ObjectiveThe lifetime direct and indirect costs of spinal injury and spinal cord injury (SCI) increase as the severity of injury worsens. Despite the potential for substantial improvement in function with acute rehabilitation, the factors affecting its cost have not yet been evaluated. We used a proprietary hospital database to evaluate the direct costs of rehabilitation after spine injury.MethodsA single-center, retrospective cohort cost analysis of patients with acute, traumatic spine injury treated at a tertiary facility from 2011 to 2017 was performed.ResultsIn the 190 patients  identified, American Spinal Injury Association impairment scores on admission were 32.1% A, 14.7% B, 14.7% C, 33.2% D, and 1.1% E. Surgical treatment was performed in 179 (94.2%) cases. Most injuries were in the cervical spine (53.2%). A mean improvement of Functional Impairment Score of 30.7 ± 16.2 was seen after acute rehabilitation. Costs for care comprised facility (86.5%), pharmacy (9.2%), supplies (2.0%), laboratory (1.5%), and imaging (0.8%) categories. Injury level, injury severity, and prior inpatient surgical treatment did not affect the cost of rehabilitation. Higher injury severity  and spinal level of injury  were associated with higher length of rehabilitation stay in univariate analysis. However, length of rehabilitation stay was the strongest independent predictor of higher-than-median cost  after adjusting for other factors.ConclusionsSpine injury has a high upfront cost of care, with greater need for rehabilitation substantially affecting cost. Improving the efficacy of rehabilitation to reduce length of stay may be effective in reducing cost. Spine fractures account for 3-6% of all skeletal fractures while acute spinal cord injury (SCI) has an estimated prevalence of 54 cases per 1 million people in the United States and an overall in-hospital mortality rate approaching 8% . In the Inpatient rehabilitation after spine injury and SCI is a time- and resource-intensive endeavor. Well-trained and highly specialized teams of physiatrists, physical therapists, occupational therapists, and other medical professionals are required for optimum rehabilitation outcomes . Many paPatient inclusionAfter receiving Institutional Review Board approval with a waiver of informed consent, a cross-sectional analysis of patients treated for spine injury or SCI from an internal database at the Department of Physical Medicine and Rehabilitation at our institution from January 2011 to December 2017 was performed. Patients were excluded if they were <18 years of age, if they underwent admission to rehabilitation for a chronic SCI, if they underwent treatment for a non-traumatic SCI mechanism, or if they lacked complete clinical, radiographic, and cost data. A manual chart review was performed to verify patients' inclusion and obtain demographic, clinical, and surgical data. Patients were cross-referenced in the institutional costs database to acquire subtotal costs.Surgical proceduresPatients underwent a range of surgical approaches in the cervical, thoracic, or lumbar spine at an outside hospital or at our institution. Patients transferred after initial evaluation or surgical stabilization were considered transferred patients while those directly admitted to our institution were considered admitted patients.AnalysisDemographic data included patient age and sex. Patient status before surgery was assessed with the American Society of Anesthesiologists (ASA) physical status system. The American Spinal Injury Association (ASIA) injury severity (AIS) score and injury level were identified from the clinical records. The Functional Independence Measure (FIM) was acquired from admission and discharge rehabilitation records. Length of stay (LOS) and discharge disposition were captured as well.The institutional database is an electronic resource that reports direct costs, in lieu of patient/insurer charges -11. TotaA total of 190 patients treated from 2011 to 2017 were included for analysis. Patient demographics and admission characteristics are displayed in Table A breakdown of surgical procedures by site shown in Table A graphical breakdown of total rehabilitation costs is displayed in Figure Table Subgroup analyses for individual variable contributions to overall costs are displayed graphically in Figure Overall, our results suggest that rehabilitation LOS impacts costs of rehabilitation most significantly after acute, traumatic spine injury. Strategies to improve the efficiency of treatment and reduce LOS would have the greatest cost savings for the spine injury population. In addition, injury severity and level suggested longer LOS but this did not correlate with higher cost in every case. In addition, less severe injury may allow for more rapid patient recovery and transition to home or outpatient services. Higher LOS was seen for ASIA grade B lesions compared with other groups, suggesting strategies to maximize rehabilitation in this patient group may have the biggest effect on controlling costs. Variability in cost was seen for each AIS grade and level of injury, which may suggest that perhaps other factors can account for some cost variation among patients. Understanding this variability and potentially streamlining care may produce some cost savings.Healthcare costs are a significant burden to society, with 2016 annual healthcare expenditures reaching nearly 20% of the United States gross domestic product . AlthougThere were several pertinent findings in the current study. Facility costs accounted for nearly 90% of the total rehabilitation costs in our study. When compared with other healthcare settings, inpatient rehabilitation typically requires fewer pharmaceutical interventions and laboratory studies and instead requires more facility resources for rehabilitation activities , which may explain the relative contribution of facility costs versus pharmacy and other costs. However, much of the post-hospitalization care of patients with spine injury, including medications and other services, continue during rehabilitation. Nonetheless, the acute rehabilitation by and large covers the largest component of cost. Previous studies by our group also suggest that, in cases that instrumentation or devices are not heavily used , facility costs contribute most to overall cost . No signStudy limitationsThis study is not without limitations. A single-center analysis limits analysis of rehabilitation costs across different health systems. Therefore, the results presented herein may not be generalizable across different rehabilitation facilities. Furthermore, our study was performed at an academic rehabilitation center and may not be reflective of costs in other practice models . The study population did however reflect the expected distribution of age, sex, and AIS injury grade found nationally . Our stuWe present data on our institution's experience with direct costs of acute inpatient rehabilitation after spine injury. Facility costs accounted for the vast majority of rehabilitation costs. Length of stay was the only independent predictor of increased rehabilitation costs. Spine injury continues to have a high upfront cost of care, with the great need for rehabilitation playing a key role in cost. Improving the efficacy of rehabilitation in order to reduce length of stay and streamline care may be important in reducing costs.


The aim is to investigate the relationship between a positive outcome on rehabilitation after hip fracture and behavioral psychological symptoms of dementia (BPSD) transition during rehabilitation. This study is a retrospective cohort study based on the Japan Rehabilitation Database. We recruited 756 subjects 65 years of age or older from 31 hospitals in the database. All subjects were in the hospital as patients undergoing rehabilitation for hip fracture. Functional independence measure (FIM), walking ability, Mini-Mental State Examination (MMSE), and BPSD were measured both at the beginning and at the end of rehabilitation. MMSE for 23 or under was defined as the cognitive-impaired group. MMSE for 24 or over was used as the cognitively intact group. Cognitive impaired participants were divided into four groups: participants presented no BPSD both at the beginning of rehabilitation and at the end of rehabilitation (Group (-/-)), participants presented BPSD at the beginning of rehabilitation but resolved at the end of rehabilitation (Group (+/-)), participants had no BPSD at the beginning of rehabilitation but appeared at the end of rehabilitation (Group (-/+)) and participants had sign of BPSD both at the beginning of rehabilitation and at the end of rehabilitation (Group (+/+)). The endpoints were waking ability, FIM gain. As results, one hundred thirty-seven cognitive-impaired older people patients out of 471 (29.1%) suffered from BPSD at the beginning of rehabilitation. FIM gains in cognitively intact group, Group (-/-), Group (+/-), Group (-/+) and Group (+/+) were 24.8 ± 18.7, 17.5 ± 16.9, 27.3 ± 19.7, 17.8 ± 12.2 and 12.2 ± 17.2, respectively. The Group (+/-) was significantly connected to a positive outcome for rehabilitation. The present study suggested that the management of BPSD can lead to better functional recovery during rehabilitation. Thus far, many studies have demonstrated that cognitive impairment has been one of the major factors that limit functional recovery during rehabilitation for older people with hip fracture. However, few reports focus on how rehabilitation of older people with dementia is handled.  Hip fracOlder people with cognitive impairment who fracture their hip have a greater risk of comorbidity such as delirium,  pressureAlthough rehabilitation has been reported to improve physical function for the cognitive-impaired older people with hip fracture, , 26–28 tThe number of cognitive-impaired older people is increasing in the world ; howeverBecause psychological symptoms such as delirium and depression prevent older people from functional recovery after hip fracture, we focused on psychological symptoms during rehabilitation. The aim is to investigate the relationship between a positive outcome of rehabilitation after hip fracture and behavioral psychological symptoms of dementia (BPSD) transition during rehabilitation. We researched whether improvement of BPSD might lead to a beneficial outcome of rehabilitation for older people with hip fracture.Detailed data was collected in the Japan Association of Rehabilitation Database from 2005 to 2015. The data set is separated by three categories: hip fracture, cerebrovascular disease, and spinal cord injury. The present study used the database of hip fracture. It includes the following data: age, sex, physical function , cognitive function, days from injury onset, type of fracture, information about long-term insurance, length of stay, BPSD, comorbidities, past history, and other detailed data that affects rehabilitation.All personal data was coded, deleting any information related to personal identification. Patients did not provide informed consent for inclusion of their data in the database and subsequent use of the data in research investigations. Informed consent was waived because of the anonymous nature of the data.  This stuThe inclusion and exclusion criteria of the study are shown in The data of age, sex, comorbidities , cognitive function, physical function, walking ability, BPSD, and length of stay were obtained from the Japan Rehabilitation database. Cognitive function and physical function were evaluated by the Mini-Mental State Examination (MMSE) and FIM, respectively. MMSE was evaluated three times in the database after operation, at the beginning of rehabilitation and at the end of rehabilitation. To minimize the effect of operation itself and operation-related psychological effect, we used maximum MMSE score among these three evaluations. The data classified walking ability in the following categories: walking independently, walking with cane or walker, and cannot walk. MMSE for 23 or under was defined as the cognitive-impaired group. MMSE for 24 or over was used as the cognitively intact group. BPSD was defined as presenting the following symptoms: delirium, delusion, wandering, insomnia, depression, aggression, and violence. Because BPSD was evaluated as only BPSD positive (+) or BPSD negative (-), the sign and symptom from which patients were suffering was unclear.Outcomes were assessed by FIM gain (FIM at the end of rehabilitation–FIM at the beginning of rehabilitation) and FIM gain per day (FIM gain/length of stay). FIM contains 18 items. Thirteen items pertain to motor function (motor FIM), five items concern cognitive function (cognitive FIM). Motor FIM gain, motor FIM gain per day, cognitive FIM, and cognitive FIM per day were also assessed as outcomes.The typical rehabilitation program was conducted for all patients. It was composed of 40 minutes of physical therapy and 40 minutes of occupational therapy per day, for 5 to 7 days per week. Physical and occupational therapy focused on gait and exercise related to activities of daily living. Passive range-of-motion exercise of the affected side and muscle-strengthening exercise of the unaffected side were included. t-test for continuous variables and chi-squared test for categorical variables were used to compare cognitively intact and older people with cognitive impairment. After older people with cognitive impairment group was classified into four groups according to BPSD transition, we investigated the difference of functional recovery among subgroups divided by BPSD transition. The cognitive-impaired group was divided into four groups according to BPSD present (+) or absent (-) both at the beginning and the end of rehabilitation. We evaluated the change of MMSE (MMSE at the end of rehabilitation–MMSE at the beginning of rehabilitation) based on the previous study. [Data were analyzed using SPSS software . Two-sided unpaired s study.  AnalysisWe recruited 759 of older people from the Japan Rehabilitation database . The numThe length of stay was no different between the cognitively intact and cognitive-impaired groups  . HoweverNext, we investigated the difference of functional recovery among subgroups divided by BPSD transition . The cogFIM gain in the cognitively intact group, Group (-/-), Group (+/-), Group (-/+), and Group (+/+) was 24.8 ± 18.7, 17.5 ± 16.9, 27.3 ± 19.7, 6.6 ± 17.8, and 12.2 ± 17.2, respectively . FIM gaiWhile BPSD was significantly associated with rehabilitation outcomes independent from MMSE, MMSE was also independent predictive factor for rehabilitation outcomes.The rate of individuals who could walk independently was higher in the cognitively intact group than in the cognitive-impaired group before hip fracture. Conversely, the rate of individuals who needed a cane or walker for walking or who could not walk was higher in the cognitive-impaired group. Similar results were detected at the end of rehabilitation. Walking ability was significantly higher in the cognitively intact group than in the cognitive-impaired group Tables  and 3. WThe present study showed that older people with cognitive impairment recovered less of their physical function during rehabilitation than older people without cognitive impairment. The prevalence of BPSD was 29.1% (137/471), and BPSD had disappeared in 41 (29.9%) of 137 individuals. Interestingly, BPSD was an important predictive factor of rehabilitation outcome for older people with hip fracture and cognitive impairment. Our study also suggests that the recovery from BPSD might lead to positive outcomes, such as improvement of waking ability and FIM gain, during rehabilitation.Stenvall M et al. illustrated a multidisciplinary intervention program, such as comprehensive geriatric assessments and rehabilitation, designed to prevent delirium, falls, decubital ulcers, and malnutrition, which improved rehabilitation outcome after hip fracture for older people with dementia.  Our studIn our data, the number of patients who presented BPSD at the beginning of rehabilitation was 137 (29.1% in Group (+/-) plus Group (+/+)) of 471 cognitive-impaired patients. Givens et al. measured the prevalence of depressive symptoms, cognitive impairment, and delirium in patients with hip fracture and found that 22% of patients had one cognitive or mood disorder, 30% had two, and 7% had three.  Other stOur study could not show which symptoms were present in the BPSD-positive group, but it has been reported in other studies that agitation was the most frequent symptom, followed by depression, apathy, and other behavioral disorders in patients with Alzheimer’s disease or multi-infarct dementia. , 36 The Regarding the therapeutic intervention for BPSD, non-pharmacological treatment approaches have become the preferred first-line option. –41 When Pharmacological treatment such as with quetiapine, benzodiazepines, or anti-psychotic drugs for agitation and aggression, and antidepressant drugs for depression and apathy, were reported. , 40 HoweThere are several limitations in the present study. First, this database did not evaluate which type of BPSD occurred. BPSD was defined in the database as delirium, delusion, wandering, insomnia, depression, aggression, and violence. However, each sign and symptom should be evaluated individually. Second, therapeutic intervention was not clear. Rehabilitation or exercise intervention was reported to reduce the prevalence of BPSD. , 51, 52 To the best of our knowledge, allowing these limitations, only a few studies showed positive outcomes of in-patient rehabilitation for older people with cognitive impairment after hip surgery.  Up untilThe present study suggested that BPSD management has the possibility to lead to better functional recovery during rehabilitation. A positive outcome might be expected to assess the psychological symptoms and comorbidities that occur most often in older people with cognitive impairment.


One of the psychological mechanisms that contribute to effective and efficient team actions is team cognition, defined either as shared knowledge states about game situations, teammates’ skills, and action probabilities or direct communication processes in the team action itself. Particularly in interactive team sports , characterized by highly complex, dynamic, and uncertain situations, sharing a common understanding concerning potential future actions and how to coordinate these actions may be an advantage. Otherwise, team members must communicate their thoughts and ideas on the fly, which might be impossible due to time pressure, cognitive costs or noisy environments. This study examined if shared knowledge and verbal communication change through collective training. Forty-six under-18 and under-21 youth football players performed a football task in teams of two. The task consisted of passing and running elements common in football. After a training phase, and before two testing phases, players evaluated their actions and the actions of their assigned teammate regarding action type, location, and timing. Out of these evaluations, two indices of common understanding were computed. Furthermore, verbal communication during the task was video-and audio-recorded. Data analysis showed that shared knowledge considerably increased over time and with practice. Simultaneously, overall verbal communication and verbal communication consisting of orienting information was significantly reduced. Additionally, there was a tendency for a correlation that when shared knowledge increased, orienting verbal communication decreased. Overall, the players used orienting communications the most (77%). The study revealed that shared knowledge states and verbal communication change through collective training and that there might be a relation between the level of shared knowledge and the use of orienting verbal communication. Further studies in and off the field are needed to disentangle the complex interplay of team cognitions. Whether playing a solid, insuperable defense; executing a fluid counterattack; or finishing a play with a blind no-look pass to the freestanding teammate to score, one of the psychological mechanisms contributing to effective performance in team sports like football is team cognition . Team coWhile shared knowledge issues were studied a lot in the military and I/O context  rather time-consuming procedures before a game  via explicit planning and other training forms like cross-training, training for adaptability or self-correction training see  and (ii)in game communication tends to be more relevant than out of game communication, because it is assumed more and directly related to performance outcomes . Stimulating is defined as messages motivating partners to maintain or increase/decrease activity levels with no reference to what to do and how to do it. Evaluation means positive or negative statements about owns or players’ actions or behavior. Task-irrelevant communications are positive or negative messages with no bearing on performance. The few studies about in game communication in team sports  playing for the under-18 and under-21 teams of a youth football academy of a club in the highest league in Switzerland participated in the study. The mean number of years of competitive playing and club training experience was 11.38 (SD = 1.67). Players have been in the same team at least since the beginning of the season. This means that they have known each other from the normal training and competitions for at least 5 months . For our study, dyads were formed out of the participants. These dyads were formed within their respective age groups, with the 46 participants resulting in 23 dyads (16 dyads of under-18 players and 7 dyads of under-21 players).Forty-six male elite junior football players , location , and timing  via situation-specific questions . Afterwards, players were informed about their new teammates for the real experiment. Thus, every player had to evaluate his newly assigned teammate based on the same questions, that is, at what speed, where and when he expects the teammate to play and receive the ball. These ratings were based on previous training and match experiences. Teams were randomly assigned in that way, that a “more skilled” player (rating A) was playing with a “less skilled” player (rating B). The responsible team managers/coaches made these player ratings beforehand. Then, these dyads completed three times a competitive run to finish the first measuring point. Upon termination, they independently answered the same questions for the second time (this lasted approximately 15 min in average) and completed another three competitive runs in the same teams to finish the second measuring point.The procedure before and throughout the measuring points was the following: After a welcoming, players were introduced to the standardized task and had the opportunity to practice the elements. Subsequently, they performed two competitive practice runs in teams of two. Based on their experiences in these runs, each player evaluated his actions by action type  and timing  from the pass giver’s perspective  and from the pass receiver’s perspective . In total, thus, ten self-evaluative and ten foreign-evaluative items resulted. These questions covering the two main team actions were answered on a corresponding 6-point Likert scale  in the same square” to “as soon as I/he stay(s) in the square we have to play in”). To facilitate better understanding, illustrations accompanied the questions about action location and action timing (see Appendix for an example).So far, scales developed with the aim of measuring shared knowledge about teams  and the evaluation of player A about player B as a pass receiver is: “In the overlapping run situation, he demands/desires the pass very precise into the foot” (LS = 5). In turn the self-evaluation of player B as the pass giver is: “In the overlapping run situation, I play the pass very steep into the run of my teammate” (LS = 2) and the evaluation of player B about player A as a pass receiver is: “In the overlapping run situation, he demands/desires the pass very precise into the foot” (LS = 5). Accordingly, the sum of the absolute differences between these 2 × 2 values over all ten items resulted in the team reciprocity index . In general, when generating these indices, the lower the absolute differences over all items, the more similar the mutual understanding. To analyze the changes of verbal communication, two independent persons counted the frequency of meaningful communication units per measuring point . A meaningful unit was defined as a self-consisted and closed message . Therefore, the chances to accept the alternative hypothesis were small. For all other analyses, power was good (1-β ≥ 0.80).We used the answers of the situation-specific questions to compute a meta-accuracy and a reciprocity accordance index. For the former index, self-evaluation ratings of each item from player A were calculated together with player B’s external evaluations about the same perspective  of player A and vice versa. For example the self-evaluation of player A is: “I play very fast passes” [Likert-Score (LS) = 5] and player B’s evaluation of player A is: “He plays rather fast passes” (LS = 4). In turn the self-evaluation of player B is: “I play rather less fast passes” (LS = 3) and player A’s evaluation of player B is: “He plays not fast passes” (LS = 2). The sum of the absolute differences between these 2 × 2 values over all ten items constructed the team meta-accuracy index . For the reciprocity index, self-evaluation ratings of each item were calculated together with the same player’s evaluations about his teammate concerning the complementary perspectives and actions and vice versa. For example the self-evaluation of player A as the pass giver is: “In the F = 4.43, p = 0.047, F = 34.9, p < 0.001, F = 9.71, p = 0.005, F = 21.2, p < 0.001, vely see , 2.r = 0.35, p < 0.05). Correlational analysis of the differences between t1 and t2 in the variables accordance indices and overall verbal communication revealed no significant correlations. However, a tendentious and weak relation between the indices and orienting verbal communication use could be found , in the sense that the more similar the indices became, the less orienting communication was used . None-the-less, the task was football-specific and carried out with experienced youth football players. Therefore, we assume good external validity as a strength of this study. At the same time, because of not controlling every team dynamics factor (except team age/familiarity), internal validity may have suffered, which is a potential limitation. Other limitations pertain to the small statistical power regarding the correlational analysis and the self-constructed questions, which may have led to arguable results. Although we wanted to fulfill the premise of All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cooperative strategies are predicted for repeated social interactions. The recently described Zero Determinant (ZD) strategies enforce the partner’s cooperation because the ‘generous’ ZD players help their cooperative partners while ‘extortionate’ ZD players exploit their partners’ cooperation. Partners may accede to extortion because it pays them to do so, but the partner can sabotage his own and his extortioner’s score by defecting to discipline the extortioner. Thus, extortion is predicted to turn into generous and disappear. Here, we show with human volunteers that an additional monetary incentive (bonus) paid to the finally competitively superior player maintains extortion. Unexpectedly, extortioners refused to become disciplined, thus forcing partners to accede. Occasional opposition reduced the extortioners’ gain so that using extortion paid off only because of the bonus. With no bonus incentive, players used the generous ZD strategy. Our findings suggest that extortion strategies can prevail when higher competitiveness is rewarded with extra gain. In game theory, ‘extortionate’ tactics in two-player games are predicted to give way to ‘generous’ strategies. Here, the authors show in a human experimental sample that extortion can prevail as a strategy in games in which there is a specific reward for doing better than the other player. The paradigm for studying potential cooperation through reciprocation is the Prisoner’s Dilemma (PD) game3. Each of two players can either cooperate (C) or defect (D). If both cooperate, each player earns more than if they both defect. However, if one defects and the other cooperates, the defector has the highest gain and the cooperator the lowest. Irrespective of what the other does you gain more by defection if the game is played only once, hence the dilemma. However, when the same subjects play the PD repeatedly, numerous sequences of, e.g., C, D, D, C, C and so on, are possible. Therefore the strategy that is most successful against any partner cannot be found by mathematical calculation because the array of potential strategies is too huge4. Axelrod’s3 computer tournament simulating evolution among strategies that had been proposed by theorists found ‘Tit-for-Tat’ as the winner . The next champion was ‘Generous Tit-for-Tat’4, followed by’Win-stay, lose-shift‘5, all largely cooperative strategies6, though the daily newscasts report widespread uncooperative human behaviour.Human cooperation is often based on reciprocity despite the risk of impending defection7 have dramatically changed our view on the Prisoner’s Dilemma. They found a special class of strategies, called Zero Determinant (ZD) strategies, which enforce a linear relationship between the two players’ scores. If the ZD player X chooses to extort Y, who is an ‘evolutionary' player not possessing a theory of mind and instead simply seeks to adjust his strategy to maximise his own score in response to whatever X is doing, Y would not try to alter X’s behaviour8. Extortionate strategies, reported by Press and Dyson7, grant a disproportionate number of high payoffs to X at Y’s expense. It is, however, in Y’s best interest to cooperate with X because only by doing so Y is able to increase his own score. Thus, he ends up increasing X’s score even more than his own. He will accede to X’s extortion because it pays him to do so8. Extortioners use a conditional cooperative strategy with a bias to their own advantage , disciplining has no future whereas in an iterated PD it may have. Theoretical and empirical studies thus predicted extortion turning into generous strategies15.Recently Press and Dyson17 for some time until they ‘punish' the extortioner by playing D, sacrificing their small gain for tearing down the extortioner’s larger gain, which could not coerce the computer. With evolutionary simulations theorists studied the new ZD world and predicted extortioners to switch to more cooperative, generous strategies. We thus expect again to see nice and cooperative strategies prevailing15.Experimental studies have shown that human players accede to a computer playing extortion11 because they end up with mutual defection when they meet each other. The more frequent they are the more likely it is to meet another extortioner. Thus there is a limit frequency under negative frequency-dependent selection beyond which more cooperative strategies have a higher overall payoff and can spread11. Stewart and Plotkin11 have identified a different subset of ZD strategies, called ‘generous ZD' strategies that forgive defecting opponents, but nevertheless dominate in evolving populations. An experimental confirmation of humans using generous ZD strategies is still elusive.Extortioners risk being disciplined and have problems succeeding in evolving populationsContrary to extortionate behaviour a generous ZD player always starts with C, cooperates after mutual cooperation and only mildly punishes defection  with the same game either where an asymmetric incentive was assigned to one player chosen randomly (T1), but both players knew who had been chosen, or where the incentive was assigned to both players, but only one, the finally more competitive one, gained the extra bonus (T2). Our first hypothesis assumes that the incentive to earn an extra bonus is strong enough, especially when restricted to a designated player in T1, that this player adopts extortion to be competitively superior at the expense of some losses due to the partner’s occasional sabotaging both his own and X’s score by defecting. The extorting player may become disciplined after some time. In T2 any player could profit but only one could succeed. Extortion might be less expressed than in T1. Our second hypothesis is that in comparison to T1 and T2, T0 is completely egalitarian which might pave the way for generous ZD strategies to predominate because there is no bonus to gain that renders potential extortion especially profitable. We thus expect more cooperation in T0 and predict to see generous ZD strategies.We find in egalitarian iterated Prisoner’s Dilemmas with no incentive (T0) that generous ZD strategies prevail. When one player is assigned an additional monetary incentive (bonus) (T1), if he/she is the finally competitively superior player, he/she often adopts the extortion strategy. Unexpectedly, extortioners refuse to become disciplined by their partners, thus forcing partners to accede. When both players can reach the bonus (T2), the use of extortion is less pronounced.F2,48 = 4.9, p = 0.012; mean ± s.e.m. per player: T0: 0.548 ± 0.21; T1 = 0.35 ± 0.18; T2 = 0.35 ± 0.17) with significantly highest proportions in treatment T0  and no difference between T1 and T2 (post hoc test: p = 0.99). Players with no incentive assigned (T0) were most cooperative. The payoff also differed significantly among treatments  with significantly higher payoffs in treatment T0 were players were assigned no incentive . The occurrence of conflict, depicted by the proportion of DD decisions (i.e. both players decided D), differed among treatments  where T1 and T2 had higher proportions of DD compared to T0 . Players with no incentive (T0) had the lowest rate of conflict. Thus, both the cooperation rate and the payoff were higher in T0 than in either T1 or T2 and the existence of an incentive, asymmetric or symmetric, to gain €10 extra when gaining 10% more than the partner over the 49 rounds decreased cooperation and income, and enhanced conflict.The proportion of cooperation, i.e. to play C over the 49 rounds, differed among treatments (GLM: p = 0.039). The incentive motivated competitive behaviour. Those assigned with the incentive made over all rounds of the game a higher proportion of D decisions (0.693 ± 0.043 per round) than their partners not assigned the incentive .Ten of 18 players (60%) in T1 assigned with the incentive of gaining €10 extra managed to earn 10% more than their co-players, whereas two players (10%) without that incentive earned 10% more than their co-players but did not receive €10 extra. Thus, among the 12 players who managed to earn 10% more than their co-player, more players had been assigned with the incentive .For deciding whether the generous strategy had been used in T0, we tested for linear relationships between the payoffs of either player across many rounds of play and the cooperation rate of the potential generous player X; payoff ~ F1,86 = 87.466; p = 9.33 × 10–15; Fig. F1,86 = 6.418; p = 0.013). Furthermore, the intercept was significantly lower for the potential generous player  as predicted for the generous strategy. Thus the generous players let their co-players succeed until they reached mutual cooperation and similar payoffs. The rest of the players in T0 do not seem to contain extortionate players . We found significant positive linear relationships between cooperation rate of the potential generous player X and payoff per round of either player .F1,176 = 120.42; p < 2.2 × 10–16; Fig. F1,176 = 16.57; p = 0.00011). Furthermore, there was no significant difference for the intercepts . Thus, the player X who was assigned the incentive of gaining the extra bonus and gained it, used probably the extortion strategy enforcing cooperation of her partner Y who could increase his own gain only by increasing his cooperation supplying the extortioner with an increasingly higher gain. The rest of the players in T1 deviate from expectation for extortionate behaviour , we found significant positive linear relationships between cooperation rate of the partner and payoffs per round . Furthermore, there was no significant difference for the intercepts . Thus, the player X who gained the extra bonus probably used the extortion strategy enforcing cooperation of her partner Y who could increase his own gain only by increasing his cooperation supplying the extortioner X with an increasingly higher gain. There was no significant difference in the pay-off increase with increasing cooperation rate for the rest of the players in T2; they thus deviate from expectation for extortionate behaviour , we found significant positive linear relationships between cooperation rate of the partner Y and payoffs per round , which is required for a generous ZD strategy. For a proof of the extortionate strategy, the regression of the co-player’s payoff on an extortioner’s payoff must yield a slope below the diagonal16, thus extortioners outcompete their co-players. In T1 . Thus, generous players gained a positive reputation, whereas they ranked their partners behaviour as almost neutral. In T1 extortioners received a negative score (5.125 ± 0.766 points). However, they rated their partners whom they had enforced to be cooperative as positive . In T2, where both players had the incentive to compete for the extra bonus, extortioners received almost the same almost neutral rating (3.455 ± 0.493 points) as their competing partners . As both players tried to be more successful and one of them managed to dominate the other, the similar score reflects the tension between them.A good reputation is worth having under many conditions7. However, ‘if the player has a ‘theory of mind’ about his co-player X, his only alternative to accepting positive, but meagre, rewards is to refuse them, hurting both himself and X'. ‘He does this in the hope that X will eventually reduce her extortion factor', as Press and Dyson7 suggested. Accordingly, evolutionary simulations predicted extortioners to switch to more cooperative strategies15. Extortion would no longer be expected. We confirm this prediction for the ordinary public goods game (T0). However, when one player could gain an extra bonus by being competitively superior (T1), she obviously used extortionate behaviour, which was opposed by the co-player with defection, probably trying to coerce the extortioner. It is well known that a fraction of the human population cares for equitable outcomes and exhibits a strong aversion against disadvantageous inequity19, i.e., a response known as ‘inequity aversion’20. Unexpectedly, however, X refused to be coerced and was steadfast extortionate, thus forcing her co-player to accede  with no promised incentive. When there was extra money to gain , extortioners unexpectedly resisted becoming disciplined. Thus extortioners with incentive were steadfast and reached the bonus despite losing money due to their co-players ‘occasional’punishment’. Finally they earned much more with the bonus added than did generous players in the control treatment. The message of the present study seems to be that extortion strategies may be expected, when there is an incentive to gain an extra bonus through being more competitive. To resist being disciplined paid off through the extra money they actually gained. In reality, many opportunities exist to gain from being competitively superior, such as for collaborating colleagues in companies to become promoted to a rare better-paid higher position. Our competitive societies may favour extortion. Previous studies found extortion when either a power asymmetry between partners existed that prevented opposition23. Each desk was equipped with paper and pencil, and subjects were encouraged to take notes. Subjects knew they would receive their earnings anonymously under their pseudonym and in cash directly after the game. Sessions took approximately 90 min including the initial instruction phase. For a detailed description of each treatment, a translation of the instructions is provided in the Experiments were conducted in November 2016 with 102 first-year biology students at the University of Kiel, Germany. All subjects gave their informed consent to participate. We invited 6 volunteers to each of 17 experimental sessions . Before each session, subjects were orally informed about how to operate the computers, and about the measures that were taken to ensure the subjects’ anonymity. During the experiment, subjects had a pseudonym, were separated by opaque partitions and they were instructed not to talk to each other during or after the experiment. Subjects made all their decisions through a computer interface based on z-Tree3 In all treatments each player of the PD group would receive a potential bonus payment of €5.00 if the pair had earned more than €0.30 per round throughout the experiment to reduce the risk of the pair to get stuck in DD, which is not informative.Treatment T0 is an Iterated Prisoner’s Dilemma game with 49 rounds. In addition, in T1 one randomly chosen player was assigned the incentive to earn extra money, in T2 both players were assigned the incentive but only the competitively superior one gained the extra money. The three treatments have in common that each player interacts with only one partner in an iterated PD over 49 rounds . To avoid end-round effects, participants were not informed about the number of rounds to be played. The three PD groups of two players each of an experimental session played the same treatment. The three PD games of a session were synchronised: after all six players had made their decisions in each round, the subjects of each pair were informed about their partner’s decision, their own and the partner’s payoff. Our statistical unit is the two players forming the prisoner’s dilemma group since they could not interact with the other participants of the experimental session in any way. In each round of the iterated PD the two partners had to simultaneously decide whether to cooperate (C) or to defect (D). If both cooperated, they each received a reward (R = €0.30). If one defected and the other cooperated, the defector got the temptation payoff (T = €0.50) and the cooperator obtained the sucker payoff (S = €0.00). However, if both defected they each received a low payoff (P = €0.10).The control treatment (T0) consisted of 15 PD groups in five experimental sessions with 49 rounds of iterative PD. The asymmetric treatment (T1) consisted of 18 PD groups in six experimental sessions with 49 rounds of iterative PD. One of the two players was randomly assigned the incentive to be able to gain an extra bonus of €10 if he/she had earned at least 10% more compared to the partner after all PD rounds. Both players were informed about who could win the extra bonus. The symmetric treatment (T2) consisted of 18 PD groups in six experimental sessions with 49 rounds of iterative PD. Again one player could earn the extra bonus of €10 if he/she had earned at least 10% more compared to the partner after all PD rounds. Different from the asymmetric treatment, where only the randomly chosen player could earn the extra bonus, both players in T2 had the potential to earn the extra bonus, but they knew that only one could achieve it.α payoff X + β, where α is the slope and β the intercept. A strict ‘extortion’ strategy arises when β = 0 and 0 < α < 1 because in this case payoff X > payoff Y, and X extorts Y by forcing her to accept a lower payoff than X, or else receive 0 payoff. Note, however, that if one chooses β = (1 −α) R, where R is the payoff for mutual cooperation in a given round of the IPD, that the relationship between payoffs is inverted with payoff X < payoff Y. This is a ‘generous’ strategy under which a player X does worse than her partner Y if they deviate from mutual cooperation.Extortion strategies belong to a class of iterated game strategies known as ZD, under which the probability of a player cooperating in a given round of the iterative PD is a function of the payoffs received by a player and her opponent. The result of a player X using such a strategy against an opponent Y is to induce a linear relationship between her expected payoff across many rounds of play (payoff X) and that of her opponent, (payoff Y), payoff Y = Thus, the generous strategy is observed whenαgenerous > 0,αpartner > 0,αgenerous > αpartner > 0βgenerous < βpartner.and The extortion strategy is observed whenαextortion > 0,αpartner > 0,αextortion > αpartner > 0βextortion = βpartner.and We used linear mixed effect models (LME) to test for potential generous in T0 and potential extortionate players in T1 and T2 using treatment-specific models  using the lmerTest package25. R2 were calculated using the r.squaredGLMM function of the MuMIn package26.For this statistical analysis we used the average payoff and the average cooperation of the 10 rounds of each period (9 rounds for the last period). So for each individual we had five data points for the payoff and the cooperation, respectively. We ran treatment-specific tests where we added pair (prisoners dilemma pair) nested within group  as random effect to all models . Statistical tests were performed in R  and positively bound. We used also GLMs with family Gamma (link = 'log') for the comparisons of the probability to cooperate, the proportion of DD decisions (i.e. both players decided D), and payoff per round and player between treatments. To determine differences between the individual treatments, we used posthoc tests with corrections for multiple comparisons with the multcomp package27.We used treatment-specific generalised linear models (GLMs) with family Gamma (link = 'log') for the correlations between payoffs of the ZD player and the other player Fig. . We usedAll experimental procedures follow German regulations. For behavioural experiments as ours, neither approval from an ethics committee nor written consent to participate is required according to German regulations. However, we have received the subjects’ written informed consent to participate; full anonymity and true information was guaranteed, and subjects had the option to stop participating at any time.Further information on experimental design is available in the Supplementary InformationPeer Review FileReporting Summary


The Earth is constantly bombarded by meteoroids of various sizes. During hypervelocity collisions a large amount of energy is coupled to the Earth’s atmosphere leading to disruption of decimeter to hundred meter-sized meteoroids. Smaller meteoroids may form meteorite strewn fields while larger initial bodies and high-strength iron meteoroids may form impact crater strewn fields. Impact crater strewn fields are ephemeral and none documented to date are older than about 63,500 years. Here we report on a newly discovered impact crater strewn field, about 280 Myr old, in tilted strata of the Rocky Mountains near Douglas, Wyoming. It is the oldest and among the largest of impact crater strewn fields discovered to date, extending for a minimum of 7.5 km along a SE-NW trajectory. The apparent width of the strewn field is 1.5 km, but the full extent of the crater strewn field is not yet constrained owing to restricted exposure. We probably see only a small section of the entire crater strewn field. The cascade of impacts occurred in an environment that preserved the craters from destruction. Shock lithification aided this process. The Earth´s atmosphere protects us from the majority of hypervelocity collisions with such small bodies2. Once the aerodynamic stresses exceed the strength of a meteoroid it will disrupt while passing through the atmosphere4. Decimeter to meter-sized meteoroids mostly disintegrate into a number of small fragments that decelerate to terminal velocity and form a meteorite strewn field5. The Carancas impact event is an exception where a meter-sized chondritic meteoroid survived atmospheric entry and created a 14 m diameter crater6. Decameter-sized objects disrupt violently at high altitudes and can create airbursts that damage infrastructures, but then slow down to terminal velocity when passing through the lower atmosphere. An example is the recent Chelyabinsk event7 that formed a 60 km long and less than a kilometer wide meteorite strewn field. Perhaps the largest meteorite strewn field, Gibeon, Namibia, was formed by an iron octahedrite that dispersed over an elliptical area 275 kilometers long and 100 kilometers wide5. Atmospheric protection of Earth becomes less effective for more massive meteoroids, and especially for high strength iron meteoroids. Bland and Artemieva1 calculated that over the mass range of 103–107 kg, iron impactors transfer ∼3 orders of magnitude more energy to the Earth´s surface than stony meteoroids. While disruption still takes place, a significant portion of their cosmic velocity remains to create impact crater strewn fields8. The currently known impact crater strewn fields are Sikhote-Alin9, Wabar10, Henbury11, Kaali12, Morasko13, and Odessa14. A comparison of them is given in Table 4. The craters and crater pits range in size between 10 and 150 m, cluster and overlap, and the biggest is usually situated toward the downrange end of the strewn field ellipse. The term crater pits is referred to those cavities that are formed by strongly decelerated meteoroid fragments that impact at sub-sonic velocity and are not capable of shocking the target. Impact craters formed in quartz rich targets that are larger than 30 m may contain shocked rocks with planar deformation features (PDFs), planar fractures (PFs), and, if the target is porous, impact melt and high pressure polymorphs15. Examples are Wabar craters A and B with diameters of 64 and 116 m respectively16 and the Gebel Kamil crater with a diameter of 45 m. The hypervelocity impact at Gebel Kamil generated shock characteristics consistent with shock pressures from 30 to 60 GPa17. The proper documentation of a shock metamorphic overprint in minerals such as quartz is key for the proof of impact craters19 and this paper carefully presents such shock features. Meteorites are frequently found in the area of the crater strewn fields  event, atmosphere disrupted fragments may impact close to each other and contribute to the formation of a single impact crater21.Meteoroids ranging in size from a few tens of centimeters to several tens of meters impact Earth much more frequently than larger meteoroids22. The exposed strewn field on Sheep Mountain has a minimum length of 7.5 km oriented SE-NW  along with shock lithification are shared features. In a porous sand target we also expect to find impact melt rock as well as coesite and stishovite similar to Wabar16, Kamil17, or Meteor crater15. Future investigations will focus on this subject. The lack of shock indicators in other strewn fields  The craters represent a section of a secondary crater field around a large, yet unknown crater, that should exist several hundreds of kilometers SE of the current exposure. Secondary craters are intensely studied on the Moon and on Mars36. Their origin is obvious if the fields of small craters surround large primary craters and exhibit distinctive morphologies such as shallow, irregular shapes and occurrence in chains and clusters, sometimes with distinctive herringbone patterns. Secondary impact velocities can generate stress pulses exceeding elastic limits of rocks and may cause shock. However, secondary crater fields on Earth are as yet unknown and the geophysical evidence of a large buried crater is so far lacking in the suspected region. (ii) Multiple airbursts of a single meteoroid at a very high altitude may form a very wide, unprecedented impact crater strewn field. The fragmentation process would be affected by the atmospheric density at the 280 Myr impact event. It is known that the Earth experienced a major O2 pulse during the Late Paleozoic with an O2 concentration of 35%37. (iii) A paired meteoroid or an asteroid break-up prior to atmospheric entry or an asteroid shower may have been responsible for a wider extent of the crater field. There are recent examples of asteroids with companions. The Ries-Steinheim impact38 is an example of a terrestrial paired impact. As long as the extent of the impact crater field is not fully constrained, modeling of the event is not meaningful. Additional craters on Sheep Mountain and outlier areas must demonstrate shocked rocks. This ongoing study will also apply geophysical and geochemical means to address these intriguing questions.The potential outliers of the NW-SE stretching strewn field would increase the width of the impact crater strewn field across the trajectory to 16 km and pose an unprecedented crater strewn field dimension that needs to be addressed in future studies. It is fortuitous that the strewn field orientation based on crater orientation matches with the narrow exposure of the uppermost Casper sandstone. Numerous craters may still be buried under the Opeche muds and younger strata east and north of the exposure. A clustering of the largest craters of the impact crater strewn field is expected downrange to the NW but limited exposure may prevent their identification. Interestingly, the buried 7 km diameter Cloud Creek39. The accuracy of U-stage measurements is estimated at ± 5°. Figures were prepared with Adobe© Photoshop and Illustrator CS3.Our investigation is based on geological field work, microscopic analysis, and remote sensing using drone and satellite imagery. We used a DJI Phantom 4 drone. The map Fig.  is a com


Towards mapping α1D-AR complex architecture, biolayer interferometry (BLI) revealed the α1D-AR C-terminal PDZ ligand selectively binds the PDZ protein scribble (SCRIB) with >8x higher affinity than known interactors syntrophin, CASK and DLG1. Complementary in situ and in vitro assays revealed SCRIB PDZ domains 1 and 4 to be high affinity α1D-AR PDZ ligand interaction sites. SNAP-GST pull-down assays demonstrate SCRIB binds multiple α1D-AR PDZ ligands via a co-operative mechanism. Structure-function analyses pinpoint R1110PDZ4 as a unique, critical residue dictating SCRIB:α1D-AR binding specificity. The crystal structure of SCRIB PDZ4 R1110G predicts spatial shifts in the SCRIB PDZ4 carboxylate binding loop dictate α1D-AR binding specificity. Thus, the findings herein identify SCRIB PDZ domains 1 and 4 as high affinity α1D-AR interaction sites, and potential drug targets to treat diseases associated with aberrant α1D-AR signaling.Many G protein-coupled receptors (GPCRs) are organized as dynamic macromolecular complexes in human cells. Unraveling the structural determinants of unique GPCR complexes may identify unique protein:protein interfaces to be exploited for drug development. We previously reported α Typically these medications compete with endogenous ligands for orthosteric binding sites, hindering drug selectivity due to the similarity of binding pockets amongst closely related GPCRs. Thus, there is great interest in identifying novel sites to modulate GPCR signaling. To this end, a growing body of research has focused on identifying and characterizing the functional roles of GPCR interacting proteins. Two prominent examples are the β-arrestins2; and PDZ (PSD95/Dlg/ZO-1) domain containing proteins, which typically interact with C-terminal PDZ ligands4. Since the discovery that rhodopsin interacts with inaD5 and β2-adrenergic receptor (AR) with NHERF6, significant effort has been put forth to understand GPCR:PDZ protein interactions and their potential as drug targets11. For example, pharmacological disruption of the nNOS:NOS1AP:PSD95:NMDAR protein complex provides an alternative approach to NMDAR antagonists for treating neuropathic pain14 and neuronal excitotoxicity15, demonstrating the therapeutic potential of targeting PDZ protein interactions to selectively modulate membrane protein function.G protein-coupled receptors (GPCRs) account for ~4% of the human genome and are targets for ~30% of FDA approved drugs1-AR GPCR subtypes  that respond to the endogenous catecholamines epinephrine and norepinephrine, only the α1D-AR subtype contains a C-terminal Type I PDZ ligand. Yeast 2-hybrid16 and tandem-affinity purification/mass spectrometry17 screens initially revealed the α1D-AR PDZ ligand interacts with the syntrophin family of PDZ domain containing proteins. Syntrophins enhance α1D-AR function via recruiting the Dystrophin Associated Protein Complex (DAPC) and signaling effectors, α-catulin, liprin and phospholipase-Cβ18. Improved proteomic analyses subsequently revealed that, in addition to syntrophins, α1D-ARs also interact with the multi-PDZ domain containing protein scribble (SCRIB); and that α1D-ARs are expressed as modular homodimers, with one α1D-AR protomer bound to SCRIB, the other to syntrophin, in all human cell lines examined to date19. Strikingly, the α1D-AR:SCRIB:syntrophin complex is highly unique – no other GPCRs containing C-terminal Type I PDZ ligands have been shown to interact with both SCRIB and syntrophins20. Without significant expression of necessary PDZ proteins, α1D-ARs are retained intracellularly and produce weak functional responses23, suggesting this protein:protein interaction site has the potential for pharmacological modulation. Indeed, numerous diseases are associated with aberrant α1-AR function, including hypertension24, benign prostate hypertrophy25, bladder obstruction26, schizophrenia27, and post-traumatic stress disorder29. Unfortunately, deleterious side effects  are frequently observed with chronic use of non-selective α1-AR antagonists. For example, the doxazosin portion of the ALLHAT anti-hypertensive study was prematurely halted due to increased morbidity30. Thus, selectively targeting the α1D-AR:SCRIB:syntrophin complex may provide therapeutic benefit, minus the toxicities associated with non-selective α1-AR ligands.Of the three α1D-AR:PDZ protein complex. Together, our data implicate SCRIB PDZ domains 1 and 4 as the primary anchor sites for the α1D-AR. We further highlight differences in α1D-AR:PDZ1 versus α1D-AR:PDZ4 interactions by identifying unique residues in PDZ4 that are critical for α1D-AR binding.Herein, we employed a combination of biophysical, biochemical and cell-based approaches to acquire structural insights into the α1D-AR interacts with multiple PDZ proteins with cell-type specificity: scribble (SCRIB), α1-syntrophin (SNTA), human discs large MAGUK scaffold protein 1 (DLG1), and calcium/calmodulin-dependent serine protein kinase (CASK)19. With the goal of elucidating the molecular architecture of this unique, modular GPCR:PDZ protein complex, we employed BioLayer Interferometry (BLI) to quantify equilibrium dissociation constants (KD) for α1D-AR PDZ ligand:PDZ protein interactions. cDNAs encoding for the PDZ domains of these proteins were subcloned into a modified pGEX vector (pCOOL), expressed in E. coli and purified. Immobilized biotin-labeled peptides containing the distal 20 amino acids of α1D-AR (α1D-CT) were incubated with purified PDZ proteins and subjected to BLI analysis , as α1D-ARs were found to interact with both PDZ proteins in all human cell lines examined19. Remarkably, α1D-CT bound SCRIB , SCRIB mutants containing single-PDZ domains , and SCRIB lacking all 4 PDZ domains (ΔPDZ) were subcloned into the pGlue vector to add N-terminal tandem affinity purification (TAP) epitope tags. HEK293 cells were transfected with TAP-SCRIB constructs and cell lysates were subjected to immunoblotting to verify expression , we suspect CASK and DLG1 are recruited to the α1D-AR complex indirectly by SCRIB. For example, DLG1 can be indirectly recruited to SCRIB via GUKH, which interacts with SCRIB PDZ2 in Drosophila synaptic boutons33, or LGL − a known interactor with both DLG1 and SCRIB35. Additionally, DLG1, CASK, and LIN-7A are expressed as a tripartite complex in vitro and in vivo38, suggesting DLG1 may be recruiting CASK and LIN-7A to the α1D-AR complex via indirect interactions with SCRIB.Taken together, these data implicate SCRIB PDZ1 and PDZ4 as the central scaffolds of the α1D-CT binding affinity for SCRIB containing all 4 PDZ domains (70 nM) relative to each individual SCRIB PDZ domain (1.14–44.16 μM). The divergent α1D-CT:SCRIB binding affinities are suggestive of a co-operative binding mechanism, in that the binding of a single α1D-CT PDZ ligand to SCRIB enhances the affinity of subsequent intramolecular α1D-CT:SCRIB PDZ binding events. We tested this model by quantifying the affinity of SCRIB C-terminal truncation mutants missing PDZ4 (ΔPDZ4) or PDZ3 and PDZ4 (ΔPDZ34) with BLI. α1D-CT bound SCRIB ΔPDZ4  with substantially lower affinity  and anti-myc . As shown, successive C-terminal SCRIB deletions produced progressive decreases in α1D-AR monomer  and multimer  signal, whereas SCRIB ΔPDZ produced no detectable α1D-AR interaction. Of note, the most dramatic decrease in α1D-AR binding was observed with SCRIB containing only PDZ1 . PAGE near infrared (NIR) analysis of HEK293 cell lysates transfected with SNAP-α1D-CT displayed protein bands of expected size 21.7 kDa  signaling assays were performed using HEK293 cells stably expressing SNAP-α1D-AR alone or transiently co-expressing SCRIB WT, SCRIB PDZ4 or SCRIB ΔPDZ4. Concentration-response curves were generated for the selective α1-AR agonist phenylephrine to facilitate efficacy comparison between transfection conditions. As shown, phenylephrine efficacy was enhanced by all SCRIB constructs with rank order WT > PDZ4 > ΔPDZ4 > pGLUE vector control  was used to predict α1D-CT:SCRIB PDZ4 interactions. Our model identified D571α1D-AR:R1110PDZ4 of the carboxylate binding loop and T570α1D-AR:H1170PDZ4 of α-helix B as possible α1D-CT PDZ ligand interaction sites within SCRIB PDZ4 . Constructs used for bacterial expression were sub-cloned into a modified pGEX vector to add GST-tags. For mammalian expression, constructs were inserted into pGLUE to add streptavidin binding protein/TEV/calmodulin binding protein tags; or pSNAPf to add SNAP-epitope tags; or pcDNA3.1 to fuse MYC tags. BG-782 SNAP substrate was from New England Biolabs . PageRuler Prestained NIR Protein Ladder was used for all PAGE NIR .1D-AR stable cell line, G418 was added to the media 24 h post-transfection. [3H]-Prazosin saturation radioligand binding (data not shown) and PAGE NIR (described in 57) were used to verify SNAP-α1D-AR protein expression.Human embryonic kidney (HEK) 293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 2 mM L-glutamine. Cells were transfected with 1 mg/ml polyethyleneimine (PEI) and used ~48 h post-transfection. For the development of the SNAP-α57. Data were analyzed with GraphPad Prism software .DMR assays were performed in 384 well Corning Epic sensor microplates  using the protocol described previously600 = 0.6–1.0 was reached; followed by induction with IPTG (1 mM) at 18 °C for 18 h. Cells were harvested by centrifugation and lysed . GST-tagged protein was immobilized on Pierce® glutathione agarose beads  and washed (20 mM Tris-HCl pH 8.0 and 200 mM NaCl). Bound protein was eluted from the beads in wash buffer supplemented with 10 mM glutathione and concentration was determined using Bradford assay. Immobilized protein for crystallography was incubated with TEV at 4 °C for 18 h and subjected to size exclusion chromatography using a Superdex 75 Increase 10/300 GL  on an AKTA FPLC  in lysis buffer. The peak 215 nm fractions were collected. SDS-PAGE analysis was employed to determine purity, and protein was flash frozen and stored at −80 °C until needed.Recombinant proteins were expressed in Rosetta™ (DE3) competent cells  in Miller LB supplemented with 100 μg/mL Ampicillin and 34 μg/mL Chloramphenicol at 37 °C until an OD1D-C terminal domain (SNAP-α1D-CT) was created by subcloning cDNA encoding the distal 16 amino acids of the human α1D-C terminal domain into the 3′ MCS of pSNAP. SNAP and SNAP-α1D-CT were then subcloned into a modified pGEX vector to add N-terminal GST tags, expressed in, and purified from E. coli using the previously described method  for 30 min @37 °C in the dark. Serial dilutions of BG-782:SNAP-α1D-CT were subjected to SDS-PAGE and near infrared fluorescence (NIR: λ = 800 nm) was quantified with the LI-COR Odyssey CLx . Samples were boiled in SDS-sample buffer, and 10 μL aliquots were subjected to PAGE NIR.SNAP-αhod Fig. . Followions Fig. . For GST20. 5 μL of 25 μM BG-782 was included in the 1st overnight solubilization step with 0.5% digitonin to label SNAP-α1D-ARs. PAGE NIR was used to observe SNAP-α1D-AR protein levels. Gels were then transferred to nitrocellulose and blotted for anti-HA  or anti-MYC , then anti-mouse Alexa-Fluor 2° antibodies in the 700–800 nm range . Gels and blots were imaged with the LI-COR Odyssey CLx.TAP purification was performed using the protocol described previously1D-CT  was immobilized to streptavidin coated probes, followed by biocytin. The immobilized peptide was incubated in serial dilutions of target proteins until steady-state binding was reached. Biocytin was used to determine non-specific binding. For reverse BLI, GST-SCRIB was immobilized using anti-GST probes, and then incubated in serial dilutions of biotin labeled α1D-CT.BLI was performed using the Octet Red 96 system . All steps were performed in 20 mM Tris-HCl pH 8.0, 200 mM NaCl, and 0.1% bovine serum albumin. 50 nM of biotin labeled peptide containing the last 20 amino acids of the α1D-AR was quantified with cell impermeable SNAP-substrate BG-782 using the method described previously57. TO-PRO-3 nuclear stain was used to normalize samples according to cell number. Data were analyzed with GraphPad Prism software.HEK293 cell surface expression of SNAP-α58, and scaled with AIMLESS60. Phases were determined by molecular replacement using Phaser61 and SCRIB PDZ4  as a search model. The Phaser solution was manually rebuilt over multiple cycles using Coot62 and refined using PHENIX63. All images were generated using the PyMOL Molecular Graphics System, Version 1.74 Schrödinger, LLC. Coordinate files have been deposited in the Protein Data Bank under the accession code 6EEY.SCRIB PDZ4 R1110G was concentrated to 11 mg/mL in 20 mM Tris at pH 8.0, 200 mM NaCl, and 5 mM DTT and screened against crystallization conditions using a Mosquito Liquid Handler . Final crystals were obtained in 21% PEG 3,350 and 0.25 M Ammonium Nitrate. Crystals were flash frozen in mother liquor supplemented with 15% glycerol. All diffraction data was collected at the Advanced Light Source at Berkeley on beam line 8.2.1, integrated with XDS1D-AR C-terminus, LRETDI, was modeled into the canonical βB and αB binding pocket of Scribble PDZ439 using PyMOL and submitted to FlexPepDock server51. Models with scores greater than −131 were analyzed for hydrogen bonding (1.5–2.5Å) between peptide and PDZ4. Only interactions identified in greater than 5 models are reported.The distal 6 amino acids of the αSupplemental data


Acalculia is an acquired disorder in calculation abilities, usually associated with left posterior parietal damage. Two types of acalculic disorders are usually distinguished: (1) primary acalculia or anarithmetia, where the patient presents a loss of numerical concepts , and (2) secondary acalculia due to a different disturbance in cognition and affecting mathematical abilities. Secondary acalculias are associated with aphasia, alexia, agraphia, executive function disorders, or visuospatial difficulties. This paper is a proposal for clinical intervention to rehabilitation of acquired primary and secondary acalculias. It is important to emphasize that rehabilitation is based in both targeting restitution of the original impaired function and reorganization of functions employing other less or unimpaired cognitive functions , 22.Research about acalculia rehabilitation has been limited . FrequenIn a review of the research devoted to the rehabilitation of acalculia published from 1980 to 2007, only 7 papers were found, all of them single case studies . RegardlIn one of the few studies analyzing the evolution of acalculia, Caporali et al.  reportedFew studies have analyzed the brain changes associated with acalculia recovery . Claros-Major strategies used in the rehabilitation of primary and secondary acalculias will be presented. However, frequently cognitive rehabilitation procedures have been implemented in single cases; few studies have included large groups of acalculia patients.Primary acalculia is found associated with left posterior parietal lesions , usuallyIt has been observed that anarithmetia usually appears simultaneously with the so-called semantic aphasia , 33, 34;According to Tsvetkova , the iniNoteworthily, primary acalculia can be observed without language disturbances, but its correlation with aphasia is significant . The rehIn conclusion, rehabilitation of the patient with primary acalculia should begin by relearning the basic concepts of number. Later, the patient should move to the acquisition of more sophisticated concepts that imply the relationship between numbers and finally to solving arithmetical operations and numerical problems.Patients with acalculia due to a language defect  and receiving language therapy usually present a significant improvement in their calculation abilities in a parallel way to the language recovery . In otheSometimes brain-damaged patients can mentally solve mathematical operations, but they fail in recognizing numbers due to visuoperceptual impairments. The intervention in those cases should be directed to recover the visual perception . PerceptThe technique known as “number reconstruction”  is basedPatients with pure alexia—alexia without agraphia—have difficulties for the visual integration of stimuli (deficit that is known as simultanagnosia) and defects in visual-motor coordination (known as optic ataxia). Consequently, the intervention should include exercises that require the visual exploration of space. Frequently used tasks included in rehabilitation programs suppose a progressive increase in their level of difficulty, beginning with simple movements such as reaching objects, followed by tasks that require more complex movements such as copying figures in two dimensions to conclude with the copy of three-dimensional designs . TraininWhen there is a visual exploration defect (ocular apraxia) impairing the ability to normally scan quantities, practicing eye-tracking exercises can help to overcome this defect. Rosselli et al.  describePatients with frontal damage due to diverse conditions, such as head injuries and tumors, usually present executive function disturbances. These patients present particularly significant difficulties in solving mathematical problems. Written calculations are easier than mental calculations. Attentional difficulties significantly contribute to the calculation difficulties. Tsvetkova  suggesteSpatial acalculia is usually observed in cases or right hemisphere damage associated with spatial neglect . SpatialRosselli and Ardila  describeBenavides-Varela et al.  reportedIn primary acalculia, the fundamental therapeutic goal is to reconstruct the understanding of the numerical system. Patients with primary acalculia seemingly do not understand how the numerical system is organizedIn aphasic acalculia, numerical impairment and recovery in general parallelize aphasia impairment and recoveryWhen acalculia is due to visuoperceptual disturbances, the fundamental aim of therapy is to recover visual perceptionIn frontal dysexecutive acalculia, providing the patient with external support  and attentional control strategies  may result in improving the numerical defectNeglect represents probably not the only but the fundamental defect responsible for the written calculation defects observed in patients with right hemisphere damage. Some simple strategies, such as using clear marks to indicate where to begin to read, can successfully improve the calculation defectsThe available information about the cognitive rehabilitation of acquired calculation disturbances is limited. However, some general conclusions can be drawn.


Castration‐resistant progression of prostate cancer is a major cause of prostate cancer mortality, and increased expression and activity of the full‐length and the splice variants of androgen receptor (AR) have been indicated to drive castration resistance. Consequently, there is an urgent need to develop agents that can target both the full‐length and the splice variants of AR for more effective treatment of prostate cancer. In the present study, we showed that raddeanin A (RA), an oleanane‐type triterpenoid saponin, suppresses the transcriptional activities of both the full‐length and the splice variants of AR. This is attributable to their decreased expression as a result of RA induction of proteasome‐mediated degradation and inhibition of the transcription of the AR gene. We further showed the potential of using RA to enhance the growth inhibitory efficacy of docetaxel, the first‐line chemotherapy for prostate cancer. This study identifies RA as a new agent to target both the full‐length and the splice variants of AR and provides a rationale for further developing RA for prostate cancer treatment. The growth and survival of prostate cancer cells rely on androgen receptor (AR), which is activated by androgens  consists of three major domains, the N‐terminal transactivation domain followed by the DNA‐binding domain and the C‐terminal ligand‐binding domain. The ligand‐binding domain is connected to the DNA‐binding domain by a flexible hinge region  is an oleanane‐type triterpenoid saponin extracted from the root of 22.1DU145, 22Rv1 and PC‐3 cells were purchased from the American Type Culture Collection. C4‐2 and C4‐2B cells were obtained from Dr Shahriar Koochekpour at Roswell Park Cancer Institute, and LNCaP95 cells were provided by Dr Alan Meeker at Johns Hopkins University. All cell lines are cultured in RPMI1640 supplemented with penicillin, streptomycin, and 10% foetal bovine serum. All cell lines were within 20 passages, authenticated, and tested negative for mycoplasma contamination. RA was purchased from Yuanye Biological . For RA treatment in the presence or absence of R1881, a synthetic androgen, the cells were cultured in medium containing charcoal‐stripped serum. Otherwise, the cells were cultured with normal serum during the course of the experiments. Cell growth was assessed by the sulforhodamine (SRB) assay as described,2.2® Infrared scanner (LI‐COR Bioscience). The following antibodies were used: anti‐AR  and anti‐GAPDH . The Western blot analysis was performed at least three times, and AR levels were normalized by GAPDH levels.Western blot analysis was performed with a standard protocol. Briefly, ~20 μg of protein samples were resolved over 10%‐15% SDS/PAGE and transferred to a polyvinylidene fluoride membrane. After blocking in blocking buffer , the membrane was incubated with a primary antibody overnight at 4°C, followed by incubation with a fluorescent‐labelled secondary antibody for 1 hour at room temperature. Membranes were scanned and analysed using an Odyssey2.3Transfection was performed with the use of the Turbofect reagent (Thermo) according to the manufacturer's protocol. Three luciferase reporter plasmids were used: ARR3‐luc  and UBE2C were purchased from Sangon Biotech . The primer sequences are: AR‐FL , AR‐V7 , PSA , UBE2C  and 36B4 . The qRT‐PCR analysis was performed at least three times in triplicate, and AR, PSA and UBE2C levels were normalized by 36B4 levels.The Quantitative reverse transcription‐PCR (qRT‐PCR) analysis was performed as described.2.5t test was used to determine the mean differences between two groups. P < 0.05 is considered significant. Data are presented as mean ± SD from at least three independent experiments.The Student's two‐tailed 33.1We first assessed the effect of RA on the growth of CRPC cells by the SRB assay. The assay was conducted in the presence or absence of 1 nmol/L R1881, a synthetic androgen, for the AR‐expressing 22Rv1, C4‐2, C4‐2B and LNCaP95 cells and in androgen‐deprived condition for the AR‐null PC‐3 and DU145 cells. The doses of RA that we tested were from 0 to 6 μmol/L. This was because RA can reach a maximum plasma concentration of ~4.5 μmol/L in rats after a single intraperitoneal administration at 0.75 mg/kg.3.2trans‐activating activity. To specifically assess the effect of RA on AR‐V transcriptional activity, we transfected 22Rv1 cells, which express both AR‐FL and AR‐Vs, with the UBE2C‐luc construct in which the luciferase gene is driven by an AR‐V‐specific promoter element of the UBE2C gene.trans‐activating activity (Figure We therefore investigated the effect of RA on AR transcriptional activity by reporter gene assay. We first transfected C4‐2 and 22Rv1 cells with the ARR3‐luc luciferase construct, which contains three tandem repeats of androgen‐response elements, and treated the cells with 3 μmol/L RA. As shown in Figure 3.3To understand the mechanism by which RA inhibits AR transactivation, we examined AR protein levels after RA treatment. C4‐2 and 22Rv1 cells were treated with RA either in the presence or absence of androgen. RA down‐regulated both AR‐FL and AR‐V proteins Figure . The eff3.4To determine whether the decrease in AR proteins was due to increased protein degradation, we treated C4‐2 and 22Rv1 cells with cycloheximide to stop protein synthesis and monitored the decay rates of AR‐FL and AR‐V proteins in response to RA treatment either in the presence or absence of androgen. As shown in Figure 3.5To investigate whether RA could modulate AR at the RNA level in addition to inducing AR protein degradation, we measured AR‐FL and AR‐V7 mRNA levels by qRT‐PCR. Interestingly, RA reduced the levels of both AR‐FL and ‐V7 mRNA Figure A and B. 3.6As the first‐line chemotherapy for patients with metastatic CRPC, docetaxel has been shown to inhibit nuclear translocation and transcriptional activity of AR‐FL.4The present study represents the first to characterize the activity of RA in prostate cancer, particularly CRPC. We showed that RA inhibits the growth of CRPC cells in a dose‐ and time‐dependent manner. While independent of androgen, the inhibition appears to be dependent on AR, as AR‐null cells are not impacted by RA treatment. Mechanistically, we showed that RA suppresses the transcriptional activities of AR‐FL and AR‐Vs, and this is attributable to decreased expression of AR‐FL and AR‐Vs. The decreased expression is a result of RA induction of proteasome‐mediated degradation of AR‐FL and AR‐V proteins and reduction of the transcription of the AR gene.The AR‐FL protein is known to suppress the transcription of the AR gene, producing a negative feedback on the expression of AR mRNA.While supporting the anticancer activity of RA that has been indicated in other cancer types,These compounds may also have the potential to improve the efficacy of the first‐line chemotherapy for prostate cancer, docetaxel. Androgen‐induced translocation of AR‐FL to the nucleus, which is required for the transcriptional activity of AR‐FL, has been reported to use a microtubule‐facilitated pathway.The authors confirm that there are no conflict of interest.HX and CH performed the research. HX, CH, SB, JL, BYZ, LZ, XY, YZ and YD contributed to research design and data analysis and interpretation. HX, YZ and YD wrote the paper, BYZ revised the paper and all the authors approved the submitted manuscript.