsy misdiagnosis,” “bell's palsy mimic,” “facial palsy misdiagnosis” were used to identify potentially relevant reports. Non‐English language publications were excluded as were any publication for which the full text was not available for review. Candidate publications were then screened for relevance based on title and abstract and relevant papers were reviewed in full with attention to the documentation of the patients’ evaluation on presentation and final diagnoses.33.1A 50‐year‐old female with no ocular history and a medical history of diabetes mellitus presented to the oculoplastic service for management of right eye lagophthalmos due to Bell's palsy. The patient reported that 4 months prior to presentation her right face became swollen and painful following a tooth extraction. This facial swelling was attributed to a dental abscess, and she went on to require intensive care unit (ICU) level care at an outside hospital for cellulitis in the setting of diabetic ketoacidosis. Her swelling resolved with antibiotic therapy, and she followed with her primary medical doctor for the next four months  for persistent complaint of right‐sided facial pain and weakness. Concurrently, she was followed by an ophthalmologist (three visits) for difficulty closing her right eye. Lagophthalmos and corneal exposure with inferior corneal scarring were noted but neither the patient's visual acuity nor the function of cranial nerves other than the facial nerve were documented at these ophthalmologic evaluations. Both providers documented concern for “Bell's Palsy,” and the patient was treated with oral corticosteroids. After 4 months, her medical doctor recommended neuroimaging (magnetic resonance imaging [MRI]) but she was referred to oculoplastics for further management of her persistent lagophthalmos prior to obtaining this study.On presentation, she had no light perception visual acuity in the right eye. Her examination was also significant for House‐Brackmann grade 4 palsy of the right facial nerve and loss of sensation in the V1 distribution of the right trigeminal nerve.3.2A 59‐year‐old female with a medical history of chronic hepatitis C was referred for management of a right nonhealing neurotrophic ulcer due to Bell's palsy. One‐year prior, she reported experiencing “lightning bolt” pains across the right side of her face. She was referred to a neurologist who did not note any abnormality of facial sensation or motor function at that time; a brain MRI was obtained and was unremarkable . She was diagnosed with trigeminal neuralgia and treated with carbamazepine. Subsequently, she noted a decrease in visual acuity and was evaluated by optometry where a serological workup for temporal arteritis was ordered and returned negative. She went on to develop right hearing loss and noticed right temporal wasting. She developed a right‐sided facial paralysis and returned to her neurologist who now documented right fifth and seventh nerve palsies. As a result, she underwent a lumbar puncture with normal protein and cell counts as well as negative results for HSV, West Nile, and tuberculosis. At this time, her ophthalmologist independently documented right facial weakness, decreased facial sensation, and neurotrophic keratitis consistent with fifth and seventh nerve palsies. The patient was told her symptoms were a result of Bell's palsy. No repeat neuroimaging was obtained after the onset of her facial weakness. She was treated with pressure patching of the affected eye but, due to a lack of resolution, was ultimately referred for management of the neurotrophic ulcer.On presentation to the oculoplastic service, visual acuity was count fingers and she demonstrated 6 mm of lagophthalmos, a poor Bell's reflex, and a large central corneal epithelial defect Figure  Incomple43015 unique publications were identified and screened as described in the Methods section. Eighty‐seven records related to the misdiagnosis of Bell's palsy and were further reviewed. Twenty‐four of these papers reported cases of patients misdiagnosed with Bell's palsy  with sufficient detail to document that a dedicated examination of the patient's cranial nerves had been performed at presentation. The patient information for the twenty‐five patients reported is summarized in Table 5The appropriate triage of patients with acute facial paralysis is a common challenge for physicians in a variety of specialties and care settings. Bell's palsy is the single most common etiology for this presentation but other diagnoses must be excluded.Factors leading to diagnostic error may be multiple and difficult to specifically identify. Review of the cases presented in this manuscript suggests that failure to perform, or incorrect interpretation of, the physical examination contributed to diagnostic error in both cases. This factor, as well as the factors suggested by the authors identified by our literature review, has been noted as contributors to diagnostic error by other reports as well. While advanced imaging studies may be appropriate in select cases, they are not infallible and up to 12.5% of malignant causes of facial nerve palsy have been missed by MRI.Multiple cranial neuropathy, like Bell's palsy, may be idiopathic but is more commonly caused by tumor and infection.The term Bell's palsy must be used with care in communication with other providers and in the literature as it implies a thorough and negative examination to exclude other entities. In the absence of documentation of this examination, referral centers must view this diagnosis with caution. A recent report from a facial nerve referral center may support this notion by reporting significantly lower proportions of patients with Bell's palsy than historical case series.None declared.CB: designed the work; acquired, analyzed, and interpreted the data; and drafted the work. NF: acquired and analyzed the data and drafted the work. LKL: conceived the work and critically revised and approved the final published version.


Prostate cancer (PCa) is one of the most frequent tumor types in the male Western population. Early‐stage PCa and late‐stage PCa are dependent on androgen signaling, and inhibitors of the androgen receptor (AR) axis represent the standard therapy. Here, we studied in detail the global impact of darolutamide, a newly approved AR antagonist, on the transcriptome and AR‐bound cistrome in two PCa cell models. Darolutamide strongly depleted the AR from gene regulatory regions and abolished AR‐driven transcriptional signaling. Enhancer activation was blocked at the chromatin level as evaluated by H3K27 acetylation (H3K27ac), H3K4 monomethylation (H3K4me1), and FOXA1, MED1, and BRD4 binding. We identified genomic regions with high affinities for the AR in androgen‐stimulated, but also in androgen‐depleted conditions. A similar AR affinity pattern was observed in healthy and PCa tissue samples. High FOXA1, BRD4, H3K27ac, and H3K4me1 levels were found to mark regions showing AR binding in the hormone‐depleted setting. Conversely, low FOXA1, BRD4, and H3K27ac levels were observed at regulatory sites that responded strongly to androgen stimulation, and AR interactions at these sites were blocked by darolutamide. Beside marked loss of AR occupancy, FOXA1 recruitment to chromatin was also clearly reduced after darolutamide treatment. We furthermore identified numerous androgen‐regulated super‐enhancers (SEs) that were associated with hallmark androgen and cell proliferation‐associated gene sets. Importantly, these SEs are also active in PCa tissues and sensitive to darolutamide treatment in our models. Our findings demonstrate that darolutamide is a potent AR antagonist blocking genome‐wide AR enhancer and SE activation, and downstream transcription. We also show the existence of a dynamic AR cistrome that depends on the androgen levels and on high AR affinity regions present in PCa cell lines and also in tissue samples. Schematic model showing how darolutamide blocks androgen (R1881)‐mediated AR signaling by inhibiting the function of normal enhancers and super‐enhancers, and impairing downstream gene transcription. ARandrogen receptorFDRfalse discovery rateGSEAgene set enrichment analysisNEnormal enhancerPCaprostate cancerROSEranking of super‐enhancersSEsuper‐enhancer1et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., The androgen receptor (AR) is a clinically validated target for the treatment of early‐ and late‐stage prostate cancer (PCa). Androgen deprivation therapy (ADT) represents the preferred initial pharmaceutical treatment for PCa and is used for nearly half the men with this disease  or 2 µm (high), and the cells were harvested 8 or 22 h post‐treatment.VCaP and LAPC4 cells were purchased from the American Type Culture Collection  and the Deutsche Sammlung von Mikroorganismen und Zellkulturen . Cells were routinely cultured as described previously . RNA integrity was measured, and samples with values above eight were further processed. RNA library preparation was performed after mRNA purification using poly‐T beads, as described by the manufacturer . Five biological replicates per condition were sequenced on a hiSeq2500 device via single‐end, 50 base‐pair reads with an average depth of 21 million reads per sample .2.3et al., N = 19 276 in VCaP and N = 18 310 in LAPC4). Differentially expressed genes were identified with DESeq2  under GSE148397. Gene expression differences of SE and normal enhancer (NE) gene groups were compared by a two‐sample t‐test.FASTQ reads were mapped via STAR aligner to the human genome GRCh38 and quantified with featureCounts from the Subread package , three replicates of eight million cells were seeded in 15‐cm plates and treated with 2 µ2.5https://www.ncbi.nlm.nih.gov/geo/) under GSE148358. Sequencing reads were mapped to human genome hg19 using the Burrows–Wheeler alignment tool with default settings  were excluded from further analysis. Peaks present in at least two replicates were used for further analysis. BAM files were used individually or merged and converted to bigwig format via Deeptools2 bamCoverage with default parameters and reads per kilobase of transcript per million mapped reads normalization     and 2 µm (high), and for two timespans (8 and 22 h) in each case. The concentrations selected corresponded to the IC50 and IC90 values previously determined in cell proliferation assays  with that of the darolutamide‐ plus R1881‐regulated genes. Altogether there were less genes up‐ or down‐regulated by R1881 in LAPC4 cells than in VCaP cells . Interestingly, in VCaP cells, there were 2575 AR peaks present in the DMSO‐only condition that were not seen in the R1881 condition, suggesting that androgen treatment can redistribute part of the AR cistrome  Heatmaps showing signals of single AR ChIP‐seq replicates centered at R1881‐induced AR‐binding sites including a 2.5 kb region up‐ and downstream. (B) Overlap of the defined AR‐binding groups in LAPC4 and VCaP cells sorted by the ratio given in percentage of overlapping regions relative to all regions in the respective LAPC4 group. (C) Motif analysis of AR‐binding clusters shown in a word cloud.Fig. S3. AR ChIP‐seq signals in healthy prostate and PCa tissue samples shown with heatmaps and centered around the AR groups identified in VCaP cells.Fig. S4. (A) ChIP‐seq signals of replicates for the DMSO, R1881 and darolutamide plus R1881 conditions centered at AR‐binding regions plus 2.5 kb up and downstream for AR groups identified in VCaP cells. (B) Heatmaps of ChIP‐seq signals from LAPC4 cells with averaged profiles for DMSO‐, R1881‐ and R1881‐ plus darolutamide‐treated samples at genomic regions bound by the AR after R1881 induction. (C) Heatmaps of ChIP‐seq signals from VCaP cells with averaged profiles for DMSO‐, R1881‐ and R1881‐ plus darolutamide‐treated samples at genomic regions bound by the AR after R1881 induction.Fig. S5. (A) Average signal plots of FOXA1 ChIP‐seq data in the identified LAPC4 cell groups. (B) BRD4 ChIP‐seq signals in VCaP cells at defined AR‐binding groupsTable S1. List of antibodies used in ChIP‐seq experiments.Table S2. GSEA output of hallmark gene sets enriched among the genes differentially expressed between darolutamide 2 µm + 1 nm R1881‐treated VCaP vs 1 nm R1881‐treated samples at 22 h post treatment.Table S3. GSEA output of hallmark gene sets enriched among the genes differentially expressed between darolutamide 2 µm + 1 nm R1881‐treated VCaP vs 1 nm R1881‐treated samples 8 h post treatment.Table S4. GSEA output of hallmark gene sets enriched among the genes differentially expressed between darolutamide 500 nm + 1 nm R1881‐treated VCaP vs 1 nm R1881‐treated samples 22 h post treatment.Table S5. GSEA output of hallmark gene sets enriched among the genes differentially expressed between darolutamide 500 nm + 1 nm R1881‐treated VCaP vs 1 nm R1881‐treated samples 8 h post treatment.Table S6. GSEA output of hallmark gene sets enriched among the genes differentially expressed between darolutamide 2 µm + 1 nm R1881‐treated LAPC4 vs 1 nm R1881‐treated samples 22 h post treatment.Table S7. GSEA output of hallmark gene sets enriched among the genes differentially expressed between darolutamide 2 µm + 1 nm R1881‐treated LAPC4 vs 1 nm R1881‐treated samples 8 h post treatment.Table S8. GSEA output of hallmark gene sets enriched among the genes differentially expressed between darolutamide 500 nm + 1 nm R1881‐treated LAPC4 vs 1 nm R1881‐treated samples 22 h post treatment.Table S9. GSEA output of hallmark gene sets enriched among the genes differentially expressed between darolutamide 500 nm + 1 nm R1881‐treated LAPC4 vs 1 nm R1881‐treated samples 8 h post treatment.Table S10. Location of constitutive AR‐binding sites found under all treatment conditions in VCaP and LAPC4 cells and corresponding genes identified based on AR peak location within 20 kbp upstream of the TSS or in the gene body.Table S11. Analysis of genes proximal to the AR‐binding regions identified in VCaP and LAPC4 cells by GREAT analysis.Table S12. MSigDB sets of genes associated with SEs in VCaP cells analyzed by GREAT.Click here for additional data file.