The ideal treatment for choledocholithiasis should be simple, readily available, reliable, minimally invasive and cost-effective for patients. We performed this study to compare the benefits and drawbacks of different laparoscopic approaches (transcystic and choledochotomy) for removal of common bile duct stones.A systematic search was implemented for relevant literature using Cochrane, PubMed, Ovid Medline, EMBASE and Wanfang databases. Both the fixed-effects and random-effects models were used to calculate the odds ratio (OR) or the mean difference (MD) with 95% confidence interval (CI) for this study.n = 1,222) and laparoscopic transcystic common bile duct exploration (LTCBDE)  regarding stone clearance , conversion to other procedures , total morbidity , operative time , and blood loss . However, the LTCBDE group showed significantly better results for biliary morbidity , hospital stay , and hospital expenses  than the LCCBDE group.The meta-analysis included 18 trials involving 2,782 patients. There were no statistically significant differences between laparoscopic choledochotomy for common bile duct exploration (LCCBDE) (LTCBDE is safer than LCCBDE, and is the ideal treatment for common bile duct stones. Approximately 10% of gallstone patients have concomitant common bile duct (CBD) stones , which aLaparoscopic surgery for CBD stones could be categorized into transcystic and choledochotomy approaches . In lapaLTCBDE has not been universally accepted because the operation is relatively complex , and mulA systematic search was carried out using Cochrane, PubMed, Ovid Medline, EMBASE and Wanfang databases using the following keywords: bile duct exploration, laparoscopic CBD exploration, laparoscopic cholecystectomy, CBD stones, LCBDE, choledochotomy, laparoscopic transcystic CBD exploration, or LTCBDE. The latest search was updated on May 20th, 2016. The eligible studies were independently selected by two reviewers. Disagreement on article inclusion between the two reviewers was resolved by discussion with a third reviewer.All controlled experimental studies about LTCBDE + laparoscopic cholecystectomy (LC) compared with LCCBDE+ LC were selected. The following were the inclusion criteria for the selected studies: (1) Patients with no contraindications for the laparoscopic approach, with no requirement of additional procedures; (2) Patients with confirmed or suspected CBD stones with gallstones; (3) Studies that included data on stone clearance, conversion to other procedures, total and biliary morbidity, blood loss (ml), length of hospital stay (days), operative time (min) and hospital expenses (wan renminbi).The total morbidity , bile duct clearance and biliary morbidity (bile leakage and biliary stricture), conversion to other procedures , operative time, length of hospital stay, hospital expenses, and blood loss were included.The exclusion criteria were non-experimental trials, articles not reporting outcomes, editorials, review articles, and nonhuman studies. The names of the authors and the journals containing the literature did not influence our decision on the articles.2 test. P < 0.05 was considered significant. If heterogeneity was significant, we used the random-effects model. Otherwise, we used the fixed-effects model. If data reported a median and range rather than a mean and standard deviation (SD), then the mean and SD were estimated as described previously [The analyses were performed using Review Manager version 5.1 . The results of this study were expressed as the odds ratios (ORs) for dichotomous data and mean difference (MD) for continuous data, with 95% confidence intervals (CIs) for both. The inverse variance method was used for continuous variables, while the Mantel-Haenzsel method was used for dichotomous variables. Statistical heterogeneity was evaluated by χeviously .P = 0.11)  in the LCCBDE group and in 88.9%  in the LTCBDE group. There was no significant difference between the two groups  .P = 0.38)  and 10.9% (105 of 959) of patients in the LCCBDE and LTCBDE groups, respectively. There was also no statistically significant difference between the two groups  .P = 0.09)  and 10.3%  in the LCCBDE and LTCBDE groups, respectively. There was also no significant difference between the two groups  .P<0.001).  of patients in the LCCBDE group versus 1.3%  in the LTCBDE group. The biliary morbidity for LTCBDE was significantly lower than in the LCCBDE group . .P = 0.05)  .P<0.001)  .P<0.001)  .P = 0.74)  .In this meta-analysis, the funnel plot shapes for postoperative complications and postoperative biliary complications showed basic symmetry . No signCurrently, there are three options for treatment of CBD stones: (1) simultaneous classic CBD exploration and cholecystectomy using the laparotomy approach, (2) combined endoscopic and LC treatment in a two-step procedure, or (3) simultaneous cholecystectomy and CBD exploration using the laparoscopic approach. Surgeons should be familiar with the limitations and benefits of all these treatment strategies. We performed this study to compare the benefits and drawbacks of different laparoscopic approaches (transcystic and choledochotomy) for removal of CBD stones. We found that LTCBDE is safer than LCCBDE, and is the ideal treatment for CBD stones.Endoscopic choledocholithiasis surgery is a mature technology; however, it is associated with high complication (19%), failure (10–15%), and mortality (3%) rates . It can Conventional management of CBD stones included open laparotomy and bile duct exploration, which were first performed by Courvoisier and Thomton separately in 1889 . ClassicLCBDE is substantially better compared to endoscopic and open surgery with regard to hospital stay, postoperative pain, and cosmesis. LCBDE was first reported in 1991, and has been performed in combination with new technologies. It is considered safe and efficient , 8. HoweThe cystic duct connects the gallbladder and the bile duct. Its length is generally 3 cm and diameter is 0.2–0.3 cm. It consists of two parts, which including 5–12 consecutive half-moon mucosal folds called the Heister spiral valves and a smooth portion close to the CBD. Its elasticity and the smoothness of its interior are similar to the CBD. The muscles of the spiral folds are arranged like an annular valve, which can drive the bile flow by contraction and relaxation. Furthermore, the cystic duct itself functions like a sphincter and can coordinate gallbladder filling. The diameter of the confluence between the cystic and hepatic ducts is wider than the diameter of the CBD. The diameter of the cystic duct can expand to 1 cm or more when the CBD is obstructed, and the expansion is more obvious at the confluence. The anatomical features of the cystic duct and CBD create favorable conditions for LTCBDE.The advantage of LTCBDE is that the integrity of the CBD is protected. Less injury to the CBD results in fewer postoperative complications. This explains the reduced length of hospital stay and lower hospital expenses for LTCBDE. For a large stone, a microincision at the enlarged confluence between the cystic duct and CBD can be performed; the diameter of the CBD has no significant effect-on the extent of suturing. Our results also showed that biliary morbidity, hospital expenses, and hospital stay in LTCBDE were significantly decreased, compared with LCCBDE. However, there was no significant difference in operative time between the two groups, which could be attributed to advances in suturing techniques. Previously, interrupted suturing was performed; however, suturing speed has greatly improved and the operative time has reduced by using barbed line or continuous sutures.Preoperative magnetic resonance cholangiopancreatography can not only reveal the size and number of stones but also any variations in the biliary tract. LTCBDE can fail if the angle between the cystic and hepatic duct is small. Pulling the mucosa from the confluence of the cystic duct and CBD using an electrocautery hook and cutting it with scissors can reduce the failure rate caused by the small angle. However, if the incision along the cystic duct towards the CBD is too long, the advantage of using the LTCBDE approach is lost.The results of our study should be interpreted with caution due to limitations. First, LTCBDE and LCCBDE utilize different levels of technology. Second, although we tried to identify all relevant data, potential publication bias was unavoidable and some data could have been missed . Finally, since this study was restricted to reports published in Chinese and English, publication bias could not be completely ruled out.Although many experts have suggested criteria for LTCBDE, we believe that there are no fixed standards. The standards should be different for different teams, because they are affected by differences in the level of technology and the anatomical conditions. Based on our surgical experience, we have also developed corresponding standards.LTCBDE provides a new understanding of biliary anatomy and the concept of choledocholithiasis treatment. Furthermore, it is a successful extension of laparoscopic biliary surgery technology. Although a longer learning time and more laparoscopic skills are required, it should be performed because of its good clinical efficacy.S1 PRISMA Checklist(DOC)Click here for additional data file.


With the increase in the elderly, stroke has become a common disease, often leading to motor dysfunction and even permanent disability. Lower-limb rehabilitation robots can help patients to carry out reasonable and effective training to improve the motor function of paralyzed extremity. In this paper, the developments of lower-limb rehabilitation robots in the past decades are reviewed. Specifically, we provide a classification, a comparison, and a design overview of the driving modes, training paradigm, and control strategy of the lower-limb rehabilitation robots in the reviewed literature. A brief review on the gait detection technology of lower-limb rehabilitation robots is also presented. Finally, we discuss the future directions of the lower-limb rehabilitation robots. Stroke is an illness that has a high potential of causing disability in the aged . With thNowadays, there have been several published review papers on lower-limb rehabilitation robot. However, very few details of control strategies, driving modes, training modes, and gait perception were given to the lower-limb rehabilitation robot.In this paper, we systematically reviewed the current development of lower-limb rehabilitation robot, providing a classification, a comparison and a design overview of the driving modes, training paradigm, control strategy, and gait perception. The rest of the paper is organized as follows. In recent years, various types of lower-limb rehabilitation robots have been developed to enhance the motor function of paralyzed limbs in stroke patients. In general, lower-limb rehabilitation robots can be divided into two categories, that is, exoskeleton robots and end-effector robots . For exaThe Lokomat, LokoHelp, Lopes, and Active Leg Exoskeleton (ALEX) belong to the typical treadmill-based exoskeleton robots. Treadmill-based exoskeleton robots are usually composed of a weight support system and runs on a treadmill through the lower-limb exoskeleton frame.In 2001, the Swiss Federal Institute of technology in Zurich  developeLokoHelp is a gait-training robot, which was developed and produced by a German company, consisting of three parts, a leg brace device, treadmill system, and suspension weight system. It can achieve the basic gait rehabilitation training and help patients complete the downhill exercise. In addition, the equipment adopts a modular design method, which is easy to assemble, disassemble, and adjust, in order to realize the training of different slope. Clinical experimental studies on LokoHelp have proved that , 35 the The Biomedical Engineering Laboratory at the University of Twente , HollandThe School of Mechanical Engineering, Delaware University, has developed an active walking training robot called ALEX. It consists of a moving bracket, lower extremity exoskeleton orthosis, and a control system. Each leg has four degrees of freedom, two degrees of freedom of the hip joint, and one degree of freedom of knee and ankle joints. The back of ALEX, using mechanical mechanisms to balance the gravity of the human body, can help patients achieve gravity balance and altitude adjustment , 40.The Active Ankle-Foot Orthosis (AAFO) , Knee-AnYonsei University, Seoul, Korea, developed a single degree of freedom hinge ankle-foot orthoses AAFO. The orthosis uses a polypropylene material, which is lightweight and has a certain degree of flexibility. Moreover, the joint uses a hinge structure; the driving part adopts the series elastic actuator. The contact between the foot and the ground is determined by installing a contact switch on the foot  and usinIn 2004, Dr. H. Kazerooni of the University of California-Berkeley  designedIn 2005, the Department of Mechanical Engineering of the Ottawa University  in CanadHybrid Assistive Limb (HAL) is a wearable lower-limb rehabilitation robot developed by the University of Tsukuba, Japan. The original purpose of the device was to assist patients with lower-limb motor dysfunction to complete the routine activities such as walking, standing, sitting, and going up- and downstairs . At presThe foot plate-based end-effector devices  consist Gait Trainer (GTI) is a suspension weight loss gait rehabilitation robot, developed by the Free University Berlin, Germany. It was based on the movement of the lower limb to stimulate the muscles of the lower limb orderly and assist the patient to complete gait training. However, because of the interaction between the foot pedal and the patient's foot, the force feedback of the lower limbs was weak, and the feeling of walking was larger than that of natural walking. In addition, the robot's gait training strategy emphasized repetitive passive motion, while ignoring the importance of active participation. GTI was an early device for lower-limb rehabilitation, and there were many clinical trials in the world –54; the In 2003, Hesse et al. proposed the concept of Haptic Walker based on virtual reality technology. They developed as a foot motion simulator 6 degrees of freedom, with the use of hanging weight loss to realize the arbitrary trajectory and the attitude motion in the sagittal plane, such as walking on the rough surface or the lawn, tripping, and so forth. In the virtual reality control mode, the patient wore a helmet display and a six-degree-of-freedom force sensor was installed on the foot pedal; the patient felt the virtual reality scene and interacted with the virtual scene. The virtual scene and music can also improve the monotonous training atmosphere and enhance the training interest of patients, to achieve the purpose of psychotherapy. The virtual walking rehabilitation training robot was the first device to realize the foot walking along the programmable free trajectory, and redundant hardware and software emergency stop circuits were set up on security as measures.Compared to other sports platform, a robot actuated by foot in Italy is driven by the pedal with lower-limb movement. The robot added a new way of walking, such as obstacle, step, and slope road. The training rich mode and the active and passive control mode can be a more effective targeted training . The comThe Ruegst ankle, ARBOT, and parallel ankle robots belong to the platform based on end-effector robots.The first truly fully used for ankle rehabilitation robot system was the “Rutgers ankle” proposed by Girone et al. of Rutgers University . It was In comparison, exoskeleton robots are usually fixed in various parts of the human limb, while producing different forces/torques. However, for different patients, these exoskeleton robots may not be able to restore the patient's limb function due to its disadvantages and poor adaptability. The end-effector robot is usually at a certain point in contact with the patient's body. Because there is no restriction on the movement of human, the end effector is easier to adapt to different patients .The choice of driving mode directly effects the system scheme of the exoskeleton robot, such as structure design and control system, and it is the basis of exoskeleton robot design. At the moment, the common drive modes of an exoskeleton robot are hydraulic drive, motor drive, pneumatic drive, and SEA (series elastic actuator) , 62. TheNowadays, the rehabilitation exoskeleton robot mostly used motor drive mode; the robot just needs to bear the body weight and assist hemiplegic patients in common activities, such as walking and going up and down the stairs. Compared with other drive modes, motor drive mode has many advantages, like easy control, no pollution, low noise, and so forth. Hydraulic drive mode is much simpler, smaller, and lighter than other modes. Under the same load, the hydraulic drive is much better than the other driving methods.In summary, the drivers, such as hydraulic, motor, pneumatic, and SEA (series elastic actuator) are limited by the power, mass, and volume, and the consequence of noise on people in the work is serious. Although the development of artificial muscles plays an important role in the problems, there are some technical challenges to overcome. Another important aspect is the drivers' energy problem. The usable energy, such as nonrechargeable battery, rechargeable battery, and small internal combustion engine, has both merits and limitations, so the potential and perpetual method to solve these problems is to develop new technologies, like electrochemical fuel cell and wireless energy transmission.Position controlForce signal controlBiological medical signal control.According to the different signals that are obtained from the initiative intention, the control strategy between robot and patients is divided into three parts:The position control method is trajectory-tracking control, which is to drive the lower limbs to walk on the fixed mode. The gait is formed by a proportional position feedback controller and joint angles and suitable for lower limb muscle strength. Hornby confirmed the efficacy of trajectory tracking control, which can increase the speed and durability of patients with incomplete spinal cord injury. Zhang et al. established the trajectory tracking control of the 5 connection model, which can enhance the participation of the patients and make the training more personalized .In this control strategy, force signal is produced by limb contraction and interactions with mechanical structure. The interaction force can be directly measured by force and moment sensor in the elegant mechanical structure design, which can be evaluated by the kinetics models of the human-computer interactive system. Compared with biological medical signal, force signal has a better determinacy, which can better reflect the motion intention of the patient, so the control based on force signal is feasible and relatively steady. However, the acquisition of interaction force usually requires mechanical structure, which is less available than biological medical signal detection, so the applicable range of interactive controlling are limited. In the interaction control strategy, between rehabilitation robot and patient, there are two most widely used methods: hybrid force/position control and impedance control .To resolve the control problems of robot in a constrained environment , RaibertLokomat achieved a new cooperative gait training strategy by using force/position hybrid control method . The conImpedance control is different from force/position hybrid control. It focuses on realizing the flexibility of the rehabilitation robot, which avoids excessive force between the mechanical structure and limbs. This method could provide a natural, comfortable, and safe touch interface and avoid secondary damage effectively. An additional advantage of impedance control is that the achievement of impedance control is independent of the prior knowledge . In the In the robot control field, the theory of impedance control was first proposed by Hogan , and it Surface electromyogram (sEMG) and electroencephalogram (EEG) are mostly used in interactive controlling of lower-limb rehabilitative robot. Since these signals are both using nonintrusive ways to get, the ways of obtaining the sEMG and EEG are operable and do not need a medical expert and its performance can get guarantees.The acquisition of sEMG is simple and does not require a complex mechanical structure design.The force signal is just the embodiment of all muscle groups, and sEMG can reflect the degree of activity of specific muscle groups, which can be more detailed monitoring and control of the movement of the limbs.The interactive control based on sEMG has more flexibility, which can realize the control of the healthy limb to the diseased limb according to the coordination of the body movement.sEMG has higher sensitivity and resolution than the active force signal, and it is more suitable to use sEMG to detect active motion intention for the patients with lower limb autonomy.EMG signal is the electrical activity produced by the skeletal muscle , 84. AccThe challenges of interactive control method based on sEMG are as follows. First, through the human skin, to collect sEMG signal has great randomness, and in order to obtain the signals, which have high signal-to-noise ratio and can truly reflect the muscle activity, we need to find an effective way to filter out the interference of sEMG. Secondly, the single channel sEMG only reflects the activity of specific muscles, in order to obtain the active motion intention of the patients, which is usually necessary to combine multiple muscle activities. In contrast, the response of the force signal to the active intention is more direct.Using the remaining EMG of diseased limbs. This method can not only stimulate the patients' awareness of active participation but also encourage patients to control the contraction of limb muscles during exercise. But for severely paralyzed patients, their diseased limbs have almost completely lost their motor function and cannot complete muscle contraction independently; the sEMG signal is so weak that it is difficult to be detected. The first scheme is not applicable in this case.Using the motion coordination of the left and right limbs or upper and lower limbs and EMG signals of the healthy limbs control the movement of the paralyzed limb. This method in active participation of patients is less than the first strategy, but it provides an active training program for severely paralyzed patients.The interactive control strategy based on sEMG can be divided into two categories:The EEG signal is the electrical activity of the brain , which iThe most important advantage of interactive control based on EEG is that it is limited to the extent of physical disability; even if the patient has completely lost the motor function of the lower limb, as long as the brain can produce motion control signals, the method is equally applicable. This method is particularly suitable for patients with complete spinal cord injury, and their brain function is normal, but the control signal transduction pathway is cut off, so the muscles of the limbs completely lost control. The interactive control based on EEG is equivalent to the reconstruction of the brain control signal transmission path outside the body, and the motor and functional electrical stimulation device are used as the actuator to regain the control of the limb motor function.This method is limited to the paralyzed patients whose brain motor control function is normal, but it is not suitable for patients with brain damage caused by stroke and other reasons, because the brain motor function area of the patients has been damaged and it has not been able to produce the EEG signal of normal limb movement control. Secondly, compared with the sEMG signal, the resolution of EEG on limb movement intention is low and the EEG signal has a greater randomness, in which changes in expression, mood, and attention will easily effect the EEG signal generated by the brain.At present, the research in this area is mainly focused on offline classification knowledge and regression analysis; the knowledge pointed out the potential of the interactive control of lower-limb rehabilitation robot based on the EEG, but the actual application and the experimental results are almost none. Compared with offline research, real-time interactive control is facing more challenges. First, the real-time acquisition of the EEG signal is not possible to have the integrity of the data used in offline research; the accuracy of the identification may be affected. Secondly, it is necessary to ensure the real-time performance of interactive control, which requires the use of EEG signal for motion recognition, more importantly, to predict. Finally, in real-time interactive control, the patient will not be able to complete the actual physical movement independently, the acquisition of the EEG signal corresponds to the movement of the brain, and this has not been considered in the study of the existing lower-limb rehabilitation robot.The effectiveness of lower-limb rehabilitation robots and treatment depends largely on its training mode , which wThe rehabilitation-training mode is divided into four kinds, which includes the passive mode, the active assist mode, active mode, and active resist mode.In the passive mode, the patient lost muscle strength and could not complete the active movement. We can only rely on the help of external forces to achieve the patient's passive training. The robot's legs drive people's legs for rehabilitation training, and the lower-limb rehabilitation robot should provide sufficient strength for passive training. The advantage of this model is through the repeated exercise to promote the recovery of limb motor function and reduce muscle atrophy, but the patient lacks motivation.In the active mode, the muscles of the patient have certain strength and the active motion of the smaller torque can be performed on the rehabilitation equipment. When the patient wants to move his joint or limb, the robot device will use an external assist force as needed. It requires the robot to perceive the state of the patient and the force/torque when following the patient's movement. This model can be modified according to the patient's intention, thereby greatly enhancing the initiative of patients.In the active assist model, the muscles have certain strength, but without the help of the robot legs, patients cannot be fully trained. This allows the patient to move without the help of a robot, which can improve the patient's ability to exercise independently.In the active resistance model, the mechanical leg provides a certain force, which is opposite to the direction of the leg to achieve the purpose of strengthening muscle training. This model is suitable for patients with high recovery, and resistance makes the movement more challenging and can enhance muscle strength in patients.At present, there are a number of other training methods, such as mirror motion and isotonic and isokinetic exercise patterns. Although these new training patterns are similar from the therapist's view, they are also trying to provide assistance or resistance to the patient in the course of robotic therapy.Accurate signal is the foundation of control; the quantitative feedback information is helpful for developing reasonable rehabilitation strategy according to the state of patients. Therefore, the choosing of sensor, which can detect the information of human computer interaction, is crucial. Gait detection technology consists of three primary parts: plantar sensing technology, limb sensing technology, and mixed sensing technology.Plantar sensing technology: it can judge the different gaits by detecting the man-machine forces or the ground reaction forces of foot using sensor.The sensing technology based on angle sensorThe sensing technology based on EMG  sensorThe sensing technology based on BCI .Limbs sensing technology: it uses sensors to detect motion intention of the lower limbs or the torso:Mixed sensing technology can be applied to identify and judge the human gaits using two or more sensors together.Combining of the detection information of all kinds of sensors, the control system can obtain accurate movement information to make sure that the exoskeleton robot will work effectively and reliably.At present, there are two main ways for detecting motion intention, as shown in The exoskeleton rehabilitation robot uses many kinds of sensors to detect gait, but the detection methods still have many problems, such as vulnerability to interference, inaccurate judgment, and poor adaptability. Therefore, the development of BCI technology and sensor technology are crucial to solve the current problems.The mechanical structure and control system of rehabilitative robot need to be improved. During rehabilitation training, it lacks exact control in real time for patients' joints angles, torque, speed, etc.The recently developed robots in domestic and abroad are mainly on motor rigid drive. The system is lacking in flexibility, and the actuator structure of rehabilitation robot is overly complex and large with low portability. At the same time, the security and comfort also need further improvement.The feedback mechanism of rehabilitative effect should be consummated. It could not give an accurate feedback to the limbs' position and force during rehabilitation training, which causes low training efficiency and directly effects the evaluation of rehabilitation training.For a flexible robot, we need to develop a more advanced high polymer as flexible material. Moreover, the driving force still needs to be improved.Patients' motivation involved in the training plays a very important role in stroke rehabilitation. However, most training paradigms are rigid and boring. Task-oriented training paradigm with interesting games such as whack-a-mole can make the training more enjoyable.The lower-limb rehabilitation robot still faces numerous technological challenges, including the biomechanics, neurophysiology, human-computer interaction (HCI), and ergonomics.In this paper, the development of lower-limb rehabilitation robot, training mode, driving mode, control strategy, and gait detection technology are reviewed. The lower-limb rehabilitation robot has many advantages, and it has shown encouraging clinical outcomes and rehabilitation efficiency. Although most of the lower-limb rehabilitation robots can provide systematic and long-term treatment, there are still some disadvantages and deficiencies summarized as follows:System design of lower-limb rehabilitation robot: the mechanical structural design is the foundation of robotic system, which needs to achieve some major objectives, such as compact, multi-DOF, great flexibility, various kinds of training methods and motions, better comfort, and high matching between human and computer.The control strategies and motion pattern design of lower-limb rehabilitation robot: due to the individual difference of the patients, the robot should perceive state information of patient's force and position, to adopt corresponding training mode and control strategy. Future researches, such as adaptability and stability of control system, the applications of sensor technique, and the design of control algorithm, are required. Therefore, the robot should not only meet the demand of low weight, fast response, and large output torque but also have some characteristics similar to animal skeleton muscles, such as pliability and reliability. Therefore, it is important to research the optimizing design method for energy saving based on active and passive mode, the energy technology of high energy density, and wireless transmission technology.The design of gait detection system: the lower-limb rehabilitation robot should be able to detect and perceive the information of interaction forces and motion position between the patient and rehabilitation robot. On the one hand, the robot should provide appropriate assistance, when the patient could not complete motion by himself. On the other hand, the robot should decrease the assist force or increase the resistance properly, when the motor ability of paralyzed lower extremity improves remarkably.Security protection mechanism: the robot must be designed to meet the safety requirements of clinical rehabilitation training, while preventing damage. In order to ensure security of rehabilitation training, two important issues should be considered when designing the lower-limb rehabilitation robots: mechanism design (hardware) and control system (software).Rehabilitation effect assessment system: by combining the detection of EMG signals and EEG signals. We should explore the inherent relationship between the rehabilitation effectiveness and the train parameters and develop new assessment strategies to verify the effectiveness of the lower-limb rehabilitation robot.The exact factors in the design of VR, which stimulate patients' motor cortex mirror neurons, should be explored in the future.The vertigo problem of VR, which limits the application of VR system, must be solved.The VR technology has been proved to be an effective tool in neurorehabilitation. On the one hand, the interesting and varied virtual scene in VR improves more motivation of patients comparing with the training course in traditional training. On the other hand, the immersive VR environment can effectively stimulate human brain mirror neurons in the motor cortex and promote the recovery of the nerve. However, VR cannot provide physical feedback to the paralyzed limb; the robot can compensate for this defect. Therefore, the combination of rehabilitation robot and VR technology is the future development direction. However, before the application, the following core issues must be addressed:The current lower-limb rehabilitation robots, to some extent, can provide a simple training program for patients and has a certain effect on rehabilitation. In our opinion, future researches on lower-limb rehabilitation robot should focus on the following aspects:


These receptors are implicated in the pathophysiology of psychiatric illness, particularly those associated with affective, psychotic, and cognitive symptoms. Indeed, non-selective α2-AR blockade is proposed to contribute toward antidepressant  and atypical antipsychotic  drug action. Both α2C- and α2A-AR share autoreceptor functions to exert negative feedback control on noradrenaline (NA) release, with α2C-AR heteroreceptors regulating non-noradrenergic transmission . While the α2A-AR is widely distributed throughout the CNS, α2C-AR expression is more restricted, suggesting the possibility of significant differences in how these two receptor subtypes modulate regional neurotransmission. However, the α2C-AR plays a more prominent role during states of low endogenous NA activity, while the α2A-AR is relatively more engaged during states of high noradrenergic tone. Although augmentation of conventional antidepressant and antipsychotic therapy with non-selective α2-AR antagonists may improve therapeutic outcome, animal studies report distinct yet often opposing roles for the α2A- and α2C-ARs on behavioral markers of mood and cognition, implying that non-selective α2-AR antagonism may compromise therapeutic utility both in terms of efficacy and side-effect liability. Recently, several highly selective α2C-AR antagonists have been identified that have allowed deeper investigation into the function and utility of the α2C-AR. ORM-13070 is a useful positron emission tomography ligand, ORM-10921 has demonstrated antipsychotic, antidepressant, and pro-cognitive actions in animals, while ORM-12741 is in clinical development for the treatment of cognitive dysfunction and neuropsychiatric symptoms in Alzheimer’s disease. This review will emphasize the importance and relevance of the α2C-AR as a neuropsychiatric drug target in major depression, schizophrenia, and associated cognitive deficits. In addition, we will present new prospects and future directions of investigation.α Howeverd α2C-AR –16, the  and MDD –21. Genen in MDD , ADHD  superfamily, belonging to the rhodopsin-like or Class A GPCR receptors , serotonin , and NA receptors, the latter via inhibition of vagal (parasympathetic) activity through presynaptic α2 receptors  as opposed to that in humans and rabbits (α2A)  , 51. Theprofiles . While afunction . The α2A the CNS , 53, 54,2-ARs in the CNS are contributed by the α2A-AR, the expression of the α2C-AR is more discrete, constituting approximately 10% of the total   , 59–61.2C-ARs in human, monkey, and rodent brains are analogous . This contradicts recent findings described in the social isolation rearing (SIR) and N-methyl-d-aspartate (NMDA)-antagonist models of schizophrenia where selective α2C-AR antagonists, improved sensorimotor gating deficits  were shown to be increased in the brains of α2C-KO mice , glutamate, and acetylcholine, as will be discussed.Despite a number of new theories that have been put forward to explain the underlying biology and development of mood and psychotic disorders, targeting monoaminergic transmission as a construct toward understanding and treating these disorders remains a relevant subject of investigation [reviewed in Ref. ]. The la-KO mice . Deactiv2A-AR and α2C-AR are the main autoreceptors involved in presynaptic feedback inhibition of NA, with the α2B-AR making no significant contribution to NA feedback inhibition , while its effects on NA’ergic transmission cannot be underestimated. Along with the α2A-AR, α2C-ARs are involved in the presynaptic negative feedback loop on NA release in the cortex, although α2C-AR-mediated presynaptic inhibition occurs more slowly than that mediated by α2A-ARs  in contrast to the α2A-AR   . These a the CNS . This steceptors . α2C-AR 2C-ARs in the striatum allows it to modulate presynaptic DA release and DA-mediated behaviors  to show increased in vitro α2C-AR potency and selectivity ratios in the presence of DA as agonist  in the frontal cortex but not in the striatum compared to wild-type controls, whereas α2C-KO animals showed lower HVA concentrations in the striatum  precursor, 5-hydroxytryptophan (5-HTP), via the rate-limiting enzyme tryptophan hydroxylase, seems to be dependent on both α2A-ARs and α2C-ARs in the rodent, with α2A-ARs emerging as the main α2-AR modulating 5-HT synthesis . These effects were not mirrored in the hippocampus, although α2B/C-AR specific antagonism decreased hippocampal 5-HTP levels in this brain region  rats  rats ), which 2C-AR is an important mediator of striatal, but not hippocampal GABA release  is completely blocked in α2C-KO mice, while enhancement of striatal GABA release by an α2-AR agonist is maintained in these mice, suggesting that inhibition of striatal GABA release is strongly mediated by the α2C-AR , and the role of GABAergic transmission in mania and the action of mood stabilizers  in attenuating symptoms induced by hypoglutamatergic states, although the mechanism is uncertain.Although it is known that α neurons , 78, vert models , while nairments –82. Cont illness –19, indi2 heteroreceptors on glutamatergic neurons by NA reduces glutamate release in various brain areas implicated in MDD and schizophrenia, including the frontal cortex, ventral tegmental area, hippocampus, and nucleus accumbens  might be attributed to the location of α2C-ARs on striatal cholinergic neurons. These authors have also reported that the α2C-AR mediates inhibition of striatal adenylyl cyclase and acetylcholine release, while these effects might be related to tonic activation of the α2C-AR by DA  and mirtazapine in rodents and depressed humans (brain and platelets), although regional differences in α2-AR downregulation have been noted in the CNS  to establish the role of the α2C-AR modulators in appropriate models.Considering the high comorbidity of anxiety in these illnesses, and that it can significantly affect prognosis and treatment response , 242, toRecent developments and the current state of knowledge support the therapeutic potential of selectively targeting the α2C-AR subtype ligands, and specifically α2C-AR selective antagonists, have been developed over the past decade. Before that, drugs with marginal selectivity were used to delineate pharmacological effects of the α2-AR subtypes. For example, although BMY7378 is mainly an α1D-AR antagonist, it also presents with a 10-fold selectivity for α2C-ARs vs. α2A-ARs -diazepan-1-yl]-ethyl}-2-phenoxy-nicotinamide was synthesized, and found to display >100-fold selectivity for α2C-AR vs. α2A-AR, with excellent binding affinity and functional activity at the α2C-AR in rats. Although low selectivity vs. α2B-ARs was shown, the α2B-ARs have negligible distribution in the CNS. This compound displayed excellent binding affinity and functional activity for α2C-ARs in rats, with adequate CNS penetration   . Both JPd humans , 25 and 2C-AR which is amenable to labeling with 11C and has been successfully used as a α2C-AR PET tracer that readily enters the CNS  and antidepressant  agents at clinical doses thus enabling a better understanding on the mode of action of these drugs. The tracer could also be of potential value to investigate disease-related changes in receptor density and effects on neurotransmitter activity. The latter aspect has been investigated and in line with evidence that the α2C-AR is sensitive to low synaptic concentrations of NA, [11C]ORM-13070 shows increased CNS binding in response to decreased synaptic NA -2H-1,4-benzoxazin-6-yl]-N-ethyl-N′-methylurea] or “Compound A” and a chemically similar “Compound B” were found to be highly selective for the α2C-AR, albeit with poor brain penetration. These compounds are being investigated for effects on peripheral vasoconstriction (On the other side of the spectrum, novel αtriction , 247.2C-AR in neuropsychiatric disorders, genetic and molecular biology driven approaches will also be critical. Mice overexpressing or lacking the α2C-AR have been generated but have been phenotyped to a limited extent. Further behavioral and biological characterization, for example using -omics type molecular profiling, as well as regionally restricted genetic manipulation using genetic deletion technology in rats, would yield valuable data. The zebrafish is another platform of discovery that may provide a powerful model in which to study developmental and genetic factors that underlie human disease (2-AR subtypes are markedly conserved compared to mammalian α2-AR subtypes with similar pharmacological profiles and functional effects compared to human α2-AR subtypes (2-AR ligands.With the aim to stimulate further investigation into the value of the α disease . Work insubtypes , 250. Th2C-AR in neuropsychiatric illness and therapy, with specific focus on the effects of α2C-AR antagonism in cognition, MDD, and schizophrenia. Targeting this receptor could present with beneficial therapeutic effects and decreased noradrenergic side effects when used alone or as augmentation strategy in the treatment of these diseases, as well as disorders presenting more specifically with cognitive decline, such as Alzheimer’s disease. The recent advent of clinical grade subtype-selective α2C-AR antagonists has contributed toward delineating the neuropsychopharmacology of this receptor. Studies employing these novel highly selective α2C-AR ligands in putative translationally relevant animal models of psychiatric illness, to inform further experimental medicine evaluation in humans, will be vital in strengthening our understanding of the α2C-AR as a therapeutic target.This review has provided an overview of recent developments and future direction in research investigating the role of the αMU prepared the first draft of the manuscript, prepared all the figures and tables, as well as managed all subsequent changes and formatting. MS reviewed the manuscript and provided input on the manuscript design and content, as well as on the figures and tables. BH was the study leader and student supervisor to MU, developed the article concept and design, and finalized the manuscript for submission.®, and has received research funding from Servier® and Lundbeck®. ORM-10921, which was used in recent studies by the authors and cited in this paper was sponsored by Orion Pharma. BH declares that, except for income from the primary employer and research funding from the below-mentioned organizations and agencies, no financial support or compensation has been received from any individual or corporate entity over the past 3 years for research or professional services, and there are no personal financial holdings that could be perceived as constituting a potential conflict of interest. The authors declare no other conflicts of interest.MS is an employee of Orion Pharma. No funding was received by Orion Pharma for this, or previous work by these authors. The authors declare that over the past 3 years, BH has participated in advisory boards and received honoraria from Servier


Molecular and histopathological profiling have classified breast cancer into multiple sub-types empowering precision treatment. Although estrogen receptor (ER) and human epidermal growth factor receptor (HER2) are the mainstay therapeutic targets in breast cancer, the androgen receptor (AR) is evolving as a molecular target for cancers that have developed resistance to conventional treatments. The high expression of AR in breast cancer and recent discovery and development of new nonsteroidal drugs targeting the AR provide a strong rationale for exploring it again as a therapeutic target in this disease. Ironically, both nonsteroidal agonists and antagonists for the AR are undergoing clinical trials, making AR a complicated target to understand in breast cancer. This review provides a detailed account of AR’s therapeutic role in breast cancer. Over 240,000 women will develop breast cancer and ~40,000 will die from the disease in the United States in 2016 , a subseFor therapeutic purposes, breast cancer has been historically classified based on the expression or lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (HER2) . Breast In 2000, Perou et al. completed a genome-wide molecular analysis of patient specimens to classify breast cancer based on cell-type and molecular signature . Breast The luminal A subtype is characterized by the expression of ER, lack of HER2, and a lower expression of the proliferative marker, Ki67 (ER+/HER2-/Ki67 low). The luminal A subtype is an indolent disease that is typically treated with hormonal therapies that either antagonize or degrade ER or inhibit aromatase, an enzyme critically involved in biosynthesis of estradiol.The luminal B subtype is characterized by the expression of ER, lack of HER2, and high Ki67 (ER+/HER2-/Ki67 high). Although luminal B is predominantly HER2-negative, a subset of it expresses HER2 while still retaining other characteristics of HER2-negative luminal B. Markers of proliferation such as cyclin B1 (CCNB1), Ki67 (MKI67), and Myb proto-oncogene like 2 (MYBL2)  and prolThe HER2 subtype is comprised of tumors that are ER-negative and HER2-positive . This suThe BLBC subtype is the most aggressive subtype of breast cancer and is associated with high mortality in women. While 75%–80% of the basal subtype is TNBC, the remaining 20%–25% express ER and/or HER2 . It is sGenome-wide studies to understand the underlying mechanisms for the aggressive phenotype of TNBC and to identify new therapeutic targets led to the classification of TNBC into six subtypes , includiThis subtyping provides an opportunity to develop focused therapeutics and conduct clinical trials in which the subjects belong to a particular subtype.The AR is a member of the nuclear hormone receptor family of ligand-activated transcription factors that is activated by androgen . The AR gene is located on the X chromosome at q11 and contains eight exons encoding for an N terminus domain (NTD), a DNA binding domain (DBD), a hinge region, and a ligand binding domain (LBD). The NTD contains the activation function 1 domain (AF-1) that retains most of the AR activity . The DBDThe unliganded AR is maintained in an inactive complex by heat shock proteins, HSP-70 and HSP-90. Upon ligand binding, the HSPs dissociate from the AR enabling it to translocate into the nucleus and bind to DNA elements that are located both proximal and distal to the transcription start site . Once boPerhaps surprisingly, the AR is the most widely expressed nuclear hormone receptor in breast cancer with about 85%–95% of the ER-positive and 15%–70% of the ER-negative breast cancers expressing AR. In a study conducted with 2171 patient specimens, AR was found to be expressed in 77% of invasive breast carcinomas . About 9PR is an ER-target gene, PR is most likely to align with ER expression pattern and hence was logical to exclude from the list of prognostic markers. These results were reproduced in other studies conducted in different patient cohorts around the world [A review of a database containing data from 19 studies with a total of 7693 women demonstrated that the AR is expressed in 61% of the patients . While 7he world ,27,28,29Noh et al. included 334 ER-negative HER2-positive or -negative breast cancers in a study to evaluate the expression of AR and clinical outcome . Most ofOne of the breast cancer subtypes where AR’s prognostic value was debated is the molecular apocrine type . MoleculIn addition to measuring AR expression, some studies measured the expression of androgen-synthesizing enzymes such as 17βHSD5  and 5α-reductase. 17βHSD5 converts the weaker androgen, androstenedione, to a more potent testosterone, while 5α-reductase further amplifies the activity by converting testosterone to the more highly potent DHT . McNamarA recent study evaluated the expression of AR and other genes in 1141 patient specimens . NuclearAn overwhelming number of publications demonstrate that the AR is a favorable prognostic marker , regardless of the tumor subtype, and suggest that in most, if not all, cases AR expression is inversely proportional to tumor size, aggressiveness, pathological grade, and directly proportional to DFS, progression-free survival (PFS), and OS. However, a few reports have identified a subset of cancers where AR expression is directly proportional to Ki67 staining and correlates with poorer OS and DFS ,40. For While the above studies strongly suggest that AR expression predicts favorable prognosis, AR expression also provides information on the treatment response. In a study evaluating 913 patients, AR expression was associated with a favorable outcome to treatment . PatientLoibl et al. evaluated 673 core primary breast cancer biopsies from patients who have received neoadjuvant chemotherapy . AR was Intracrine hormone synthesis in breast and prostate cancers has been recognized in the recent years as a vital but previously unrecognized driver of continued tumor growth ,47,48. FThe activation and inactivation of steroid hormones are influenced by a class of enzymes called hydroxysteroid dehydrogenases (HSD), which catalyze the NAD(P)(H)-dependent oxidoreduction of the hydroxyl/keto groups of androgens, estrogens and their precursors ,60 and tEstrogens in pre-menopausal and post-menopausal women are synthesized from their adrenal androgen precursors, dihydroepiandrosterone sulphate (DHEAS) and dihydroepiandrosterone (DHEA) . DHEAS aTogether, these lines of evidence suggest that intracrine androgen synthesis, higher androgen concentrations, and AR expression are strongly associated with a better prognosis, favorable therapeutic outcome, and a reduction in tumor in patients with AR-positive breast cancers.Steroidal androgens were the mainstay of clinical treatment for breast cancer before the discovery of tamoxifen or other ER antagonists and AIs ,71. EarlDespite or perhaps because of plentiful historical evidence, a controversy remains with respect to whether an AR agonist such as an androgen or an AR antagonist will be effective in treating breast cancer. The conflict is primarily due to the skewed outcome of experiments performed with preclinical immortalized cell line models. Below we summarize the clinical and preclinical evidence supporting the use of both AR agonists and antagonists as treatment options for breast cancer.Preclinical models to evaluate the role of the AR in breast cancer are highly variable. ZR-75-1 is an ER-positive luminal A breast cancer cell line that expresses high levels of the AR. Treatment of this cell line with DHT resulted in significant growth inhibition . In thisTilley and colleagues using MCF-7 and T47D ER- and AR- positive luminal breast cancer cell lines demonstrated that two steroidal androgens (DHT and mibolerone) inhibited the cell proliferation . AlthougMore convincing results evolved from the dimethylbenzanthracene (DMBA)-induced mammary carcinogenesis rat model . Rats beWhen analyzing the preclinical data in TNBC or BLBC models, the landscape is complex and inconclusive. Most of the data were generated in one ER-negative apocrine breast cancer cell line, MDA-MB-453. The proliferation of MDA-MB-453 cells or growth of MDA-MB-453 xenografts are stimulated by androgens and inhibited by AR antagonists ,79. It iBarton et al. used TNBC cell lines to evaluate the effect of DHT . Treatme50 values between AR-positive and -negative prostate cancer cell lines, it inhibited TNBC cell lines at comparable concentrations regardless of the AR expression. These results suggest that the effect of AR antagonist enzalutamide in TNBC cell lines could be AR independent.Ince and colleagues evaluated the effect of DHT in various ER-negative and TNBC cell lines ,83. WhilMultiple lines of evidence suggest that the AR is a favorable prognostic indicator in breast cancer and that AR agonists would be the preferred approach for choice of androgenic treatment for ER-positive breast cancer. However, data is conflicting in TNBC. With multiple players involved in TNBC, the action of the AR in TNBC appears to be influenced by cross-talk with other pathways that differ between cell types and cancer subtypes.Clinical evidence supporting the use of steroidal androgens for breast cancer dates back to the 1940s when testosterone and DHT were used to treat women with breast cancer ,84. SeveInitial evidence of synthetic steroidal androgens showing growth inhibitory effects in breast cancer came from the use of fluoxymesterone  and medroxyprogesterone acetate ,90,91. TDespite the historic and positive clinical results achieved with androgens in breast cancer, there have been few controlled clinical trials. As such, it remains unclear which subtypes respond best to androgens and the magnitude of response that can be expected. Ongoing clinical trials with newer nonsteroidal SARMs and nonsteroidal antiandrogens are poised to fill this knowledge gap. DHT, testosterone, and fluoxymesterone are steroidal androgens that have androgenic effects not only in breast, but also in other tissues including uterus, ovaries, skin, and hair follicles. SARMs were first reported in the late 1990s and subsequently shown to tissue selectively activate the AR in breast, muscle, and bone, without side effects associated with steroidal androgens ,96,97,98Abiraterone acetate is an inhibitor of Cyp17A1 enzyme, an enzyme upstream in the steroidogenesis pathway. An intriguing result was obtained in a clinical trial with abiraterone in ER-positive breast cancer patients . The cenThe results obtained in MDA-MB-453 cells provided an impetus to evaluate antagonists in breast cancer, TNBC in particular. Two AR antagonists, bicalutamide and enzalutamide, and a CYP17A1 inhibitor, abiraterone, are currently used in the clinical treatment of prostate cancer. Repurposing these drugs to treat TNBC should prove straightforward if they are found to be effective in the clinic. An investigator-initiated clinical trial was conducted to evaluate the efficacy and safety of bicalutamide . Out of A follow-up case report of one patient with AR-positive TNBC who relapsed after chemotherapy and progressed after multiple treatments and surgery and responded to treatment with 150 mg bicalutamide has also been published . The patBased partially on the modest success achieved with bicalutamide, clinical trials in TNBC and ER-positive breast cancer were initiated with a second generation AR antagonist, enzalutamide. Enzalutamide has a unique mechanism of action where it blocks AR nuclear translocation and is more potent than bicalutamide . AlthougAbiraterone, the CYP17A1 inhibitor, was tested in 34 AR-positive TNBC patients . PatientStudies from several groups support the concept that AR elicits anti-proliferative effects in ER-positive breast cancers by antagonizing ER action. Data also suggests that the AR in the presence of agonists binds to estrogen response elements (EREs) by competing for common binding regions  Figure . LikewisWhile the mechanism is, safe to say, modestly clear in an ER-positive setting, it is still ambiguous in TNBC, especially considering that only one cell line MDA-MB-453 was used for mechanistic studies. The AR has been shown to cross-talk with several proteins in MDA-MB-453 cells. FOXA1 regulates AR and ER DNA binding and has significant overlapping binding regions in MDA-MB-453 . SimilarThe AR is a favorable prognostic marker and a promising therapeutic target in breast cancer. In ER-positive breast cancer, the landscape is clear suggesting that androgens and in particular nonsteroidal AR agonists may provide beneficial effects. On the other hand, data on TNBC is conflicting with historical data favoring the use of agonists, data from enzalutamide clinical trials supporting antagonists, and data from abiraterone clinical trials suggesting that inhibition of AR signaling is not beneficial. This is likely to come down to the subtypes in TNBC where a subtype might respond to agonists, while another subtype might respond to antagonists. A clear picture can be obtained only with new preclinical translational models such as the patient-derived xenografts (PDXs) that will provide clarity. Even in this case, the outcome and mechanisms might vary between patient specimens and exposure to prior treatments. In addition, the evolving AR splice variants (AR-SVs) have to be taken into consideration while planning a strategy . ConsideOverall the next few years, when results from clinical trials with enobosarm and enzalutamide will be available, are critical to provide greater clarity on the role of the AR in ER-positive and –negative breast cancers. Considering that new agonists and antagonists for the AR are available, the emergence of nonsteroidal drugs targeting the AR as a new hormonal treatment for breast cancer is almost certainly on the horizon.


Xenopus oocytes. This allowed us to correlate the nuclear AR concentration to its capacity for specific DNA binding and transcription activation in vivo. AR variants with polyglutamine tracts containing either 25 or 64 residues were expressed in Xenopus oocytes by cytoplasmic injection of the corresponding mRNAs. The intranuclear AR concentration was monitored in isolated nuclei and related to specific DNA binding as well as transcriptional induction from the hormone response element in the mouse mammary tumor virus (MMTV) promoter. The expanded AR with 64 glutamines had increased capacity for specific DNA binding and a reduced capacity for transcriptional induction as related to its DNA binding activity. The possible mechanism behind these polyglutamine-induced alterations in AR function is discussed.Expansion of a polyglutamine-encoding trinucleotide CAG repeat in the androgen receptor (AR) to more than 37 repeats is responsible for the X-linked neuromuscular disease spinal and bulbar muscular atrophy (SBMA). Here we evaluated the effect of polyglutamine length on AR function in  •Spinal bulbular muscular atrophy is caused by a polyQ expanded androgen receptor.•Xenopus oocytes.Function of AR with expanded polyQ tract was analyzed in •AR with expanded polyQ tract has increased DNA binding but reduced gene activation. They exert their role Xenopus oocytes to look more closely at the function of the AR with an expanded polyQ tract. The large size of these cells allows quantification of intranuclear receptor concentration, sequence specific DNA binding and AR target gene activation Although the cause of SBMA is expansion of the CAG repeats in the AR gene the exact disease mechanism remain unclear. We decided to use Xenopus oocyte system is that proteins may be expressed in variable amounts by injection of corresponding in vitro transcribed mRNAs Xenopus oocytes leads to second-strand DNA synthesis coupled to assembly of a tightly organized chromatin structure in vivo footprinting An advantage of the Here we show that AR with a pathological polyQ tract of 64 residues (ARQ64) has increased capacity for specific DNA binding. Interestingly, this increase did not correlate with an increase in transcription induction at the MMTV promoter. Hence the transcriptional activity of ARQ64 was significantly reduced in comparison to the wild type ARQ25 as related to its DNA binding activity. The possible mechanism for this effect is discussed.22.1−3 M in EtOH and MDV3100  as 1×10−2 M in DMSO . The reporter pMMTV:M13 contains the 1.2 kb MMTV LTR fused to the HSV TK gene and its transfer to M13 was described XmaI and EcoRI restriction sites was excised from pβhAR and replaced with fragments containing 0, 13, 25, or 64 CAG repeats. The AR-Q64 cDNA was shown to be contaminated with AR variants containing shorter repeat(s) than Q64. The contaminants were removed by re-transformation of the pβAR-Q64 plasmid in E-coli cells with reduced recombination activity .AR ligands used were R1881 , as 1×102.2Xenopus laevis oocytes have been described previously 3H]-R1881 for quantification of intranuclear AR (see below). Oocytes were incubated at 21 °C after mRNA injections and until they were harvested ∼28 h later The animal experiments were conducted according to a defined protocol approved by a local ethical committee . DNA and mRNA injections into 2.33H]-R1881 . Since Xenopus oocytes contain endogenous androgens 3H]-R1881 concentration above 50 nM was enough to fully saturate the expressed AR with radioactive ligand  was used to estimate the efficiency for tritium .We previously used Western blot (WB) to monitor the amount of AR in H]-R1881 B. The noXenopus oocytes are filled with yolk protein and lipophilic substances. Steroids such as [3H]-R1881 are also rather lipophilic. We addressed this potential source of error in quantification of AR based on radioactive ligand by measuring the amount of [3H]-R1881 ligand in extracts from whole oocytes and from manually isolated nuclei that were either not injected or injected with ARQ25 mRNA .Our finding of a reduced capacity by ARQ64 to drive MMTV transcription as related to its specific DNA binding activity c.f. D encouraOocytes injected with the three AR variants Q0, Q13 and Q25 followed by the ssDNA injection, 3 ng MMTV: M13, three independent experiments did not generate any consistent difference in expression , DNA bin3.5in vivo. This difference was distinct when comparing DNA binding as a function of nuclear AR concentration (We observed a difference in sequence-specific DNA binding by ARQ64. Unexpectedly, this AR variant showed increased capacity to bind specific DNA sequence in a chromatinized template ntration , but it ntration . In the Interestingly, inhibiting SUMOylation of AR with an expanded polyQ repeat counteracted the significant loss of transactivation caused by polyQ expansion and ameliorated the mutant AR-mediated disease in a mouse model in vivo. This important step in the mechanism of action of androgens is confined to the chromatin target and the transactivating factor, i.e. AR. It remains to be determined whether this result also applies to other AR driven enhancers and in other cellular context.We are the first to report increased specific DNA binding capacity of the ARQ64 The authors have declared no conflicts of interest [23].