Wrist rehabilitation is needed to help post-stroke and post-surgery patients recover from wrist fracture or injury. Traditional rehabilitation training is conducted by a therapist in a hospital, which hinders timely treatment due to the corresponding time and space constraints. This paper presents the design and implementation of a soft parallel robot for automated wrist rehabilitation. The presented wrist rehabilitation robot integrates the advantages of both soft robot and parallel robot structures. Unlike traditional rigid-body based rehabilitation robots, this soft parallel robot exhibits a compact structure, which is highly secure, adaptable, and flexible and thus a low-cost solution for personalized treatment. The proposed soft wrist-rehabilitation robot is driven by six evenly distributed linear actuators using pneumatic artificial muscles and one central linear electric motor. The introduced parallel-kinematic mechanism design enables the enhancement of the output stiffness of the soft robot for practical use. An electromyography sensor is adopted to provide feedback signals for evaluating the rehabilitation training process. A kinematic model of the designed robot is derived, and a prototype is fabricated for experimental testing. The results demonstrate that the developed soft rehabilitation robot can assist the wrist to realize all the required training motions, including abduction-adduction, flexion-extension, and supination-pronation. The compact and lightweight structure of this novel robot makes it convenient to use, and suitable rehabilitation training modes can be chosen for tailored rehabilitation at home or in a hospital. Stroke will induce damage to the central nervous system. One of the injuries is loss of wrist motion. The wrist is an important joint that connects the hand and arm. Abnormal and frequent use of the wrist could also induce a wrist fracture or injury. Thus, rehabilitation training is essential for patients with stroke and wrist injuries, with the goal of helping patients recover to normal status2. The traditional rehabilitation training method is based on one-on-one training between a therapist and a patient. The therapist helps the patient perform repeated training; however, this approach has many inconveniences, and the cost is high3. For instance, during the COVID-19 epidemic, face-to-face treatment in hospitals has been suspended. Alternatively, wrist rehabilitation robots provide a solution to assist patients in rehabilitation training. Such robots make it possible for patients to freely enjoy rehabilitation treatment at home, which can reduce the workload of therapists and expenditure of the patients. Generally, wrist rehabilitation robots are characterized by safety, flexibility, efficiency, and practicality6.Stroke has become the main cause of abnormal human death. According to the global burden of disease research, more than 10 million strokes occur every year worldwide, and the number of people disabled by stroke is up to 116 million7. This full-featured upper limb rehabilitation robot can perform rehabilitation training in arm mode and wrist mode with its a haptic drive. Krebs et al. designed a robot called MIT-MANUS, which assists patients in quantifying exercise and allows patients to complete interactive video games for realizing rehabilitation8. However, this robot structure has a large footprint size, high cost, and complicated operation. Moreover, this robot is suitable for use only in hospitals; it is not appropriate for patients to perform rehabilitation training independently with this robot. Later, Squeri et al. designed a progressive wrist rehabilitation robot that uses an adaptive control method to achieve wrist rehabilitation by vibration. It achieves three-degrees-of-freedom (3-DOF) motion of the wrist, including extension, pronation, and flexion9. However, such a rehabilitation robot must be worn by the patient, and it has a rigid structure, which exhibits a certain degree of risk. Aabdallah et al. designed a compact wrist rehabilitation device for achieving two kinds of motion of the wrist, which detects muscle activity by using electromyography (EMG) sensors10. However, limited by its low mobility, the robot cannot drive the wrist to achieve most exercises and requires the patient’s self-motion. Similarly, Allington et al. designed a wrist rehabilitation robot with only two DOF11. The cylinder drive needs to account for the nonlinear effects caused by static friction and must reduce the effects of friction. Lan et al. designed an upper limb exoskeleton robot using series elastic actuators (SEAs)12. It adopts spherical and crank slider mechanisms, which have a light weight and can be precisely controlled. However, the rigid-body exoskeleton structure has the disadvantage of being unsafe and may cause secondary injury to the patient to a certain extent. Abhishek et al. designed a 3-Revolute Prismatic Spherical (RPS) RiceWrist parallel rehabilitation robot that can achieve kinaesthetic feedback during rehabilitation. It has a compact structure and low friction and can achieve 4-DOF movement. Due to the rigid structure of the exoskeleton, there is also a certain degree of insecurity13. In addition, Krebs et al. designed another wrist rehabilitation robot14. However, it cannot fit the actual movement tracking of the wrist, and the rehabilitation effect is not satisfactory. Moreover, Spencer et al. developed a 3-DOF wrist rehabilitation robot15, which is prone to lock-up, but the motion control is not precise enough. Additionally, several 5-DOF exoskeleton rehabilitation robots have been developed in the literature16. For patients with wrist dysfunction, a 3-DOF exoskeleton, i.e., a portable wrist rehabilitation robot, was designed in the literature17. However, such devices have relatively large footprints.In previous work, several wrist rehabilitation robots have been proposed. For example, Oblak et al. designed a rehabilitation robot for the motion training of wrists and arms18. Unlike traditional rigid-body robots, soft robots deliver motion or force with soft materials, resulting in safer interactions with humans19. Nevertheless, soft robots usually exhibit a large compliance with relatively low stiffness21. It is challenging to devise a soft robot with sufficiently large stiffness for use in wrist rehabilitation. To this end, a new soft parallel robot is designed in this work by integrating the concepts of soft robots and parallel robots. Unlike the serial-kinematic robot with multiple links and joints connected in series (such as a robotic arm), the end-effector of a parallel robot is connected to the base by multiple limbs in parallel, providing a larger output stiffness23. Hence, the proposed soft parallel robot exhibits the advantages of both soft robots and parallel robots for wrist rehabilitation. To the best of our knowledge, this is the first of its kind presented in the literature.To overcome the shortcomings of previous designs, including complex structures, safety risks, complicated operations, and limited-DOF motions, a new wrist rehabilitation robot for practical use is needed. Specifically, soft robots provide a solution25. As a type of soft-body actuator, a PAM requires only air pressure to produce its movement26. The control and operation of PAMs are easy to realize, and the structure is compact and very safe. The main structure of the wrist rehabilitation robot employs the 6-SPS/PS parallel mechanism 27. Compared with the serial kinematic mechanism, the parallel mechanism exhibits a more compact structure, lower inertia, and higher precision. Unlike traditional rigid-body wrist rehabilitation robots, the designed soft rehabilitation robot provides a safe interface, which increases the motion range of rehabilitation movement. Moreover, it can adjust the training range of the bending angle, which enables higher flexibility and safety.In particular, the soft parallel wrist rehabilitation robot presented in this work was designed based on pneumatic artificial muscles (PAMs). PAM is an emerging soft actuator that has been used in soft robotics29.According to different mechanical structures, wrist rehabilitation robots can be divided into terminal-driven rehabilitation robots and exoskeleton rehabilitation robots. This work focuses on terminal-driven rehabilitation robots owing to their better flexibility. As shown in Fig. 30.To describe the movement of the wrist, the three rotational movements are represented by three-dimensional coordinates in space see Fig. a. The ceThe purpose of the wrist rehabilitation robot is to drive the human wrist to perform the three major motions to help the patient recover to normal health status.Figure Considering the elasticity of the PAM and steel wire, the structure can be categorized as a 6-SPS/PS parallel mechanism. The central PS limb is driven by an electric cylinder, which is used to constrain unnecessary movement, such as external swing movement. The upper end of the PS limb is a spherical joint, which can support the handle to realize motion in various directions. The advantage of this design is that it is more stable, along with better limb support and stability. To express more clearly the expected working state of the wrist rehabilitation robot, the expected rehabilitation motions are illustrated in Fig. The developed prototype is shown in Fig. 19. The moving platform is connected to the six actuators by elastic steel wires. The hinge point Ai (i = 1 to 6) on the moving platform and hinge point Bi (i = 1 to 6) on the fixed platform are located in the corresponding coordinate planes. In addition, d represents the length of the electric cylinder. The circumscribed circle radii of the moving and fixed platforms are ra and rb, respectively. Based on the above settings, the kinematics and workspace of the designed 6-SPS/PS parallel structure are analysed below.To facilitate the kinematic analysis, the coordinate systems of the moving platform (o-xyz) and fixed platform (O-XYZ) are established, as shown in Fig. li (i = 1 to 6) of the six branches and length d of the central electric cylinder for the required rehabilitation training action. In contrast, given the length of the seven branches, the objective of the forward kinematics is to calculate the pose of the moving output platform31.Kinematic modelling involves both inverse and forward kinematic analyses. The purpose of inverse kinematic modelling is to solve the length 34. Although the analytical method can find all the possible solutions of the pose for the output platform, the solving processes are rather complicated for practical applications. Hence, the forward kinematics of the 6-SPS/PS parallel mechanism are solved by resorting to the numerical iterative method35.Concerning a parallel mechanism, the inverse kinematics problem is straightforward to solve based on geometric analysis. In contrast, the forward kinematics problem is more complicated to solveli (i = 1 to 6) of the six branches and length d of the central electric cylinder for the given rehabilitation training action. Referring to the geometric relationship and initial pose values  and d =|Concerning the 6-SPS/PS soft wrist rehabilitation parallel robot, the purpose of inverse kinematic analysis is to solve the length see Fig. a, the leIn addition, the objective of the forward kinematic analysis is to calculate the pose of the moving platform given the length of the seven branches. Usually, solving a series of nonlinear equations with an analytical method or a numerical method is a complex problem. Theoretically, the analytical method requires a large number of matrix operations. Although the analytical method can find all the possible solutions of the pose, the solving processes are extraordinarily complicated for practical applications. As the inverse kinematics of the 6-SPS/PS parallel mechanism have been derived easily, the forward kinematics of the 6-SPS/PS parallel mechanism are solved by an iterative method based on the inverse kinematic solution, which is explained as follows.0, β0, γ0,  x0, y0, z0). This initial pose is very different from the desired pose. According to the inverse kinematic solution, the assumed initial length of the branch li of each branch is known, the difference between the length of the ith branch at the assumed initial pose and the corresponding length with actual input is recorded by δ li. That is,First, it is assumed that the initial pose is , which can be calculated byThe change in the pose of the moving platform is expressed by  can be obtained by δli, and the pose value can be updated after the first correction:The first step of pose variation  and inverse kinematic solution, a new branch length li2 can be obtained. The new value of branch length will gradually approach the input length of the branch. In the same way, after the second correction, an updated branch length is obtained and compared with the input value. The above iterative process is then repeated.Then, according to the updated pose  is the desired forward kinematic solution.During the iterative process, when max |δThe workspace of the 6-SPS/PS parallel wrist rehabilitation robot is a collection of working points that can be reached by the reference point of the moving platform. The size of the workspace determines the range of motion of the terminal handle, which is the motion range of the wrist. Unlike the serial-kinematic robot, the workspace of the 6-SPS/PS parallel rehabilitation robot is more complicated. It is influenced by many parameters, such as the diameters of the moving platform and the fixed platform and the variation range of the branches.36. In particular, the contraction range of each branch meets the following condition:lmax and lmin represent the maximum and minimum lengths of the branch, respectively. In addition, the length variation of the electric cylinder and the rotation angle of the branch should meet the following constraints:dmax and dmin denote the maximum and minimum lengths of the electric cylinder, respectively, and φmax represents the maximum angle of the branches.Since the physics and structure of the parallel mechanism constrain the pose of the mechanism, the range of branch lengths should be considered for obtaining the workspace of the end-effector (moving platform)V) can be calculated by taking into consideration the resulting discrete points:θ is the torsion angle and ρi is the polar radius value of the ith boundary calculation. According to the inverse kinematic solution and the constraints of the workspace, the numerical method with boundary values is used to generate the workspace of the 6-SPS/PS parallel rehabilitation robot. The workspace consists of a myriad of discrete points. If the corresponding inverse kinematic solution satisfies the constraint conditions, the discrete point is considered to be located in the workspace. With the given kinematic parameters of the 6-SPS/PS parallel wrist rehabilitation robot, the spatial workspace is simulated using MATLAB software based on the boundary numerical method. The result is shown in Fig. After a series of polar radius and polar angle search calculations, the volume of the sub-workspace  in the internal chamber governs the length of the PAM and determines the external load40. The length change is defined as w, the PAM actuator volume is v, and the load force is T. Under static conditions, the PAM actuator’s static load characteristics can be derived asIn particular, the air pressure .41. Specifically, a 3-D model of the actuator is built in ANSYS software; additionally, the properties of the rubber material are defined, and the thin layer of the reticular fibre outside the model is restricted. As the working principle of the PAM is based on the Mc-Kibben muscle, a simulation study is performed with a one-end fixed, one-end free condition by applying different pressure values (0–400 kPa) at the input end. For instance, the simulation result of total deformation is shown in Fig. To reveal the feasibility and efficiency of PAM materials functioning as actuators in soft wrist rehabilitation robots, a simulation study with FEA was performed by considering their material propertiesAccording to the total deformation displacement, it is observed that the PAM actuator undergoes an expansion of the thin air pressure, an increase in diameter, a shortening of the axial length, and a gradual shortening with an increase in the pressure value. Based on the simulation results, the relationship between the PAM displacement and air pressure value and the relationship between the PAM strain and air pressure value are obtained, as shown in Fig. To further evaluate the performance of the PAM for driving wrist movement, experimental testing of the PAM was conducted by applying pressure values of 0–500 kPa. Experimental results are also shown in Fig. q is the percentage of contraction. The pressure-contraction relationship indicates the quantitative correspondence between the pressure value and PAM contraction, which provides fundamental information for controlling the wrist rehabilitation robot in experimental implementation. Thus, the degree of contraction of the PAM actuator can be adjusted by applying different pressures, which adjust the wrist motion according to the forward kinematic solution given in Eqs. . This allows extreme rehabilitation training to be performed with the designed wrist rehabilitation robot. The achievement of multiple rehabilitation modes enhances the versatility of the rehabilitation robot.N is the number of sampling points in the concerned time period and Xi is the amplitude of the EMG signal at the ith sampling point42.To evaluate the training process, the time-domain analysis of the myoelectric signals is conducted by using the average absolute value (AAV) and variance (VAR) of the eigenvalues of the EMG signal. They are calculated below.Q is defined as a parameter to measure the strength of the wrist muscle state. Under different training modes, the Q value is different, and the isometric and isotonic motion of the muscle can be recognized. The equation for the calculation of the Q value is given below.F is the power spectrum, F0 is the power spectrum component at the peak, g0 is the frequency corresponding to the maximum power spectrum, and δf is the unit frequency increment value.In addition, the power spectrum ratio Q results are shown in Table Q values are different. The Q value does not change under the same rehabilitation training mode. Essentially, Q indicates the peak area of the power spectrum and the total area occupied by the power spectrum. Since the energy of the EMG signal is concentrated at the peak value, regardless of the maximum value of the subject’s EMG signal at the frequency, its characteristic value Q will remain stable. This characteristic is very meaningful for general applications because different people produce different frequencies at which the power spectrum peaks appear, while the trend of the variation in the surface EMG signal during muscle movement is stable. Thus, the Q value can be adopted for evaluation in general.The foregoing two methods need to only amplify, rectify, and filter the EMG signal, which are easy to calculate and can be widely used in feature extraction of EMG signals. For illustration, the AAV, VAR, and Depending on the degree of wrist injury, different modes of rehabilitation training can be selected. The system implements multiple sets of training programs with LabVIEW software, which makes the rehabilitation robot more versatile and improves the patient’s autonomy in training mode selection.In this work, a new soft parallel robot dedicated to automated wrist rehabilitation tasks was designed. The robot employs an improved 6-SPS/PS parallel mechanism structure, which is driven by six PAM actuators and a linear electric cylinder. An EMG sensor is adopted to provide feedback for the rehabilitation robot system. Experimental studies verify that the developed robot meets the expected requirements. It is able to drive the patient’s wrist to achieve abduction–adduction, flexion–extension, and supination-pronation motions. In addition, different training programs, such as minor rehabilitation training and extreme rehabilitation training, have been developed and stored, allowing patients to choose suitable training modes based on their specific injuries. Such flexibility improves the versatility and autonomy of rehabilitation training. Compared with previous work, the developed soft wrist rehabilitation robot exhibits prominent superiority in terms of safety, adaptability, flexibility, cost, convenient operation, etc. The compact and lightweight structure of this robot makes it easy to use at home or in a hospital. In future work, more evaluations will be conducted to assess the rehabilitation training process. An EMG sensor-based feedback control system will be implemented, and a human–robot remote interaction will be designed to make the wrist rehabilitation robot more intelligent and convenient for practical use.Soft materials are becoming increasingly widely used in robots due to their high safety and flexibility. As a representative example of such materials, PAM actuators are made of rubber material, which can be expanded and contracted by applying air pressure. A PAM control scheme is easy to realize. The designed rehabilitation robot is driven by six PAM actuators. This robot exhibits a high degree of flexibility, adaptability, safety, and mobility, is easily operated, and has a compact footprint. To demonstrate the superiority of this soft wrist rehabilitation robot, its performance is qualitatively compared with those of typical wrist rehabilitation robots described in the literature. The results are shown in Table 43, and human–machine interface technology will be introduced to realize remote control of the mechanism based on the electroencephalogram (EEG) signal44. In addition, the adaptation feedback mechanism will be combined with clinical rehabilitation treatment to achieve a better wrist rehabilitation effect. Further development of relevant sensing, virtual reality, and navigation technologies will continue to promote the application of wrist rehabilitation robots.With the improved mechanical design of this work, the required training motions are achieved in a given space. This approach overcomes the disadvantages of traditional rehabilitation equipment, which has bulky, expensive, and heavy structures with limited mobility. Notably, open-loop control is implemented in the present work, which is sufficient for realizing the required wrist rehabilitation motion. In future work, closed-loop control will be realized to achieve more accurate control results45 and one central electric cylinder. One end of the PAM is fixed at the base, and another end is connected to the movable platform by a short elastic steel wire (diameter: 0.7 mm). The central electric cylinder along with an upper passive spherical joint is introduced as the support and constraint limb (seventh limb) with a linear drive. The PAM actuator does not require a piston rod and has a shrinkable diaphragm. It exhibits fine dynamic performance, and the shrinkage length can reach up to 25% of the rated length. Moreover, it provides sufficient load, which is highly suitable as a soft actuator for wrist rehabilitation robots. The PAMs are controlled by using a pneumatic controller . As a linear actuator, the electric cylinder provides a trust force of 200 N and no-load speed of 45 mm/s, with a stroke of 100 mm and diameter of 20 mm.The novel wrist rehabilitation soft robot is fabricated by adopting six PAMs 46. The sensor module integrates a filtering and amplifying circuit to amplify the weak myoelectric signal (in the range of ± 1.5 mV) of the human body. The output signal is provided in analogue form with the range of 0–3.0 V, and the reference voltage is 1.5 V. The signals from surface-bonded EMG sensors are acquired with a sampling frequency of 2 kHz during the wrist training process. The EMG signal is obtained using a single-board microcontroller  with a serial port connection. The obtained analogue voltage signal is then transmitted to a personal computer running LabVIEW software for the Arduino toolkit.To detect the healing effect for the wrist, a myoelectric sensor  was used to monitor the activity of the wrist and upper limb muscles


Targeting the androgen receptor (AR) signaling axis has been, over decades, the mainstay of prostate cancer therapy. More potent inhibitors of androgen synthesis and antiandrogens have emerged and have been successfully implemented in clinical practice. That said, the stronger inhibition of the AR signaling axis has led in recent years to an increase of prostate cancers that de-differentiate into AR-negative disease. Unfortunately, this process is intimately linked with a poor prognosis. Here, we review the molecular mechanisms that enable cancer cells to switch from an AR-positive to an AR-negative disease and efforts to prevent/revert this process and thereby maintain/restore AR-dependence. In prostate cancer, androgen steroid hormones bind to the androgen receptor (AR) and thereby trigger a key lineage-specific, oncogenic transcriptional program . For manOver the last decade, more effective androgen receptor signaling inhibitors  have shown additional clinical benefit in patients with CRPC, indicating that CRPC remains largely dependent on the activation of AR signaling . In lineThe stronger inhibition of AR signaling through ARSi in recent years has led to an increase of metastatic prostate cancers that de-differentiate into AR-negative disease and consequentially no longer respond to the inhibition of the AR signaling axis Fig. . While bThere is little consensus about the treatment of AR-negative disease. The situation is further complicated because many patients with therapy-induced AR-negative disease are not diagnosed as the confirmation requires the assessment of current tumor tissue. That said, the treatment of confirmed AR-negative disease with neuroendocrine differentiation, the most common subtype of AR-negative mCRPC, is a platinum-based regimen similar to those employed for the treatment of other neuroendocrine small cell carcinomas. Unfortunately, response rates to cisplatin/carboplatin combinations with either docetaxel or etoposide are relatively high, but not durable –18. ConsThe loss of AR expression during prostate cancer progression occurs as part of a larger cellular rewiring process that is paralleled by dramatic changes in cellular differentiation . As AR-nAmong the heterogeneous group of AR-negative tumors, the historically best-known subtype is neuroendocrine prostate cancer (NEPC), also referred to as small cell PCa , 23. ThiTMPRSS2-ERG gene fusions [More recent functional studies have demonstrated that NEPC typically arises from AR-positive conventional adenocarcinoma through a transdifferentiation process. Most notably, Zou et al. showed in lineage tracing mouse model studies that the neuroendocrine features arise from transdifferentiation of luminal cells . Genetic fusions , 29–31.Interestingly, there is an emerging link between the activation of Wnt/β-Catenin signaling and AR-negative disease as the Wnt/β-Catenin target genes FOXA2 and MYCN have been shown to promote neuroendocrine transdifferentiation –36. MoreBesides NEPC, other subtypes of AR-negative disease have emerged. A double-negative subtype (negative for both AR and neuroendocrine markers) that is more intimately linked to the use of new-generation inhibitors of the AR-pathway has been described more recently . The perRecently, a new subtype of mCRPC has been identified by chromodomain helicase DNA-binding protein 1 (CHD1) loss. Cancer cells with CHD1 deficiency have strong chromatin landscape alteration, which allows the outset of ARSi-resistant clones if challenged with enzalutamide . This suRecently, Labrecque et al. distinguished five different subtypes of mCRPC based on RNA expression of AR and the most common neuroendocrine markers: AR-high tumors (ARPC), AR-low tumors (ARLPC), amphicrine tumors with both the expression of AR and NE markers (AMPC), double-negative tumors (DNPC), and tumors with small cell and NE features without AR expression (SCNPC) . DespiteAs mentioned above, the different subtypes are not always clearly separated from each other and likely also not stable over time . BecauseThe de-differentiation from AR-positive to AR-negative disease involves cellular plasticity and extensive rewiring of transcription , 44–46. Multiple oncogenes and tumor suppressor genes have been linked with lineage switching from AR-positive to AR-negative prostate cancer Fig. . Importa45Notably, there is not a complete concordance between RB1 and TP53 combined loss and AR-negative diseases . In factAs mentioned above, also the activation of oncogenes can induce the de-differentiation to AR-negative prostate cancer. The oncogene NMYC is a key regulator of prostate tumorigenesis involved in de-differentiation to AR-negative prostate cancer , 53. IndThere is an intimate interplay between NMYC and AURKA, a critical kinase and oncogene involved in cellular division. Different groups have shown multiple positive feedback loops linking AURKA and NMYC and sustaining one another’s expression , 54. In The oncogenes and the tumor suppressors cited above may be part of a larger, highly interconnected network. For example, AURKA can inactivate p53 by phosphorylation and p53 activation can decrease AURKA transcription . MoreoveMultiple transcription factors involved in cellular plasticity and AR independence have been linked to prostate cancer progression to AR-negative disease Fig. . There iAnother Yamanaka’s factor, OCT4, is implicated in the de-differentiation process to AR-negative prostate cancer. OCT4 levels increase during neuroendocrine differentiation, drive AR-independence, and consequentially decreases the sensitivity toward pharmacological AR blockage –60. MorePossibly, there is also a role for MYC in prostate cancer progression. Its specific genetic locus is predominantly amplified within the 8q-arm in CRPC  and funcNANOG is another pluripotency-inducing transcription factor that has been linked to therapy resistance and tumor progression. In prostate cancer, NANOG overexpression leads to enhanced clonal growth, tumor regenerative capacity, and resistance to castration , 66. ItsFinally, Onecut2 is another transcription factor involved in cellular differentiation that has been recently associated with lethal prostate cancer and the downregulation of the AR transcriptional program. Onecut2 expression induces neuroendocrine markers, while its inhibition or silencing decreases tumor growth and metastasis formation in mice . In the The different driver genes and transcription factors outlined above cooperate to de-differentiate prostate cancer cells to an AR-negative state Fig. . Over tiOne of the most important upregulated epigenetic modifiers during disease progression is EZH2, a histone methyltransferase whose mRNA and protein levels are strongly associated with prostate cancer progression . MoreoveFinally, other key chromatin regulators are the SWI/SNF complex members, whose functions are strictly associated with euchromatin . ChangesBecause the AR pathway represents such a key therapeutic contact point in prostate cancer, researchers have set out to search for means to restore AR signaling in AR-negative prostate cancer Fig. . As mentAlternatively, drugs that affect epigenetic silencing in different ways have been successfully used to re-express AR in AR-negative cell lines. Among these are drugs that block DNA methylation (azacytidine) and increase active (HDAC inhibitors Trichostatin A) or diminish repressive histone marks (EZH2 inhibitors) at the AR promoter , 74, 79 NTR and TrkA. p75NTR is known in prostate cancer field since its tumor suppressor features [Moreover, indirect perturbations have shown the ability to restore AR expression as well. Among these are the administering of the nerve growth factor (NGF) to DU145 cells  Fig. 2)2). NGF hfeatures . Moreovefeatures . Althougfeatures . It is tMore recently, the Forkhead Box C2 protein (FOXC2) has been shown to modulate AR in both AR-positive and -negative cell lines. Generally, its expression is negatively correlated with AR presence. Indeed, its silencing in AR-negative DU145 cells can strongly re-establish AR protein expression. The effects on FOXC2 targets are mediated by Zeb1, a known transcriptional repressor. Since FOXC2 is directly activated by P-p38 phosphorylation, inhibition of this pathway by the p38 inhibitor SB203580 can exert the same effect in restoring AR expression  At this point, the studies mentioned above provide only incomplete insights into the feasibility and relevance of AR re-expression in a few cell line models. Moreover, the clinical relevance is yet to be fully discovered. Nevertheless, these findings confirm that it is possible to revert at least in part the process that leads to de-differentiation and AR negativity.The question remains as to what the functional consequences are of AR re-expression Fig. . The AR The AR gene has been exogenously transduced into AR-negative prostate cancer cell lines to study the consequences of AR expression in this setting. Although somewhat artificial, it could be useful to understand better which functionality AR can have in an AR-negative cell context. For example, in DU145 cells exogenously expressing AR leads to the upregulation of PSA  and seveAlthough few reports describe the consequences of AR revival in prostate cancer cells, results seem to be concordant. Conversely, AR re-expression may decrease the levels of transcription factors that were initially involved in the shutdown of its expression. For example, it has been shown that AR can directly bind the enhancer element within the SOX2 promoter in prostate cancer cells, resulting in the inhibition of SOX2 transcription . Taken tProstate cancer is almost uniformly dependent on androgens at its initial presentation , while tThe development and clinical success of more efficient AR signaling inhibitors come with the drawback of an increased prevalence of highly aggressive, AR-negative prostate cancer. Lineage plasticity as a resistance mechanism will likely further increase in the future with the clinical implementation of potent chimeric small molecule AR degraders . BetweenMouse models may help to elucidate factors in the tumor microenvironment or specific cell types of the host that contribute or prevent the switch from AR-positive to -negative disease Fig. . In lineThe models mentioned above may also help develop new therapeutic approaches to treat or prevent the occurrence of AR-negative prostate cancer Fig. . Indeed,Over the last few decades, many researchers have pursued endeavors to re-activate AR expression in progressed prostate cancer cells Fig. . Most stTP53 and RB1 mutations). The development of cell line models that switch easily from AR-positive to -negative disease may be useful to search for such combinatorial approaches. Complementary, loss-of-function screens may nominate effective therapeutic targets as well. Possibly, these efforts may identify target proteins that function in both oncogenic AR signaling and the induction of cellular plasticity to AR-negative disease. For example, the AR activator TRIM24 has been recently also involved in the activation of SOX2 [In a treatment naïve setting, the upfront inhibition of key factors involved in the lineage plasticity may render responses to ARSi more durable Fig. . Such a  of SOX2 , 105.In conclusion, AR-negative prostate cancer incidence will further rise as our ability increases to abrogate AR signaling. The characterization of this currently fatal disease will hopefully contribute to the development of better treatment options for patients facing AR-negative disease and combination therapies to prevent disease progression under ARSi treatment.


Accurate and precise determination of free fatty acid (FFA) contents is essential for quality control and assurance in food production. Herein, a mass spectrometric study was performed to develop a sample preparation protocol that can minimize exogenous FFA contamination during the quantification of FFA in food. The quantities of exogenous FFAs were measured using various combinations of seven pretreatment methods for a sample tube, three extraction methods, and four types of sample tubes. Methanol washing could effectively reduce exogenous palmitic acid (PA) and stearic acid (SA) by 73 and 64%, respectively, in contrast to furnace baking resulting in a decrease in the amount of PA and SA contaminants by 50 and 37%, respectively. A lower amount of FFA contaminants was extracted from glass tubes during comparative analysis of the four different sample tubes studied. A combination of a methanol-washed glass tube and chloroform extraction solvent was determined to be the optimal method for minimizing the error in FFA quantification. The optimized sample preparation protocol used for FFA quantification can lower the amount of foreign palmitic acid and stearic acid to the sub-nanomolar level in the analysis of FFAs in skimmed milk. Thus, the accurate quantification of FFAs in food is critical for quality control and assurance in the food industry5. However, it is challenging to accurately and reliably determine the FFA contents in food because FFAs typically exist at low concentrations in the presence of highly complex matrices consisting of other nutrients, such as fats, proteins and carbohydrates7.Fatty acid (FFA) present in food has diverse effects on health and nutrition as well as sensory characteristics of food, such as flavor and taste8, followed by gas chromatography (GC) combined with either flame ionization detector (FID) or mass spectrometry (MS)9. Very recently, Mannion et al. proposed the conversion of FFAs into fatty acid butyl esters as a novel esterification strategy for the accurate quantification of FFAs in dairy foods10. Supercritical fluid chromatography12 and high-performance liquid chromatography (HPLC)17 have also been widely exploited to quantify either derivatized or non-derivatized FFAs.Various analytical techniques have been employed for the quantification of FFAs in food. The most commonly used method to quantify FFAs involves the chemical derivatization of FFAs into fatty acid methyl esters (FAMEs)18 and Bligh/Dyer (B/D)19 liquid–liquid extraction (LLE) methods, employing a mixed solvent consisting of chloroform, methanol, and water at various ratios, have been used as the standard procedure to extract FFAs. Matyash and coworkers suggested methyl-tert-butyl ether (MTBE) as an alternative organic solvent, which can occupy the top layer in two-phase LLE and facilitate the recovery of FFAs, while avoiding contamination with the aqueous phase20. LLE is an easy and efficient method used for the extraction of FFAs, but it may also be used to extract exogenous FFAs used as additives to improve the surface properties of plastic consumables21, such as pipette tips, sample tubes22, and barrels of solid-phase extraction columns23. Exogenous FFA contaminants are also found in soft polyethylene frits containing a plasticizer24, chromatographic papers used for blood collection25, glass products and even high-purity organic solvents26. Unfortunately, the amount of foreign FFA contaminants introduced during the sample preparation procedure is high enough to be detected in MS used for the quantification of FFA and complete elimination of these contaminants remains a significant challenge. The presence of FFA contaminants eventually leads to an overestimation of the endogenous FFA content, resulting in inaccuracies during the quantitative measurement of FFAs. Thus, it is of great importance to determine the amount of FFA contaminants originating from experimental reagents or apparatus and to set up an experimental procedure that can reduce the impact of exogenous FFAs on the quantification of endogenous FFAs present in food samples.Regardless of the analytical method used for quantification, the extraction of FFAs from food samples is a prerequisite step to reduce any interference from the food matrix in the downstream analysis steps. FolchHerein, we have conducted a systematic study to develop an optimized experimental protocol that minimizes the amount of foreign FFA contaminants negatively affecting the accurate quantitative analysis of FFAs. The amounts of exogenous FFAs detected in LC–MS were compared with regard to the following three experimental parameters: (1) Pretreatment of the sample container, (2) extraction method, and (3) type of sample container. Finally, the optimal method established in this comparative study was exploited for the quantification of FFAs found in commercial milk samples using LC–MS.n-hexane, ammonium hydroxide and formic acid were from Sigma-Aldrich . HPLC-grade methanol (MeOH) and ethanol (EtOH) was from DUKSAN . Isotope-labeled [methyl-d3]-PA (d3-PA) and [methyl-d3]-SA (d3-SA) were from Cambridge Isotope Laboratories Inc. . Plastic tubes were from Eppendorf (Ep)  and SPL Life Sciences (SPL) , and glass tubes were from Glassco Laboratory Equipments (GLASSCO)  and Supelco Analytical Products (SUPELCO) .HPLC-grade water, acetonitrile (ACN), and chloroform were from Thermo Fisher Scientific . Palmitic acid (PA), stearic acid (SA), HPLC grade MTBE, 26. In the second method, a glass tube was rinsed three times under ultrasonication for 10 min in different organic solvents22. MeOH, EtOH, hexane, and a mixture of EtOH and hexane, which are commonly used for solvation of fatty acids27, were tested. The mixture of EtOH and hexane was chosen at the azeotropic concentration  because it possesses a constant composition during evaporation. In addition, to investigate the effect of pH on solvation of exogenous FFAs, 0.1% (v/v) of formic acid or ammonium hydroxide were added to EtOH as a pH modifier. These experiments were conducted with GLASSCO glass tubes that are durable at very high temperature. After the pretreatment steps, the residual FFAs were extracted using the Folch method, as shown in Table Different pretreatment methods of sample containers were evaluated in terms of their efficiency toward eliminating background FFA contaminants in an empty tube. The first method was to bake a glass tube overnight at 450 °C in an electric furnaceTo compare the level of exogenous FFAs extracted from commercially available consumables, two plastic tubes (Eppendorf and SPL) and two glass tubes (GLASSCO and SUPELCO) were subjected to three different FFA extraction methods  with some modifications. The composition and ratio of the solvents used for each extraction method are listed in Table 4 with some modifications. Briefly, 15 mL of the milk sample was sonicated for 15 min to disrupt the micelle structure of milk and 100 μL of the sample was then transferred to an Ep tube or MeOH washed GLASSCO tube. The FFAs were finally extracted using the Folch and B/D methods for triplicate samples.Skimmed milk samples were purchased from a local market in Gwangju, South Korea and stored at 4 °C prior to analysis. Two different conditions were tested for the extraction of FFAs from skimmed milk. The first was a general FFA extraction method without any pretreatment of the sample tube and the second was the optimized FFA extraction scheme that minimizes the level of exogenous FFA contaminants, which was established from the comparative experiments described in the above sections. The procedure used for FFA extraction from skimmed milk was carried out according to the method described by Scano et al.FFAs were analyzed on an Agilent 6520 quadrupole time-of-flight mass spectrometer coupled with an Agilent 1260 infinity HPLC system  after injecting 20 μL of a sample. Chromatographic separation was performed on a C8 column  using isocratic elution with a mobile phase consisting of 0.1% formic acid in water and 0.1% formic acid in ACN  for 5 min at a flow rate of 1 mL/min. The C8 column was used to eliminate the possibility of the carryover effect of long-chain FFA, instead of a C18 column, which is commonly used in lipidomics. The temperatures of the column and auto-sampler were maintained at room temperature and 4 °C, respectively. The ESI parameters were as follows: Sheath gas, 350 °C at 12 L/min; nebulizer, 60 psi; capillary voltage, 3.5 kV; fragmentor, 200 V. MS was performed in negative ion mode from 200 to 300 m/z with an acquisition rate of 2 spectra/sec.The FFAs were identified based on the accurate mass and retention time of their corresponding standards. For the accurate quantification of PA and SA, which are known as the two major FFA contaminants originating from the sample tube, d3-PA and d3-SA were spiked into the extraction solution as internal standards (ISTDs) at a concentration of 5 μM and the absolute concentrations of PA and SA were obtained using the ratio of peak area observed for STD:ISTD. Quantification of FFAs was performed using Agilent MassHunter Qualitative Analysis software (ver. B.05.00). The ratio of endogenous to exogenous FFA contaminants in the skimmed milk samples was calculated using the following equation: [Amount of exogenous FFA contaminant/ total extracted FFA (exogenous FFA contaminants + endogenous FFA)] × 100.To compare the FFA removal efficiency of different pretreatment methods, significant differences in the amounts of exogenous FFAs were tested by an analysis of variance (ANOVA). The data normality and homogeneity of variance were checked with Shapiro–Wilk test and Levene’s test, respectively. Since the amount of FFA was not normally distributed and its variance was not homogeneous, Kruskal–Wallis nonparametric one-way ANOVA was conducted. If a difference in the amount of FFAs among the conditions was found to be significant, Dunn’s post hoc analysis was conducted to reveal the pairs of significantly different conditions. For comparison of exogenous FFAs with and without pretreatment using different extraction methods and types of sample tubes, Welch’s t test for unequal variance was used. All statistical tests were conducted using R statistical software .Multiple pretreatment methods were tested to estimate how much of FFA contaminants in a sample tube can be reduced. First, GLASSCO glass tubes were baked in an electrical furnace overnight at 450 °C, which is higher than the boiling points of the FFAs analyzed, in order to evaporate the physically adsorbed residual FFA contaminants on the glass surface. Second, the glass tubes were sonicated for 10 min in various solvents three times to leach out hydrophobic FFAs.n = 18) after application of furnace baking and MeOH washing are shown in Fig. P = 1.39 × 10−1 for furnace baking and 4.53 × 10−6 for MeOH washing, Kruskal Wallis test, Dunn’s post hoc test with Bonferroni correction). The poor removal efficiency of SA upon furnace baking is in agreement with the lower vapor pressure of SA compared to PA at equal temperature28. In contrast, a significant reduction of PA or SA was observed upon MeOH washing, resulting in an average of 73% and 64% removal for PA and SA, respectively. Figure n = 6 for each lot, n = 18 for total). The variation in the concentration of exogenous FFA among different packages was minor compared to that among individual tubes from a same package . Thus, the rest of analysis was performed in a single batch, rather than three batches.The amounts of PA and SA remaining in the tubes  is displayed in Fig. P = 2.72 × 10−2, Kruskal Wallis test, Dunn’s post hoc test with Bonferroni correction). Pretreatment with n-hexane was not effective for exogenous FFA removal, in spite of its higher solubility for FFAs over MeOH and EtOH29. Although the acidified EtOH could significantly reduce PA, it was not effective for SA removal. Since only MeOH washing showed significant reductions of exogenous PA and SA, it was chosen as the pretreatment method for further experiments to minimize the amount of exogenous FFAs extracted from the sample tube.The comparison between different pretreatment solvent types for exogenous FFA removal in sample tube , respectively. For the Folch method, the MeOH washing step of GLASSCO tubes decreased the amounts of PA and SA contaminants by 37%  and 12%, respectively. Among the four different types of sample tubes studied, GLASSCO tubes exhibited the minimum absolute amount of residual PA and SA on average for the three extraction methods after the MeOH washing step. However, MeOH washing was not effective to significantly reduce the amount of FFA contaminants in SPL, Ep and SUPELCO tubes.To study the effect of the pretreatment used for the sample tubes on the conventional FFA analysis protocols studied, the MeOH washing method was applied to all combinations of the four sample tubes  and three conventional FFA extraction methods  studied, and compared with the non-pretreatment method in terms of the FFA contaminants present in the sample tubes. The amounts of PA and SA extracted from empty tubes with and without MeOH washing are displayed in Fig. It is noteworthy that only two out of the 24 combinations of the four sample tubes and three extraction methods showed statistically significant differences in the amount of PA or SA contaminants extracted after the MeOH washing step. The average standard deviations of exogenous FFAs for untreated GLASSCO tubes were 3–5 times higher than those of MeOH washed GLASSCO tube. These results imply that washing GLASSCO tubes with MeOH lowers the standard deviation of the measurement of FFA contaminants, leading to the improved precision in the quantification of FFAs. However, no significant difference in the standard deviation of the measured FFA contaminants was observed for the other types of tubes. Interestingly, no apparent tendency was observed in the extraction efficiency of the PA or SA contaminants in the sample tubes among the Folch, B/D, and Matyash extraction methods. On average, the Folch method extracted the highest amount of PA and SA contaminants from SPL tubes, while the Matyash method extracted the highest amount of PA or SA contaminants from Ep tubes and SUPLECO tubes. On the other hand, the relative abundance of exogenous FFAs extracted using the three methods was quite consistent between PA and SA within the same type of tube, which indicates that the extraction efficiencies of PA and SA were similar in each specific FFA extraction method.The presence of exogenous FFA contaminants introduced during the sample preparation step will cause an overestimation in the quantification of FFAs in food. To assess the contribution of FFA contaminants to the analysis of the FFA content in food, two different tubes were used for FFA sampling: One was a normal Ep plastic tube with no pretreatment and the other was a GLASSCO tube rinsed three times with MeOH, which resulted in the minimum amount of residual FFAs in the experiments described in the above section. The Folch and B/D protocols were employed to extract FFAs in this study because the Matyash extraction method showed the highest PA and SA contaminants in GLASSCO tubes Fig. . SkimmedThe quantification results obtained for PA and SA extracted from 100 μL of skimmed milk using different combinations of the sample tubes and extraction methods are summarized in Table The present study was designed to search for an optimized experimental procedure that minimizes the amount of foreign FFA contaminants negatively affecting the accurate quantification of FFAs in food. The quantities of exogenous FFAs detected using LC–MS were compared in terms of the pretreatment of the sample tube, FFA extraction method, and type of sample tube used. The combined use of MeOH washing and glassware proved to be the most effective way to reduce the amount of FFA contaminants. In particular, MeOH washing is helpful for further lowering the amount of PA and SA contaminants in glass tubes where the amount of exogenous FFAs is typically at the sub-nanomolar level before MeOH washing. It is strongly recommended to avoid the use of plastic consumables that are expected to be the main source of exogenous FFA contaminants during the analysis of FFAs in food.Supplementary Information


Prostate cancer is the second most common cancer in men world-wide, with nearly 1.3 million new cases each year, and over the next twenty years the incidence and death rate are predicted to nearly double. For decades, this lethal disease has been more or less successfully treated using hormonal therapy, which has the ultimate aim of inhibiting androgen signalling. However, prostate tumours can evade such hormonal therapies in a number of different ways and therapy resistant disease, so-called castration-resistant prostate cancer (CRPC) is the major clinical problem. Somewhat counterintuitively, the androgen receptor remains a key therapy target in CRPC. Here, we explain why this is the case and summarise both new hormone therapy strategies and the recent advances in knowledge of androgen receptor structure and function that underpin them.Prostate cancer (PCa) is the most common cancer in men in the West, other than skin cancer, accounting for over a quarter of cancer diagnoses in US men. In a seminal paper from 1941, Huggins and Hodges demonstrated that prostate tumours and metastatic disease were sensitive to the presence or absence of androgenic hormones. The first hormonal therapy for PCa was thus castration. In the subsequent eighty years, targeting the androgen signalling axis, where possible using drugs rather than surgery, has been a mainstay in the treatment of advanced and metastatic disease. Androgens signal via the androgen receptor, a ligand-activated transcription factor, which is the direct target of many such drugs. In this review we discuss the role of the androgen receptor in PCa and how the combination of structural information and functional screenings is continuing to be used for the discovery of new drug to switch off the receptor or modify its function in cancer cells. Prostate cancer (PCa) is the second most common cancer in men world-wide, with nearly 1.3 million new cases each year   is credited with the first true description of the prostate gland in the 16th century. However, due to the location of the prostate gland, access to surgically excise tumours was seen as particularly challenging and from the mid-18th century to the early 20th century different approaches were trialled. Due to pioneering approaches and the skill of early surgeons such as Eugene Fuller (New York), Peter J. Freyer (UK), Hugh H. Young  and H. Kuchler (Germany) radical proctectomy has been a standard of care for over 100 years (reviewed in [5 to 1569The differentiation and development of the prostate is dependent upon the androgen dihydrotestosterone (DHT), which can be derived directly from testosterone in prostate cells or by an alternative pathway in the embryo ,11,12. IThe “classical” model for androgen action, via the AR, is illustrated in AR gene is present on the long arm on the X chromosome (Xq11-12) and contains eight exons interrupted by introns of varying lengths (0.7–2.6 kb) and codes for a protein of approximately 920 amino acids. There is variation in overall length due to polymorphic amino acid repeat regions within the N-terminal region. One of these, a polyglutamine repeat, is associated with the neuronal degenerative disorder Spinal and Bulbar Muscular Atrophy (SBMA) when the glutamines number greater than 38  [The AR  is a member of the steroid hormone group of nuclear receptors along with the oestrogen receptors , glucocorticoid receptor , progesterone receptor  and mineralocorticoid receptor . The lations) . A predolations) ,33. The The AR-NTD makes up more than 50% of the receptor protein and shows little or no sequence homology with even its closest relatives, the GR and PR . ImportaIn contrast to the rest of the protein the AR-NTD is intrinsically disordered and demonstrates a high degree of structural plasticity , witIn the case of the AR the presence of the NTD has also been shown to modulate DNA binding affinity for different response elements . There i578, Ser579Val582) as part of the DNA recognition helix. Five amino acids in the second zinc cluster form the “D-box” (Ala597-Ser-Arg-Asn-Asp601), involved in receptor dimerization. The P-box residues are conserved in the GR, PR and MR leading to shared response elements (often termed hormone response elements or HREs) and potentially overlapping gene signatures. However, the AR-DBD dimerisation and the CTE are thought to contribute to specific binding to selective DNA sequences, termed androgen response elements (AREs) ([The DNA-binding domain (DBD) is the most well-conserved region between different steroid receptors and is defined by nine conserved cysteine residues. The AR-DBD has a helical-globular structure  in which (AREs)  (. These d (AREs)  ( and have (AREs)  it was found that loss of AR binding to such selective AREs resulted in infertility  due to impaired sperm maturation in the epididymis . More reThe onset, development and progression of PCa depends on androgenic hormone activation of the receptor and from the early work of Huggins and Hodges the AR is recognized as a crucial drug target in the fight against this cancer. First, PCa is treated by depriving tumours of androgens such as DHT and testosterone or blocking their actions by impeding their direct binding to the ligand binding pocket (LBP) on the LBD . The com10 helices, and four short β-strands assembled in two anti-parallel β-sheets  and detailed atomic information on how the hormones or analogous molecules were nested in the pocket. This initial information allowed more predictive models of designed compound binding and fuelled intense drug discovery programs to develop more selective competitive AR modulators (SARMs). Since these first structures of the LBD , structuThe LBP of AR exhibits numerous residues , which fhttp://androgendb.mcgill.ca) [Several point-mutations in the LBP  have beegill.ca) .Mutations in the LBP have been shown to underlie a switch in activity of some therapeutic antagonists to have agonist activity. These point mutations encode residue substitutions that increase the agonist activity of the receptor because it no longer recognizes clinical anti-androgens as antagonists, and the receptor continues being activated. Several mutations inside the LBP  have been found in advanced tumours and observed to result in the acquirement of agonist activity of anti-androgens. Most of these mutations expand the LBP cavity by concrete structural rearrangements in the surrounding helices conforming the walls of the pocket. So AR LBP, as has been shown for other related receptors, exhibits certain structural plasticity and flexibility in its LBP that allows the protein to rearrange—induced by the action of designed ligands and/or point mutations that are mostly treatment-induced .Inhibitors of AR action can be broadly categorised as drugs that target the steroid biosynthetic pathway through effects on the adrenals, gonads, hypothalamus or pituitary gland, including leuprolide, finasteride, dutasteride and abiraterone or drugs that act on the receptor protein directly, for example bicalutamide and enzalutamide  Figure .Anti-androgens have been developed that differ in their chemical structure and exhibit different efficacy and safety profiles and in the case of flutamide, nilutamide and bicalutamide were primarily developed to be used in combination with chemical or surgical castration to provide combined androgen blockade. In subsequent clinical trials with enzalutamide, significant survival benefits were demonstrated in patients with metastatic, castration resistant PCa (mCRPC). More recently developed antiandrogens such as apalutamide and darolutamide have proven to be effective at overcoming resistance to the antiandrogens bicalutamide and enzalutamide ,61.The first anti-androgen to be used clinically was the steroid cyproterone acetate CPA; , a compeFlutamide  is a comBicalutamide  was deveMalat1 has also been shown to drive enzalutamide mediated selection of AR-v7 expression and downregulation of this splice variant can be achieved by siRNA and the degradation enhancer ASC-J9 [Enzalutamide  is a secr ASC-J9 . The F87r ASC-J9 ,61. It ir ASC-J9 .Non-steroidal anti-androgens compete with testosterone and DHT for AR binding, thereby inhibiting receptor-dependent gene regulation and prostate tumour growth. However, antiandrogens also act systemically throughout the body and can therefore block negative feedback of testosterone action at the hypothalamus and pituitary. This can result in a moderate increase in the levels of plasma testosterone and oestrogens, which in PCa may eventually lead to resistance. Thus, anti-androgen withdrawal is recognized to be beneficial for a sub-set of patients, providing a short-term regression in disease (up to six months). The mechanism underlying this response or indeed identification of the patients likely to show a response is poorly understood, but mutations in the AR may play a part.Anti-androgens have now been improving outcomes in advanced PCa for decades, indeed they are moving into their third generation. Many in the field feel there is still more to be done with existing, widely-used, well-characterised anti-androgens and that moving them earlier in the pathway, or/and combining them with other drugs, will improve patients’ outcomes and quality of life further . The larLast year two further multinational trials, ENZAMET (NCT02446405) and ARCHES (NCT02677896), reported that addition of the antiandrogen enzalutamide to standard-of-care improved overall survival and radiographic progression-free survival, although with some toxicity ,85,86,87As discussed below, a number of new therapies are designed to target functional domains of the AR other than the LBD. That said, the AR LBD remains a valuable therapeutic target, both because there is compelling evidence that use of a second anti-androgen or SARM after resistance develops to a first has clinical benefit, and because the more selective ligands may reduce side-effects by acting in a more tissue-specific manner. Hence new SARMs are continually being developed and assessed for their ability to extend life and, importantly, improve quality of life in CRPC. Apalutamide has recently been approved for use in non-metastatic CRPC based on results of the SPARTAN trial (NCT01946204), again combining the new anti-androgen with standard ADT. Median metastasis-free survival increased from 16.2 months in the control arm to 40.5 months in the Apalutamide arm, again with slightly increased incidence of adverse effects . DarolutExtending the use of drugs that target the LBD, recent years have seen the development of a new class of inhibitors, which not only compete for hormone binding, but have the capacity to induce AR protein degradation  ,91. ReceGaleterone, a potent steroidal multi-target agent, has been shown to reduce AR levels by increasing degradation, reducing levels of both full-length AR and AR-v7 ,99,100. In recent developments, Narayanan and co-workers have described a number of small molecules, UT-69, UT-155 and UT-34, which inhibited AR transcriptional activity and led to ubiquitin-mediated turnover of the receptor protein ,105. WhiAlthough new antiandrogens such as apalutamide and darolutamide continue to be developed and brought to the clinic, the emergence of tumour resistance due to point mutations and, more recently, the discovery of AR variants without the canonical druggable LBD which may contribute to resistance ,80,108  has contThe structural plasticity of the AR-NTD, which underpins transactivation activity, presents several challenges in terms of identifying and developing antiandrogens targeting this domain. However, in a ground-breaking study, Sadar and co-workers identified a bisphenol A glycidyl ether derivative from a marine sponge extract that exhibited anti-androgen activity . The bisDysidea sp., and inhibited proliferation of LNCaP cells and hormone dependent AR transactivation as well as forskolin induced transactivation by the AR-NTD [Interestingly, the Sadar group has described other marine compounds reported to mediate antiandrogen activity, naphetenone B and sinkotamides A-E, which also target the AR-NTD . Sintokae AR-NTD ,116. Nipe AR-NTD ,118. Thee AR-NTD . These sIn addition to the described studies screening marine sponge extracts, on-going research into targeting the AR-NTD includes high throughput screening of compound libraries , alongside the design of polypeptide antagonists based on the AR-NTD ,120 and Ligand binding creates surfaces on the LBD such as the AF2 and BF3 pockets, which directly or indirectly are important for protein–protein interactions ,121. TheAs AR-variants lacking the LBD retain the DBD, targeting therapies to the DBD is an emerging area of interest. Surprisingly perhaps, given that its structure has been known for the longest, this is the least-targeted region of the AR, probably due to its high homology to the DBD of other steroid receptors, so cross-inhibition is a potential problem. Recently, the PROTAC approach has moved in this direction: MTX-23 is a PROTAC targeted to the DBD, which recruits Von Hippel-Linda E3 ubiquitin ligase resulting in degradation of both full-length AR and AR-v7 . Only tePyrvinium pamoate (PP) was in this way found to be a non-competitive AR inhibitor. While it did also inhibit related receptors, including GR and ER, interestingly the inhibitor activity appears to be highly tissue specific and highest in prostate, potentially limiting off-target effects [Another approach to DBD inhibition is screening for small molecule inhibitors.  effects . However effects , that alDespite the mixed or inconclusive outcomes of early studies with cytotoxic drugs  taxanes The differential trafficking of AR splice variants is intriguing, as AR-FL and AR-v7 have been found to have distinct but overlapping gene signatures (see ). HoweveUBE2C. However, in this model system docetaxel treatment did not reduce AR-target gene expression and although selective knock-down of AR-v7 resulted in decreased cell proliferation it failed to restore sensitivity to taxane treatment [In contrast to the above study , Kyprianreatment . Taken treatment ,135.With the projected rise in the number of men with PCa there will be a continued clinical need for improved diagnosis and therapeutic options. The importance of AR signalling in advanced and mCRPC ensures that the receptor will remain a primary drug target. Whilst academic research and the pharmaceutical industry continue to develop new LBD targeted anti-androgen therapy, the mechanisms of resistance that drive CRPC suggest that the provision of fundamentally different approaches to therapy may be beneficial.The AR-NTD is essential for transcriptional activity and despite the structural challenges of this domain has become the target for a number of candidate inhibitors and drug-discovery approaches. Similarly, targeting surfaces of the AR distinct from the LBP  while also posing significant challenges, offer the potential for developing “selective androgen receptor modulators” and novel inhibitors to overcome drug resistance that arises in advanced and metastatic PCa and for reducing side-effects. It is also worth remembering that, although most AR-focused drug discovery programs are aimed at tackling PCa, receptor modulators may also be of value for the treatment of emerging other conditions such as androgenic alopecia, breast cancer, melanoma, medulloblastoma, androgen insensitivity syndromes or SBMA. However, despite promising results for both SARMs and SARDs in cell-based and, in some cases, pre-clinal in vivo models none have so far been translated to the clinic. To date, only ralaniten (EPI-0506) the NTD inhibitor has made any real progress towards clinical development.The AR is the key driver of, and therapy target in, PCa at all stages including advanced CRPC. Therapy resistance can arise via alterations in the AR, but new drugs and combination strategies can overcome this. The development of effective new drugs relies on detailed knowledge of the mechanism of action of AR, its structure and how this changes when bound to ligands. In the near future we anticipate the licensing and/or clinical adoption of both new combinations of existing drugs and of new drugs targeting different regions of the AR protein.


Skeletal muscle possesses an outstanding capacity to regenerate upon injury due to the adult muscle stem cell (MuSC) activity. This ability requires the proper balance between MuSC expansion and differentiation, which is critical for muscle homeostasis and contributes, if deregulated, to muscle diseases. Here, we functionally characterize a novel chromatin-associated long noncoding RNA (lncRNA), Lnc-Rewind, which is expressed in murine MuSCs and conserved in human. We find that, in mouse, Lnc-Rewind acts as an epigenetic regulator of MuSC proliferation and expansion by influencing the expression of skeletal muscle genes and several components of the WNT (Wingless-INT) signalling pathway. Among them, we identified the nearby Wnt7b gene as a direct Lnc-Rewind target. We show that Lnc-Rewind interacts with the G9a histone lysine methyltransferase and mediates the in cis repression of Wnt7b by H3K9me2 deposition. Overall, these findings provide novel insights into the epigenetic regulation of adult muscle stem cells fate by lncRNAs. Among tmic loci .Lnc-mg has been shown to regulate MuSC differentiation by acting as a sponge for Mir125b  promoter, leading to CpG hypermethylation and silencing (Repressor of wnt induction), a chromatin-associated lncRNA conserved in human and expressed in proliferating C2C12 myoblasts.In muscle, high-throughput transcriptome sequencing (RNA-seq) and differential expression analyses have facilitated the discovery of several lncRNAs that are modulated during the different stages of skeletal myogenesis and dysregulated in muscle disorders . Althoug Mir125b . In the ilencing . In mousilencing . Among tWnt7b gene. Our data show that Lnc-Rewind expression is necessary to maintain Wnt7b repressed and to allow MuSC expansion and proper differentiation.Here, we provide evidence on the role of Lnc-Rewind in the epigenetic regulation of the WNT (Wingless-INT) signalling in muscle cells. The WNT transduction cascade has been demonstrated to act as a conserved regulator of stem cell function via canonical (β-CATENIN) and non-canonical  signalling, and dysregulation of its activity has been reported in various developmental disorders and diseases . In the pre-Mirlet7c-2 (mmu-let7c-2) and pre-Mirlet7b (mmu-let7b) genomic loci  isolated MuSC (Myog) and muscle creatin kinase (Mck) (2C12 and MuSC-derived myoblasts and significantly decreased in fully differentiated (DM) cells  . QuantitM) cells . SubcellM) cells . AccordiM) cells  and C2C112 cells  confirmeMirlet7b and Mirlet7c-2) revealed that, although the lncRNA depletion results on their concomitant downregulation (Mirlet7b and Mirlet7c-2 targets (~1% of the upregulated genes)  and troponins   progeny upon Lnc-Rewind downregulation (+-derived clusters (composed of more than two nuclei), pairs (composed of two nuclei), or single MuSCs within each myofibre revealed a reduction of activated pairs and clusters upon Lnc-Rewind depletion  or the scramble control (GAP-SCR) . A princgulation , none ofd genes) , belong d genes) . Of note, Tnnt1) , were do, Tnnt1) . Moreove, Tnnt1) , suggest, Tnnt1)  might re, Tnnt1) . Accordianscript  and protanscript . In furtgulation . Moreoveepletion , suggestWnt7b, which expression was found upregulated at both transcript and protein levels  profiles of mouse samples, available on ZENBU genome browser  analysis in proliferating C2C12 cells using antibodies against G9a and EZH2. We observed that, although both G9a and EZH2 were successfully immunoprecipitated  localization  FISH/G9a (PROTEIN) immunostaining approach showed the colocalization of both G9a and Lnc-Rewind on Wnt7b genomic locus yoblasts . Interesyoblasts . These oepletion . Moreovell types , suggestcis-acting chromatin-associated lncRNAs function by recruiting and targeting chromatin modifiers to specific genes , we found that only G9a specifically interacts with Lnc-Rewind  inhibits skeletal myogenesis through modulation of Mir715 expression, which in turn targets Wnt7b mRNA  WT mice was performed as follows: briefly, whole lower hindlimb muscles were carefully isolated, minced, and digested in phosphate-buffered saline (PBS) (Sigma) supplemented with 2.4 U/ml Dispase II (Roche), 100 μg/ml Collagenase A (Roche), 50 mM CaCl2, 1 M MgCl2, 10 mg/ml DNase I (Roche) for 1 hr at 37°C under agitation. Muscle slurries were passed 10 times through a 20G syringe (BD Bioscience). Cell suspension was obtained after three successive cycles of straining and washing in Washing Buffer consisting of HBSS containing 0.2% bovine serum albumin (BSA) (Sigma–Aldrich), 1% penicillin-streptomycin. Cells were incubated with primary antibodies CD31-PE , CD45-PE , Ter119-PE  1:25; Sca1-FITC  1:50; α7Integrin-APCVio770  1:20 for 45 min on ice diluted in HBSS containing 0.2% BSA, 1% penicillin-streptomycin, and 1% DNAse I. The suspension was finally washed and resuspended in PBS containing 2% fetal bovine serum (FBS) and 0.5 μM ethylenediaminetetraacetic acid (EDTA). Cells were sorted using a FACSAriaIII  equipped with 488 nm, 561 nm, and 633 nm laser and FACSDiva software . Data were analyzed using a FlowJo software . Briefly, cells were first gated based on morphology using forward versus side scatter area parameter (FSC-A versus SSC-A) strategy followed by doublets exclusion with morphology parameter area versus width (A versus W). Muscle satellite cells (MuSCs) were isolated as Ter119neg/CD45neg/CD31neg/a7-integrinpos/Sca1neg cells. To reduce stress, cells were isolated in gentle conditions using a ceramic nozzle of size 100 μm, a low sheath pressure of 19.84 pound-force per square inch (psi) that maintain the sample pressure at 18.96 psi and a maximum acquisition rate of 3000 events/s. Cells were collected in five polypropylene tubes. Following isolation, an aliquot of the sorted cells was evaluated for purity at the same instrument resulting in an enrichment >98–99%  with 5% horse serum (HS). Cells were counted on DAPI-stained images using the Fiji (RRID:SCR_002285) tool ‘Multi point’. Downregulation of RNA expression was performed at ∼50% confluence by transfection in Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions, with 75 nM of LNA GapmeRs (Euroclone) for the downregulation of Lnc-Rewind and with siRNA (Sigma) for G9a and Wnt7b, according to manufacturer’s instructions. Negative Control A (Euroclone) (GAP-SCR) and Mission siRNA Universal Negative Control (Sigma) (si-SCR) were used as controls. See Freshly sorted cells were plated on ECM Gel (Sigma)-coated dishes in Cyto-grow (Resnova) complete medium as a growth medium (GM) and cultured at 37°C and 5% COhttp://www.exiqon.com/ls/Pages). Negative Control A (Euroclone) was used as negative (Scramble) control. Sequences are reported in GapmeRs were designed against Lnc-Rewind sequence using the Exiqon web tool (For EdU (Invitrogen) detection cells were incubated for 6 hr and stained using Click-iT EdU Alexa Flour 594 HCS Assay (Invitrogen) according to the manufacturer’s instructions.Single myofibres were isolated from Extensor digitorum longus (EDL) muscles of C57/Bl10 mice and digested in 2 mg/ml collagenase I (Sigma) for 1 hr at 37°C, gently shacked every 10 min. Single fibres were obtained gently triturating the digested EDL muscles using a glass pipette in DMEM supplemented with 10% HS. The myofibres were manually collected under a dissecting microscope and cultured in DMEM + Pyr with 20% FBS, 2.5 ng/ml FGF (Gibco), and 1% chick embryo extract (CEE) (Life Science Production). RNAi experiments were performed 4 hr after plating with one round of transfection, as described above. The fibres were incubated for 24 hr with EdU (Invitrogen) and were analyzed 96 hr after plating.For EdU detection cells were stained using Click-iT EdU Alexa Flour 594 HCS Assay (Invitrogen) according to the manufacturer’s instructions.AB_2299243) 1:10 and Ki67  1:100) were diluted in 10% FBS–PBS and incubated overnight at 4°C. After incubation for 1 hr with the appropriate secondary antibodies , nuclei were counterstained with DAPI (Sigma), and fibres were mounted on cover-glasses. Images were taken with Axio Observer microscope (ZEISS) and processed with ZEN 3.0 (Blue edition) software.For the immunofluorescence, the single myofibres were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature (RT), permeabilized with 0.5% Triton X-100 in PBS, and blocked with 10% FBS in PBS for 1 hr at RT. Primary antibodies . RNA hybridization and signal development were carried out using Basescope assay  and BA-Mm-Lnc-Rewind probe-set (ref. 722581) designed to detect three junction regions of Lnc-Rewind . A probe specific for exon junction exon1/exon2 of human Dlc1 mRNA  was used as negative control. Confocal images were acquired at Olympus IX73 spinning disk confocal microscope equipped with a Confocal Imager (CREST X‐LIGHT) plus CoolSNAP Myo CCD camera (Photometrics) and at Olympus iX83 FluoView1200 laser scanning confocal microscope. Stacks of images were taken automatically with 0.2 or 0.3 microns between the Z-slices using a 60x NA1.35 oil objective and 405/473/559/635 nm or 405/470/555/640 nm lasers subset, respectively. Filter setting for DAPI, Alexa Fluor 488, Cy3, and Alexa Fluor 594 was used. The images were collected with MetaMorph or FV10-ASW confocal image acquisition software, and post-acquisition processing was performed by FIJI software to the entire image. 3D viewer plugin was used to perform 3D-rendering. DNA-FISH were sequentially carried out after RNA-FISH staining according to Wnt7b and negative control  for 30 min at RT. G9a primary antibody  was incubated in 1% donkey serum/PBS overnight at 4°C, while secondary antibody  were applied in the same incubation buffer for 45 min at RT.Lnc-Rewind in situ hybridization analyses were performed as previously described . BrieflyGapdh transcript.Total RNA from myoblasts/myotubes and MuSC-derived myoblasts was extracted with TriReagent (Sigma) using the Direct-Zol RNA MiniPrep Kit (Zymo Research) according to protocol specification. 0.5–1.0 µg of RNA were treated with RNase-free DNase I enzyme (Thermo scientific). Nuclear/cytoplasmic fractionation was performed using the Paris kit  according to the protocol specifications. RNA was reverse transcribed using the SuperScript VILO Master Mix (Thermo Scientific). Real-time quantitative PCRs were performed by using SYBR Green Master mix (Applied Biosystems), according to the manufacturer’s instructions. Relative expression values were normalized to the housekeeping AB_2147781), G9a , Ezh2 , Wnt7b , Cyclin D3 , and GAPDH . After washing in TBS with 0.2% Tween, membranes were incubated in HRP-conjugated specific secondary antibody (goat anti-rabbit-anti-mouse [IgG-HRP Santa Cruz Biotechnologies]) for 1 hr at RT. After washing in TBS with 0.2% Tween, blots were developed with Western lightning enhanced chemiluminescence (ThermoFisher Scientific), the signal detection was performed with the use of ChemiDoc (Bio-Rad).Total proteins were prepared by resuspending cells in RIPA buffer , 0.5% sodium deoxycholate, 1% NP-40, 1 mM EDTA, protease and phosphatase inhibitors [Roche]). Protein concentration was determined using a BCA assay (ThermoFisher Scientific). The cell lysate was denatured at 95°C for 5 min. The cell lysates were resolved on 4–15% TGX gradient gels (Bio-Rad Laboratories) and transferred to nitrocellulose membrane (Amersham). Membranes were blocked with 5% non-fat dried milk in TBS with 0.2% Tween for 1 hr at RT and then incubated with primary antibody overnight at 4°C. Primary antibodies used were against MF20 (MyHC) , 1× Protease Inhibitor cocktail (PIC). After a 15 min incubation on ice, the cell suspension was centrifuged at 2000 rpm for 5 min at 4°C to pellet the nuclei. The supernatant was collected as cytoplasmic extract. The pellet was re-washed with Buffer A for three times. The pellet was resuspended in NT2/Wash buffer , 1 mM dithiothreitol [DTT]), break with 7 ml dounce (tight pestel/B pestel), and centrifuged at 14,000 rpm for 30 min at 4°C. Protein A/G Magnetic beads (Thermo Scientific) were incubate with IgG , G9a , and Ezh2  antibody on rotating wheel ON at 4°C, while the nuclear extract (NE) was precleared with the beads. Ten percent of the NE was collected for INP. The NE was divided in each sample with the coated beads (beads + Ab) and incubates on rotating wheel ON at 4°C. The beads were washed with NT2 buffer, and ¼ was collected for protein and ¾ for RNA analyses. RIP qRT-PCR results were represented as percentage of IP/input signal (% input).C2C12 cells were washed with PBS, crosslinked with UV rays and collected in Buffer A . Cells were centrifuged at 500 x g for 10 min and the supernatant was collected as cytoplasmic extract. The nuclei pellet was re-washed with Buffer A for three times. After the last wash, the nuclei pellet was resuspended in NP40 lysis buffer , break with 7 ml dounce (tight pestel/B pestel) and centrifuged at maximum speed for 20 min at 4°C. The supernatant was collected and quantified with Bradford assay. Ten percent of the NE was collected for INP. Protein A/G Magnetic beads (Thermo Scientific) were washed with PBS Tween 0.02%  and incubated in PBS Tween 0.02% with IgG  and G9a  antibody on rotating wheel 1 hr at RT. The NE was divided in each sample with the coated beads (beads + Ab) and incubated on rotating wheel ON at 4°C. The beads were washed three times with the NP40 High Salt Buffer 0.5.  and two times with polynucleotide kinase PNK Buffer  and resuspended in 100 μl of NP40 lysis buffer. Seventy-five microlitres were collected for RNA analysis: an equal volume of 2× Proteinase K Buffer  was added, followed by the addition of Proteinase K (Roche) to a final concentration of 1.2 mg/ml and incubated for 30 min at 55°C. The RNA was recovered and analyzed through qRT-PCR. Twenty-five microlitres were heated at 95°C for 5 min, and the supernatant collected and resuspended in Protein elution buffer (4× Laemmli sample buffer [Bio-Rad]) with DTT 50 mM and analyzed by Western blot.CLIP experiments were performed on NE obtained with some modification of the Rinn et al.’s protocol . Briefly2C12 cells were washed with PBS and harvested in Buffer A . After a 15 min incubation on ice, they were centrifuged at 2000 rpm for 5 min at 4°C to pellet the nuclei. After three washes performed with Buffer A, the pellet was resuspended in NT2 buffer , broken with 1 ml dounce (tight pestel/B pestel) and centrifuged at 14,000 rpm for 30 min at 4°C. The supernatant was quantified with Bradford assay. Streptavidin magnetic beads (Promega) were incubated with biotinylated primers against Lnc-Rewind and LacZ on a rotating wheel for 30 min at RT, while the NE was precleared with the beads with the same conditions. One percent of the NE was collected for INP. The NE was divided in each sample with the coated beads (beads + primers) and incubated on rotating wheel for 2 hr at RT. The beads were washed with NT2 buffer and 1/5 was collected for the RNA. The remaining 4/5 were eluted in elution buffer  and incubated 15 min at 70° before incubation of 5 min at 90° in order to collect proteins. Pulldown (PD) qRT-PCR results were represented as percentage of PD/input signal (% input).RNA pulldown experiments were performed on NE obtained with some modification of the Rinn et al.’s protocol . C2C12 cAB_449854) or rabbit IgG  antibodies. A standard curve was generated for each primer pair testing 5-point dilutions of input sample. Fold enrichment was quantified using qRT-PCR  and calculated as a percentage of input chromatin (% Inp). Data from GAP-SCR vs GAP‐REW conditions represent the mean of six independent experiments ± SEM. For G9a ChIP experiments on proliferating C2C12, crosslinking was performed by adding DSG  at a final concentration of 2 mM for 45 min at RT. Then, formaldehyde (Sigma) was added to culture medium to a final concentration of 1% for 10 min at RT and stopped by glycine to a final concentration of 0.125 M. Chromatin was extracted as described in AB_2792982) or rabbit IgG  antibodies carried out overnight at 4°C. Sequences of the oligonucleotides used for ChIP analyses are reported in ChIP experiments on MuSC-derived myoblasts were performed on chromatin extracts according to the manufacturer's protocol  by O.N. incubation with 3 μg of immobilized anti-H3K9me2 . Libraries were prepared from 100 ng of total RNA using the QuantSeq 3′ mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen GmbH); the quality was assessed by using screen tape high-sensitivity DNA D1000 (Agilent Technologies). Sequencing was performed on a NextSeq 500 using a high-output single-end, 75 cycles, v2 Kit (Illumina Inc). Illumina novaSeq base call (BCL) files were converted in fastq file through bcl2fastq (version v2.20.0.422). Sequence reads were trimmed using trimgalore (v0.4.1) to remove adapter sequences and low-quality end bases . Alignment was performed with STAR 2.5.3a  on mm10 RNA-Seq reads from WT myoblasts cultured in GM  were dowThe statistical analyses were performed using Microsoft Excel (v16). The use of an unpaired or paired two-tailed Student’s t-test is specified in each figure legend. For the data that did not show a normal distribution, the Spearman’s rank correlation test was used. When more than two conditions were compared, one-way Anova with Tukey’s multiple comparison test was applied using Prism (v9). Statistical significance was set at p<0.05. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Senior Editor, a Reviewing Editor, and three reviewers. The following individual involved in review of your submission has agreed to reveal their identity: Ahmad S Khalil (Reviewer #2).Thank you for submitting your article "Epigenetic regulation of Wnt7b expression by the cis-acting long noncoding RNA lnc-Rewind in muscle stem cells" for consideration by The reviewers have discussed the reviews with one another and the Editors. We will not be able to offer publication of the manuscript, at least not in the present form. You'll see that the reviewers have a number of criticisms and some feel that the data are of a preliminary quality that would need to be improved through additional experiments. While we cannot accept the current manuscript, we remain open to receiving a much revised version once you have had a chance to address the substantial concerns.Summary:LncRNAs serve as key regulators of gene expression and have recently been demonstrated to be involved in skeletal muscle development and disease. In this manuscript, Cipriano et al., characterize a novel lncRNA, lnc-Rewind, that acts a regulator of muscle stem cell proliferation through a cis-acting chromatin regulation mechanism. Using experiments with mouse cell lines and primary cells, they propose that lnc-Rewind is a conserved chromatin-associated lncRNA, whose expression is associated with muscle stem cell proliferation, that directly interacts with the methyltransferase G9a to mediate repression of Wnt7b.Essential revisions:1) Please provide direct evidence that lnc-Rewind interacts with the Wnt7b locus in cis. It would be ideal to perform a CHART/ChIRP/RAP experiment to determine if lnc-Rewind interacts with the Wnt7b locus. An RNA-DNA FISH would also be good to perform to determine the behavior of the RNA relative to the Wnt7b locus. Is the RNA confined to the locus or does it diffuse?2) It is also not clear that lnc-Rewind directly interacts with G9a. Biochemical data will be required to demonstrate an interaction.3) If lnc-Rewind upregulates 332 and downregulates 756 via Wnt7b derepression, an essential experiment would be to determine if Wnt7a overexpression in muscle cells results in a similar expression change.4) Reviewer 3 questions how important Lnc-Rewind is to muscle homeostasis and differentiation; and how important is Wnt7b for the observed phenotype? Additional evidence to support the claims would be essential.5) The percent of Lnc-Rewind precipitated with antibodies directed to G9a is very low. How much of the known G9a target Kcnq1ot1 precipitated in this assay? Is this background or a specific signal? More controls are required.6) Furthermore, the H3K9me3 ChIP shows small differences between GAP-SCR (control) and GAP-REW (experiment), and the regions that were analyzed are located about 17-28 kb away from Wnt7b. We agree with Reviewer 3 that a ChIP with anti G9a would have been more informative, and a 4C analysis might show a connection between regions 91-94 and the Wnt7b gene. Essential revisions:1) Please provide direct evidence that lnc-Rewind interacts with the Wnt7b locus in cis. It would be ideal to perform a CHART/ChIRP/RAP experiment to determine if lnc-Rewind interacts with the Wnt7b locus. An RNA-DNA FISH would also be good to perform to determine the behavior of the RNA relative to the Wnt7b locus. Is the RNA confined to the locus or does it diffuse?Lnc-Rewind and Wnt7b genomic loci, gave a first piece of advice into a local, cis-acting, regulatory mechanism trough which Lnc-Rewind might modulate Wnt7b expression. In agreement with the need to provide a more direct evidence on the proposed in cis interaction, we have performed a double RNA/DNA FISH approach. Differently from CHART/ChIRP/RAP experiments, which are considered as population-based approaches, this assay allows a quantification of possible RNA/DNA proximities at single cell level. For this, we have labeled Lnc-Rewind RNA (BaseScopeTM technology) and Wnt7b locus  with different fluorophores and used these FISH probes in MuSCs-derived myoblasts. Signal acquisition by confocal microscopy evidenced that ~47% of the Lnc-Rewind nuclear signals colocalize with Wnt7b locus ). No proximity was instead found when an unrelated region  was used as negative control ). Importantly, in the revised version, we have also strengthened the evidence on the concomitant recruitment of Lnc-Rewind and G9a on Wnt7b. These data, which required the use of a new imaging set-up able to combine the RNA/DNA-FISH signals with the G9a immunofluorescence, clearly show the close proximity of both Lnc-Rewind and G9a signals with Wnt7b gene locus . Overall, we believe that the dissection of the Lnc-Rewind mechanism of action has been considerably improved as a result of these revisions in further supporting its cis-acting role into the regulation of Wnt7b expression.Our initial RNA-FISH analyses evidenced a pattern of Lnc-Rewind fluorescence which is quite confined to discrete spots rather than being diffused through the nucleoplasm . These findings, together with the genomic proximity between 2) It is also not clear that lnc-Rewind directly interacts with G9a. Biochemical data will be required to demonstrate an interaction.direct interactions between RNA and proteins as the UV wavelength (254nm) is able to induce the crosslinking only if the RNA and the protein are in close contact . Based on the reviewers’ concern on the need of other biochemical data, we further applied an RNA pull-down approach allowing the purification of the endogenous Lnc-Rewind RNA and its interacting proteins. This analysis, which is RNA-centric and complementary to our initial RIP and CLIP experiments, strengthen our initial evidence by confirming the presence of G9a in Lnc-Rewind precipitated sample. These new results have been incorporated the revised Figure 4B and discussed in the subsection “Lnc-Rewind directly interacts with the methyltransferase G9a and mediates specific in cis repression of Wnt7b in MuSCs”. Additional support was provided, as discussed above, by the RNA/DNA-FISH/IF analysis showing a G9a/Lnc-Rewind concomitant recruitment on Wnt7b genomic locus .We thank the reviewer for having raised this point on the direct interaction between Lnc-Rewind and G9a whose clarification improved our experimental strategy and the general quality of the manuscript. Several evidences available in literature highlight the high rate of false positives identified by native IP analyses as many artificial interactions between RNA and proteins can occur in vitro after cell lysis . Being aware of this limitation, in the previous version of the paper we already combined our native approach  with another, crosslinked-based, protein-centric strategy . Please, kindly note that the CLIP, assay, which confirmed the specificity of Lnc-Rewind interaction with G9a, is recognized as the safest approach to detect 3) If lnc-Rewind upregulates 332 and downregulates 756 via Wnt7b derepression, an essential experiment would be to determine if Wnt7a overexpression in muscle cells results in a similar expression change.primum movens of the regulatory cascade downstream to Lnc-Rewind. Although some overlapping might exist, Wnt7b overexpression in muscle cells is not expected to globally modulate the same subset of Lnc-Rewind target genes. An additional issue is that Wnt7b ectopic expression will not necessarily mimic the overexpression level obtained upon Lnc-Rewind knockdown. It is well known that different levels of Wnt pathway activation give rise to diverse, and sometime opposite, phenotypes . Therefore, unbalanced levels of Wnt7b overexpression might give rise to contradictory results. Nonetheless, to link the phenotype observed by Lnc-Rewind depletion to the induction of Wnt7b expression, we considered more straightforward to perform a rescue experiment. Thus, within a contest where the upregulation of Wnt7b was triggered by Lnc-Rewind depletion, we interfered with Wnt7b expression by siRNA-based knockdown. This experiment allowed us to show that restoration of Wnt7b to physiological levels is able to rescue the proliferation defects observed upon depletion of Lnc-Rewind. These results add an important piece of evidence supporting the role of Lnc-Rewind in sustaining MuSCs proliferation by controlling the expression of Wnt7b. These data are now discussed in subsection “Lnc-Rewind regulates muscle system processes and MuSCs” and included in the revised figures .While it is true that the expression of several genes is affected upon Lnc-Rewind knockdown, our transcriptomic data are not hierarchical and do not assume Wnt7b as the 4) Reviewer 3 questions how important Lnc-Rewind is to muscle homeostasis and differentiation; and how important is Wnt7b for the observed phenotype? Additional evidence to support the claims would be essential.We agree with the reviewer on the importance of clarifying the impact of Wnt7b on the observed phenotype and we thank him/her for the input to provide additional evidence to strengthen our conclusion. As also described above, to fulfil reviewer’s request we performed a rescue experiment depleting Wnt7b expression upon Lnc-Rewind KD-induced upregulation . As shown in Figure 3E, this approach allowed us to uniquely demonstrate that the proliferation/activation defects observed on MuSCs depleted of Lnc-Rewind were rescued by restoring Wnt7b expression to control levels, through si-Wnt7b treatment. Of note, we proved this on both MuSCs-derived myoblasts  and in myofibers-associated MuSCs , consolidating our conclusion on a direct role of Lnc-Rewind-mediated Wnt7b repression in sustaining MuSCs proliferation and activation. These data are now discussed in subsection “Lnc-Rewind regulates muscle system processes and MuSCs”.5) The percent of Lnc-Rewind precipitated with antibodies directed to G9a is very low. How much of the known G9a target Kcnq1ot1 precipitated in this assay? Is this background or a specific signal? More controls are required.#2, the above results unequivocally confirm a direct and specific interaction between Lnc-Rewind and G9a. We thank the reviewer for his/her pertinent comment, which has helped to improve our data and to clarify the details of the proposed model.The low percentage of Lnc-Rewind precipitation might be imputable to a general limitation of native RIP. Please kindly note that, indeed, higher levels of enrichments were obtained by CLIP assay  when the interaction between Lnc-Rewind and G9a was stabilized by UV-cross-linking. As requested, we have quantified the levels of Kcnq1ot1 precipitation and found similar levels of Lnc-Rewind enrichment . Together with the biochemical experiments discussed at point 6) Furthermore, the H3K9me3 ChIP shows small differences between GAP-SCR (control) and GAP-REW (experiment), and the regions that were analyzed are located about 17-28 kb away from Wnt7b. We agree with Reviewer 3 that a ChIP with anti G9a would have been more informative, and a 4C analysis might show a connection between regions 91-94 and the Wnt7b gene.2C12 (not shown), which revealed the presence of several G9a enriched peaks upstream Wnt7b, in particular on the region that is proximal to TSS. In agreement with the need to better specify the reason why these amplicons were prioritized, we have included the G9a ChIP analyses in the revised version  and showed by qPCR that (i) these regions are significantly enriched in G9a and that (ii) at these sites, the levels of G9a enrichment was even higher than Myogenin, known G9a-target . In further support of a role for G9a in mediating Wnt7b repression, we also provide evidence that G9a knock-down induces upregulation of Wnt7b mRNA levels on MuSCs. These new data are shown in the revised Figure 4C and commented in subsection “Lnc-Rewind directly interacts with the methyltransferase G9a and mediates specific in cis repression of Wnt7b in MuSCs”.The choice for these regions was driven by data already available in Mozzetta’s lab, and derived from G9a ChIP-seq analyses performed in C