The German welfare system follows the principle “rehabilitation rather than pension,” but more than the half of all disability pensioners did not utilize medical rehabilitation before their early retirement. A major barrier is the application procedure. Lack of information about the opportunity to utilize rehabilitation services restricts the chance to improve work ability and to prevent health-related early retirement by rehabilitation programs. The establishment of new access paths to medical rehabilitation services was, therefore, identified as a major challenge for rehabilitation research in a recent expertise. Thus, a web-based information guide was developed to support the application for a medical rehabilitation program.For this study, the development of a web-based information guide was based on the health action process approach. Four modules were established. Three modules support forming an intention by strengthening risk perception (module 1), positive outcome expectancies (module 2) and self-efficacy (module 3). A fourth module aims at the realization of actual behavior by offering instructions on how to plan and to push the application process. The study on the effectiveness of the web-based information guide will be performed as a randomized controlled trial. Persons aged 40 to 59 years with prior sick leave benefits during the preceding year will be included. A sample of 16,000 persons will be randomly drawn from the registers of 3 pension insurance agencies. These persons will receive a questionnaire to determine baseline characteristics. Respondents of this first survey will be randomly allocated either to the intervention or the control group. Both study groups will then receive letters with general information about rehabilitation. The intervention group will additionally receive a link to the web-based information guide. After 1 year, a second survey will be conducted. Additionally, administrative data will be used to determine if participants apply for rehabilitation and finally start a rehabilitation program. The primary outcomes are the proportion of applied and utilized medical rehabilitation services. Secondary outcomes are cognitions on rehabilitation, self-rated work ability, health-related quality of life and perceived disability, as well as days with sick leave benefits and days of regular employment.The randomized controlled trial will provide highest ranked evidence to clarify whether theory-driven web-based information supports access to rehabilitation services for people with prior sickness benefits.DRKS00005658, 16 January 2014).German Clinical Trials Register (Identifier: The online version of this article (doi:10.1186/s13063-015-0968-7) contains supplementary material, which is available to authorized users. Many Western welfare systems provide rehabilitation services to improve work ability and to support participation in working life. In Germany, these services are provided usually as 3-week inpatient or outpatient programs under the umbrella of the German Pension Insurance (GPI). The treatment is multi-professional and follows evidence-based therapy standards . The legOne main reason for non-utilization seems to be that medical rehabilitation requires a formal application of the person in need. A current study revealed that about one third of the disability pensioners were not informed on the possibility of claiming a medical rehabilitation program due to limitations in work ability . If needOne potential gate for information might be web-based information guides . These pAdditionally, web-based information guides have the potential to support e-health literacy if they provide evidence-based information in a comprehensible format. This might help to handle the information flood of the Internet which often is perceived to be diffuse or overtaxing –13. As aIn a qualitative study, Sillence et al.  examinedAgainst this background we developed a web-based information guide promoting the application for rehabilitation services. The information guide aims at raising the frequency of applications for and utilization of medical rehabilitation services in persons who experience restrictions in their work ability. This study protocol describes the design of a randomized controlled trial which was developed to clarify whether the anticipated higher rates of applications and utilization will be achieved.The study will be performed as a randomized controlled trial.Participants in the control group will receive a letter which informs about the general aims of medical rehabilitation and the possibility to apply for a medical rehabilitation program. The complete wording of the letter is available as Additional file Participants in the intervention group will also receive an informative letter. The first section of this letter equals the letter of the control group. In addition, the participants will be informed on a web-based information guide, obtain a link to the corresponding webpage and will be invited to use the web content in order to verify a potential need for rehabilitation and to claim a medical rehabilitation program. The complete wording of the letter is available as Additional file The development of the web-based information guide was based on the health action process approach (HAPA) . AccordiIn the case of rehabilitation claims, Zimmermann et al.  reportedThus, the web-based information guide was designed for supporting both, forming an intention and planning the application for medical rehabilitation. We therefore developed four modules. Modules 1 to 3 support the formation of an intention by strengthening risk perception (module 1), positive outcome expectancies (module 2) and self-efficacy (module 3). The realization of an actual behavioral outcome, e.g. a formal rehabilitation claim, is focused by the fourth module. This module includes structured instructions in order to support the planning of a rehabilitation claim  will be used.The WAI was developed by the Finnish Institute of Occupational Health in the mid-1980s. It is available in 26 languages and internationally the most common instrument to assess work ability. The WAI measures the degree to which persons consider their state of health as adequate to cope with their job demands . The WAIRecent studies confirmed that the WAI is a predictor for the need for rehabilitation and rehabilitation-related impairments of functional capacity also within the setting of the German welfare system , 35. TheAfter completing the WAI the individual total score will be automatically calculated, and participants will receive a brief explanation and a concluding recommendation. The feedbacks are summarized in Table Persons with good or very good work ability currently do not have a need for rehabilitation. In this case the participants will receive recommendations how to maintain their work ability in cooperation with their general practitioner or their occupational physician. Participants with moderate work ability will be informed about medical rehabilitation as an option to improve the current work ability. If the WAI indicates poor work ability, participants will also receive a recommendation to apply for medical rehabilitation. Additionally, the participants are informed about the urgent need in order to avoid a disability pension. In both cases of recommendation for rehabilitation participants will be directly forwarded to the step-by-step instructions on how to apply for a medical rehabilitation.To strengthen positive outcome expectancies, reports of successful medical rehabilitation by former rehabilitation users will be presented. These reports shall strengthen the perception that rehabilitation is an effective option for the participants to sustain their work ability.The reports are based on interviews which we conducted with former rehabilitants. Interview partners were recruited in five stationary rehabilitation centers and five ambulant rehabilitation centers. The guided interview dealt with the main diagnosis, personal pathways to rehabilitation, progress of health problems before, during and after medical rehabilitation and self-rated success factors of medical rehabilitation. Eleven guided interviews were conducted via phone and examined using content analysis . For preIn this module all relevant information on medical rehabilitation under the umbrella of the GPI and the application procedures are summarized in a clear and structured way. The content includes aims and types of rehabilitation as well as the legal framework and practical aspects, e.g. assumption of costs, continued payment of wages, additional payments and childcare facilities. In this way the participants will be prepared for the application process and be made aware of access barriers and information myths. The information section is hyperlinked to the step-by-step instructions and a frequently asked questions section (FAQ section). The FAQ section is designed dynamically. Participants may ask questions using a contact form which will be answered by mail. Questions and answers will successively complete the FAQ section. Additionally, a search function was implemented for accessibility of each section of the webpage by entering a search term.The step-by-step instructions shall reveal that the way towards an application for a rehabilitation program is manageable and that formal obstacles and the temporal expenditure can be mastered. The instruction traces the complete path of the application process until the start of the rehabilitation program and thus strengthens action planning by decomposing the complex task of a rehabilitation claim. Each required form is named and explained regardless of whether it has to be completed by the applicant herself or himself, by the attending physician, the health insurance or the employer. By this, participants will keep track of their documents and can control the application process . The description of the forms provides detailed explanations on how to complete them. Each administrative term and the function of the information that is to be given by the applicant will be explained. Additionally, a hyperlink is provided to download the corresponding form. All in all, the participants receive a comprehensive overview of the complete application process. This also includes the steps following the approval.Persons aged 40 to 59 years with prior sick leave benefits and regular employment of at least 6 months in the year before the beginning of the study will be included. Exclusion criteria are the receipt of pension benefits and the receipt of medical or vocational rehabilitation services during the last 4 years. Samples are randomly drawn from the registers of three pension insurance agencies, i.e. the federal agency and two regional agencies. Sampling will be stratified by pension insurance agency, sex and duration of sick leave benefits.Persons will be informed about purpose and intervention of the study, its duration and data protection. Participation in the study will be consented by answering the questionnaire of the initial survey. Consent for using administrative data of the participants will be obtained by signature on the informed consent form during the second survey.A postal survey on baseline characteristics will be sent to potential participants. Respondents to this first survey will be randomly allocated to the intervention and the control group. Randomization will be conducted by a statistician of the Institute of Social Medicine and Epidemiology, University of Lübeck, who is not otherwise involved with the trial, by using computer-generated random numbers. Allocation will be stratified by a pension insurance agency.To detect a 10-point higher rate of applications for a medical rehabilitation program (controls: 50 %), 1,076 persons (538 per group) are needed relying on a 2-sided alpha error of 0.05 and a power of 0.90. Response rates of 30 % to the first survey and of 50 % to the second survey were assumed to calculate the gross sample size to be drawn from the insurance registers. We finally decided to draw 8,000 persons for both the federal agency and the two regional agencies to allow segmented analyses according to the type of agency. Sample drawing was stratified by sex and the duration of sickness benefits in the year prior to the first survey (15 to 30 days versus more than 30 days).http://piwik.org).The insurance agencies generate numeric pseudonyms as unique identification numbers for all randomly selected individuals. The insurance agencies will post the pseudonymized questionnaires to the sampled individuals. Persons will be informed that in case of participating in the first survey they will be randomly allocated either to the intervention or the control condition. Participants will then send back their questionnaires to the researchers. After the first survey is completed, random assignment will be performed, and participants will obtain their corresponding intervention letter. Intervention letters for both the participants of the control group . In addition, work-related and family-related negative outcome expectancies, e.g. fear to be seen by co-workers as a slacker and uncertainty about who will keep obligations at home, will be assessed each by three items. Perceived self-efficacy to manage an application, e.g. certainty to apply for a rehabilitation even if it is complicated, will be assessed by four 4-point scaled items (1 = “agree” to 4 = “do not agree”) following Schwarzer et al. . FinallySelf-rated work ability will be assessed by the Work Ability Score (WAS). This is the first item of the WAI and compares current self-rated work ability to the lifetime’s best. The item has an 11-point scale, where 0 represents complete incapacity to work and 10 represents lifetime best work ability. The WAS correlates highly with the overall WAI score , 40. AddHealth-related quality of life will be assessed with 3 subscales of the 36-item Short-Form Health Survey of the Medical Outcomes Study . The values of the scales range from 0 to 100. Higher values represent better health-related quality of life .Information about days of sick leave benefits and days of regular employment will be extracted from the individual GPI accounts.The proportions of applied and utilized medical rehabilitation services will be compared by Pearson’s chi-squared test. Effects on continuous outcomes will be tested by analyses of covariance; thereby the baseline scores will be considered as a covariate .The study protocol was approved by the ethics committee of the Hannover Medical School, Hannover, Germany (Number: 2060-2013). The committee stated that there are no legal or ethical concerns to the planned procedures. The trial was registered in the German Clinical Trials Register on 16 January 2014 (Identifier: DRKS00005658).Our randomized controlled trial will provide highest ranked evidence to clarify whether theory-driven web-based information supports access to rehabilitation services for people with prior sickness benefits.Conception and development of the web-based information guide is finished. Recruiting has started and is ongoing.


Chronic suppurative otitis media (CSOM) is considered one of the most common causes of acquired hearing impairment in developing countries. CSOM is a multifactorial persistent inflammatory disease of the middle ear. A distinct pathophysiologic mechanism linking allergic rhinitis (AR) and CSOM remains to evolve. The purpose of this study was to investigate the association between AR and CSOM in adults.This was a case-control study.The subjects were 62 adults  with established CSOM and 61 healthy controls. CSOM was diagnosed when there was a history of chronic (persisting for at least 3 months) otorrhea, accumulation of mucopurulent exudates in the external auditory canal or middle ear and/or perforated tympanic membrane on otoscopy. All participants were evaluated for the presence of AR by clinical evaluation of allergic symptoms, and underwent a skin-prick test for 23 common regional allergens. Statistical analysis was performed using SPSS version 16.The prevalence of clinical rhinitis  was significantly higher among the cases compared with controls . The prevalence of AR (proven by positive skin-prick test) was also significantly higher among affected adults than controls . Adjusting for age, a logistic regression model showed that there was a significant difference between the two groups. Patients with AR and non-AR were at 3.27-  and 2.57- fold increased risk of developing CSOM, respectively, compared with healthy individuals.The study showed a higher prevalence of AR in CSOM patients than in controls. It may be valuable to evaluate and control this factor in these patients. Chronic supportive otitis media (CSOM) is a major problem facing health systems around the world. The condition is characterized by persistent inflammation of the middle ear and mastoid cavity associated with otorrhea through a perforated tympanic membrane, persisting for more than 6 weeks . The worThe pathogenesis of CSOM is considered multifactorial, and most patients with CSOM have a history of recent acute onset of otitis media, risk factors associated with acute otitis media, or otitis media with effusion. The pathogenesis is thought to include Eustachian tube (ET) dysfunction, immature or impaired immunologic status, upper respiratory allergy, familial predisposition, presence of other siblings, male sex, passive smoking and other factors ,5. HowevWith the prevalence of 10–30%, allergic rhinitis (AR) is the most common allergic disorder. AR occurs in association with a number of other disorders, principally sinusitis, asthma, allergic conjunctivitis , and atopic dermatitis -10. StudDespite the existence of a few studies, there remains controversy about the relationship of AR and CSOM, and more studies are needed relating to the prevalence and role of allergy in the pathogenesis of CSOM ,15,17,18In a case-control study, 62 patients who were candidates for tympanoplasty and mastoidectomy due to established CSOM were chosen, as well as 61 controls. The controls were selected from patients who were referred to the same hospital for minor head and neck trauma, with no history of CSOM or ear symptoms.One of the cases and three of the controls were excluded from the study due to an inability to discontinue current medication or the presence of dermographism.All subjects were examined by ear, nose and throat (ENT) specialists, and a thorough medical history and physical examination including anterior rhinoscopy and otoscopy, were performed.The study was performed in Amiralmo- menin Educational Hospital and ENT Research Center of Guilan University of Medical Sciences (GUMS) in Rasht, Iran. Written informed consent was obtained from all the participants. The study protocol was approved by the ethics committee of GUMS.CSOM was diagnosed when there was a history of chronic (persisting at least 3 months) otorrhea, accumulation of mucopurulent exudates in the external auditory canal or middle ear and/or perforated tympanic membrane on otoscopy.AR was defined as the presence of signs and symptoms of clinical rhinitis, including watery anterior rhinorrhea, nasal obstruction or congestion, nasal pruritus, and sneezing, especially paroxysmal (according to standard questionnaire ). Post-nAll subjects underwent a SPT for 23 common allergens  relevant in the north of Iran by a single immunologist, who was blind to both the otologic and rhinologic situation of participants. Allergens included six types of grass, four weeds, nine trees, two mites, cat allergen and Cladosporium.The positive control was histamine hydrochloride (10 mg/mL) and the negative control was diluent (AllergoPharma). The mean wheal size was evaluated after 15 minutes, and SPT was determined as positive when observing a wheal with a mean diameter at least 3 mm larger than wheals at the site of the negative control. All subjects who were pregnant or had a history of recent consumption of antihistamines, immunotherapy with a specific allergen, or dermographism were excluded from the study. A positive SPT result could confirm the robust clinical diagnosis of AR, and negative results were considered as non-AR.All data were analyzed using SPSS version 16. The χ2 and Fisher exact tests were used to test the significance of the differences between the two groups. A p-value lessthan 0.05 was defined as significant. Odds ratio and 95% confidence intervals were also calculated.A total of 61 cases  with a mean age of 37.1±14.3 years (range 15–70 years) and 58 controls  with mean age of 28.3±11.7 years (range 15–70 years) completed the study. There was a statistically significant difference between the groups in terms of age (P=0.047). Among the 61 cases with CSOM, 26 (42.6%) patients had right ear, 25 (41%) had left ear, and 10 (16.4%) had bilateral involvement. The female–to-male ratio was 1.7:1, but the difference was not statistically significant. Thirty-seven (60.7%) patients had a history of CSOM from childhood (<18 years); others developed the disease in adulthood. The exact time of presenting CSOM symptoms for those who developed CSOM from childhood was unavailable.The proportion of patients with clinical rhinitis  was significantly higher in the cases compared with the control group . The prevalence of AR  was 24.6% (n=15) and 13.8% (n=8) among cases and controls, respectively. However AR was more prevalent among patients with CSOM compared with controls, although the difference was not statistically significant (P=0.065) .Using a logistic regression model, after correcting for the age factor, the difference between the two groups became significant. Patients with AR and non-AR had a 3.27-  and 2.57-  fold increased risk of developing CSOM, respectively, compared with healthy individuals.Patients with a history of childhood CSOM were more likely to have AR than the control group .Among all participants with AR, 52.2% (n=12) had a posterior nasal drip, 34.8% (n=8) had lower turbinate hypertrophy, and 60.7% (n=14) had pallor and swelling of the turbinate mucosa. Indoor allergens, especially mites (dermatophagoides farina and dermatophagoides pteronyssinus) were the most prevalent allergens in both groups, while outdoor allergens such as grass pollen and weeds were less prevalent .As CSOM is associated with recurrent attack of otitis media and allergy and contributes to chronic otitis media with effusion, it is plausible that allergy also contributes to CSOM. Previous studies have reported wide prevalence of AR in otitis media with effusion, ranging from 24–89% ,20,21. TFliss and colleagues collected data during the children’s visit to the clinic by means of a structured interview with the parents using an appropriate questionnaire and by extracting from the records if necessary . GorguluCSOM could be a complication of acute otitis media or otitis media with effusion, both of which are more prevalent in the early childhood . Also, AAR is more frequent in CSOM patients, and may be a risk factor for CSOM. Avoidance of recognized allergens may reduce this risk and improve the outcome of surgical therapy. Further studies in this regard are warranted.


Comorbidity can affect health-care utilization and outcomes, and the results and interpretation of risk adjustment studies that attempt to predict rehabilitation utilization and outcomes are influenced by the choice of comorbidity measurement. Although the identification of an appropriate measurement has been conducted in some populations and outcomes, this information is currently lacking for the non-traumatic brain injury (nTBI) population in inpatient rehabilitation settings. As such, this is a systematic review protocol to survey the methods used to measure comorbidities in the rehabilitation setting for patients with nTBI.MEDLINE, MEDLINE In-Process, Embase, The Cochrane Database of Systematic Reviews, PsycINFO, and Health and Psychosocial Instruments will be systematically searched using the concepts ‘nTBI,’ ‘comorbidity,’ and ‘rehabilitation.’ Grey matters and the reference list of eligible articles will also be searched. Study selection will be performed independently by two reviewers based on predetermined eligibility criteria through two rounds of screening using, first, the title and abstract, followed by full-text. Extracted information will include study purpose, design, and setting; data source and type; outcomes variables; statistical methods; comorbidity measurement method, rationale, justification, or validation; and results involving comorbidity. The data will be tabulated and narratively synthesized. Meta-analyses will be performed if appropriate.This protocol has not been registered with PROSPERO.This protocol provides a systematic method for surveying current practice as well as monitoring the progress on comorbidity measurement methodology and effects of comorbidity on rehabilitation outcomes for patients with nTBI. The selection of an appropriate comorbidity measurement method has implications for the interpretation of both descriptive and risk adjustment studies, and thus, the validity of evidence used to inform planning and delivery of services.The online version of this article (doi:10.1186/2046-4053-4-14) contains supplementary material, which is available to authorized users. Non-traumatic brain injury (nTBI) is defined as acquired brain damage occurring after birth due to cancer, anoxia, infection or inflammation, toxicity, and vascular insults . ApproxiComorbidity is often considered in studies that assess rehabilitation outcomes as they can affect health service utilization  and funcAs an emerging field of study, various comorbidity measures have been compared against each other to assess their predictive power for certain populations and outcomes. For example, among the CCI, Elixhauser Index, and Johns Hopkins Aggregated Diagnosis Groups (ADGs), the Johns Hopkins ADGs showed marginally higher discrimination for predicting 1 year all-cause mortality in patients with chronic obstructive pulmonary disorder . In a saTo date, no such analysis has been published for rehabilitation outcomes after a nTBI. Since an absence of evidence is not evidence of absence, it should not be assumed that a given comorbidity measurement method is more or less appropriate for inpatient rehabilitation outcomes such as length of stay and functional outcome following a nTBI, especially as this population differs in various characteristics from those that are not referred to an inpatient rehabilitation setting . Indeed,The breadth of patients included in the definition of nTBI may preclude the discovery of important differences in the appropriateness of specific comorbidity measures between specific subgroups of patients with nTBI. It is expected that certain indexes may be less appropriate for certain patient groups. For example, since tumors are scored in the CCI, it may be less appropriate for patients with brain tumors because their comorbidity score will be inflated. Therefore, it is important to investigate subtypes of nTBI separately, as well as patients with nTBI as a whole, which reflects clinical practice where patients with nTBI are treated in the same rehabilitation unit. The inclusion of all subtypes of nTBI in this protocol will allow for investigations of comorbidity measurement methods within and between subtypes of nTBI.The primary purpose of this protocol is to systematically review the literature to identify current comorbidity measurements used for the nTBI population in inpatient rehabilitation. The secondary objective is to assess the face and predictive validity of these comorbidity measurements for various rehabilitation outcomes, including functional outcome and length of stay, specifically for this population. The final and tertiary objective is to catalogue the profile of comorbidities in patients with nTBI in inpatient rehabilitation settings using the identified studies. This information can be used to inform efforts to develop predictive multivariable models of rehabilitation outcomes for health service research, in which the selection of the most valid comorbidity measurement specific to the nTBI population in the rehabilitation setting can improve the clinical relevance and predictive power of the risk adjustment models for rehabilitation outcomes of these patients.This protocol has not been registered with PROSPERO.The search strategy was developed with assistance from a medical librarian.MEDLINE (1946 to present) and MEDLINE In-Process (present)Embase (1946 to present)Cochrane Database of Systematic Reviews (2005 to present)PsycINFO (1806 to present)HAPI (1985 to present)The following databases will be searched for relevant articles:Grey literature will also be searched using Grey Matters: a practical search tool for evidence based medicine . ResourcAdditional file All database results will be limited to English language. A limit to human only studies will be applied in MEDLINE, and limits to exclude conference material , editorials, letters, and notes will be applied in Embase.Two reviewers will independently assess all titles and abstracts for inclusion using predetermined eligibility criteria. Two iterations of screening will be performed. First, record titles (and abstracts if the title is ambiguous) will be used to select articles that study a nTBI population in inpatient rehabilitation. Second, a full-text screen will be used to identify relevant articles for inclusion in the systematic review. Articles that are included in the systematic review must (1) examine comorbidities in the nTBI population in inpatient rehabilitation and 2) state the method in which these comorbidities were measured. Several study designs will be included, such as cohort studies, case–control studies, randomized controlled trials (RCTs), or systematic reviews on the topic. Qualitative studies, case-reports, abstracts, conference material, and editorials or commentaries will be excluded because these sources of information are unlikely to present the level of detail and information required for the purposes of this systematic review. Ecological studies will be excluded because they will not provide sufficient evidence for individual-level effects of comorbidity on rehabilitation outcome in patients with nTBI. Finally, although stroke may be considered an etiology of nTBI, it will be excluded from this review to reflect current research and clinical practice in rehabilitation—for example, various national rehabilitation information systems , 21 and  state thThe presentation of results will be guided by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist .The standard data extraction table will include authors, publication year, purpose of study, year of study, study design, geographical location of study, setting , data source or type , type and definition of nTBI population, statistical methods , age of population, comorbidity measurement method , rationale, justification, or validation of the comorbidity measurement, outcome variables, and results involving comorbidity  will be listed in the column titled ‘comorbidity measurement’  checklists  will be The analyses will depend on the search results and included studies. Due to the infancy of this field of research and thus the expected lack of validation studies, a narrative synthesis, similar to Buck et al. , will beIn addition, meta-analyses will be conducted if appropriate. Since the comparison of the predictive validity of comorbidity measurement methods for risk adjustment is an emerging field, there has yet to be an established gold standard measurement for comorbidity in order to predict a given outcome. Also, it is unlikely that there will be a gold standard for risk adjustment, generally, due to the diversity of variables considered and the idiosyncratic development and validation methods used. However, if at least two studies are found to assess the predictive validity of at least two comorbidity measurement methods, a new meta-analytical technique will be applied, in which a hypergeometric test will be used to identify measurement methods with significantly superior or inferior performance for predicting functional outcome and length of stay . Meta-anTo the best of our knowledge, this is the first systematic review protocol to survey comorbidity measurement methods used in the nTBI rehabilitation population. Previous studies on the performance of comorbidity measurement methods have not focused on nTBI nor have they focused on inpatient rehabilitation , though Due to the expected paucity of literature on this topic, the search terms were developed for high sensitivity using general terms, such as ‘brain injury,’ as well as high specificity using specific terms, such as ‘glioma.’ Proxy terms were developed and included for all three concepts. For example, the terms ‘cardiac arrest,’ ‘drowning,’ ‘choking,’ and ‘suffocation’ were included for the anoxic brain injury condition, as these events often result in this type of injury , 31. SimHowever, these strengths have associated limitations. The increased sensitivity of this search strategy is expected to increase the number of irrelevant records, which requires more time for the screening process. Moreover, since the definition of comorbidity used to develop the search terms is limited to medical conditions or diagnoses, it does not include symptoms, function, or procedures, which are sometimes defined as comorbidity and may affect outcomes . FinallyThis systematic review protocol allows for a comprehensive review of comorbidity measurement in the nTBI population in inpatient rehabilitation. This will provide insights into the current state of the literature, which can be used to guide future research on comorbidity measurement and risk adjustment for rehabilitation outcomes for this population. The results of the review may also provide a synthesis of the profile of comorbidity in patients with nTBI in inpatient rehabilitation and their rehabilitation outcomes. Most importantly, identification of the most appropriate techniques for comorbidity measurement for this population informs best practices for epidemiologic or health service research. This allows for a more accurate prediction of various rehabilitation outcomes for patients with nTBI with comorbidities, which will contribute to enhanced planning and delivery rehabilitation services.Database specific search terms. Additional file Additional file 1:Example of data extraction table. Additional file Additional file 2:


Recent work has suggested that punishment is detrimental because punishment provokes retaliation, not cooperation, resulting in lower overall payoffs. These findings may stem from the unrealistic assumption that all players are equal: in reality individuals are expected to vary in the power with which they can punish defectors. Here, we allowed strong players to interact with weak players in an iterated prisoner's dilemma game with punishment. Defecting players were most likely to switch to cooperation if the partner cooperated: adding punishment yielded no additional benefit and, under some circumstances, increased the chance that the partner would both defect and retaliate against the punisher. Our findings show that, in a two-player game, cooperation begets cooperation and that punishment does not seem to yield any additional benefits. Further work should explore whether strong punishers might prevail in multi-player games. Punishment involves an individual paying a cost in order to inflict harm on a cheat ,2. This Labroides dimidiatus), have shown that males (the larger sex) punish females that cheat during joint inspections of model clients but—apparently due to the size difference—females never retaliate or punish males [Some of the puzzling findings regarding punishment might stem from the assumption in several studies e.g. ,9,24) th,24 th9,2sh males ,34. Simish males . Power ash males ,37, or bsh males –40 a pubsh males . AlthougHere, we incorporated power asymmetries into a two-player iterated prisoner's dilemma (IPD) game  with punIt was envisaged that by these mechanisms, punishment use for strong players could promote cooperation more effectively in asymmetric games than in symmetric games. We chose to focus on the behavioural consequences of punishment, rather than the payoffs accruing to punishers. In artificial laboratory settings, individual payoffs are necessarily determined by the (largely) arbitrary costs and benefits associated with the different actions available to players in the game. In addition, the time horizon of the interaction, which is again largely determined arbitrarily by the experimenter, can have a fundamental bearing on whether punishment is found to improve payoffs: punishment is least likely to be beneficial in short-run interactions . Since tThis research was approved by the University College London ethics board (project number 3720/001). All subjects remained anonymous so informed consent about the use of personal data was deemed unnecessary and was therefore waived by the University College London ethics board. The experiment took place over six sessions, one in May 2012, one in Nov 2012, two in March 2013 and two in April 2014 in the experimental laboratory in the Department of Economics, University College London. The lab consists of twenty computers, which are visually partitioned. A total of 120 participants  were recruited to play an IPD game with a punishment option Players interacted anonymously in pair-wise encounters by means of computer screens using the z-Tree  softwareThe IPD game lasted 50 rounds. To avoid end effects , playersPlayers were randomly split into two types: weak and strong. Weak players punished with a 1:1 fee to fine ratio, meaning that if they chose to punish their partner it would cost them one unit and it would also cost their partner one unit. Strong players punished with a 1:4 fee to fine ratio (as in ), meaninIn this study we were interested in the effects of power asymmetries on punishment, rather than on coercive behaviour. Coercion is similar to punishment since they are both aggressive behaviours that can induce cooperation from the target. However, coercion differs from punishment because unlike punishment which is typically aimed at producing mutual cooperation, coercion forces the target into a position where they would do better if they could avoid interacting with the aggressor altogether, but are constrained to do so .To ensure that weak partners were not being coerced into cooperating with a defecting strong partner (e.g. ), playerEach round of the game was split into two steps. In step one, both players simultaneously chose between the options of “cooperate”, “defect”, or “do not participate in this round” (opt out). In step two, players were given the option of whether or not to punish the partner. Thus, unlike Dreber et al. (2008) , choosinIn order to avoid framing effects, neutral language was used. Player types “weak” and “strong” were replaced with “type 1” and “type 2”; “cooperate” and “defect” were replaced with “option A” and “option B”; and “punish” and “don’t punish” were replaced by “option C” and “option D”. After both games had finished, all subjects were required to fill in a questionnaire to provide demographic information (see ESM for questions and demographic data).We asked how asymmetries affected (i) players' tendency to cooperate and (ii) players' tendency to punish their partner’s defection. Previous work has shown that cooperators are more likely than defectors to use punishment . Thus, in + 1 after having been punished (or not) in round n. We classed a player as retaliating if they punished a cooperative partner in round n + 1, having been punished by that partner in round n. For analyses (iii)—(v), data were restricted to instances where players had defected in round n .Next, we asked how players responded to being punished. We asked whether punishment affected the likelihood that a player would subsequently (iii) cooperate (iv) retaliate or (v) opt out in the next round in both symmetric and asymmetric games. We compared the likelihood that players cooperated and retaliated in round n + 1 could also be affected by whether their partner cooperated or defected in round n. Therefore, in analyses (iii) & (v), we controlled for whether or not the partner cooperated in round n. However, since opportunities to retaliate were relatively rare , in analysis (iv), we did not have sufficient data to control for the effects of whether or not the partner cooperated in round n , initially a three-way interaction was a component of the best model , then punishment had no meaningful effect on the target’s propensity to cooperate in the next round ; Fig. 3.Neither weak nor strong players ever retaliated against a weak partner . HoweverPrevious studies have suggested that punishment use is detrimental because it induces retaliation rather than cooperation ,26,27. HAs expected, punishment was most often directed from cooperating players at defecting partners and strong players were generally more likely to punish than weak players. While these results supported our initial predictions, we had other findings that were more puzzling. First, weak players were more likely to punish in asymmetric games than in symmetric games, which is the opposite pattern to that which we predicted. Second, players seemed to respond more to the cooperative decision of the partner than to the punitive action when deciding how to behave in the next round. In situations where the partner cooperated, there was no additional positive effect of punishment on the propensity of the target to cooperate and, in fact, in some cases the punishment made the target more likely to defect. Third, while strong punishers frequently encountered retaliation from the target, we did not record a single instance of retaliation against a weak punisher. These results are clearly counter to our predictions that punishment would operate down a dominance hierarchy and be most likely to promote cooperation when directed from strong players at weak targets. In part these findings might stem from the fact that cooperating and defecting were specified as binary, all-or-nothing responses in this study, rather than continuous variables. Thus, a player that switched to cooperation (in response to the partner's cooperation) could not, by definition, increase their investment even more in response to being punished. This is unlike the situation with cleaner fish, where, although defecting is a binary outcome (bite client / do not bite client) investment can be modelled as a continuous variable (duration of time 'cooperating' by removing ectoparasites) . In otheAnother possibility is that the effectiveness of punishment varies between two-player games and multi-player games. Previous theoretical and empirical studies ,30 have While these explanations might help us to understand why punishment did not promote cooperation, it is less clear why punishment from strong, but not weak, players was actually detrimental—being more likely to provoke both retaliation and defection. It has been suggested that the moral legitimacy of punishment is an important determinant of how the target is expected to respond ,60. AccoIn general, weak players opted out more than strong players and both player types opted out more when faced with a strong partner. Although, theoretical studies have suggested that cooperation and punishment is more likely to occur and persist if players have the option to opt out ,63, thesWeak and strong players were also more likely to opt out of rounds if their partner defected than if their partner cooperated in the previous round. This suggests that players used opting out either to avoid defectors or to signal their disapproval with the defector's action. However, opting out appeared to decrease the probability that a defecting partner would switch to cooperation .To summarise, we found no evidence that punishment promoted cooperation in this two-player game and in fact punishment use sometimes had a detrimental effect on cooperation. Future work could explore whether these results stem from using a binary response term for cooperation and whether punishment might be more effective where investments are continuous. We also propose that understanding the moral assessment of punishment will be crucial for predicting when it will promote and when it will undermine cooperation.S1 Supporting InformationColumns are as follows: Partner coop prev = whether the players partner cooperated (yes) or defected (no) in the previous round; Player C after D = 1 if player cooperated and 0 if player defected in round n + 1 after defecting in round n; Player P in response to C = 1 if the player punished and 0 if the player didn't punish in response to a cooperative partner; Partner P after D prev = 1 if the partner punished and 0 if the player didn't punish in response to a defecting player in the previous round; Player opt out after D = 1 if player opted out and 0 if player didn't opt out of round n + 1 after defecting in round n; Player P in response to D = 1 if the player punished and 0 if the player didn't punish in response to a defecting partner; Partner opt out prev = 1 if players partner opted out in the previous round and 0 if player did not opt out in the previous round; Player C after D 2 = If partner did not opt out of round n + 1; 1 if player cooperated and 0 if player defected in round n + 1 after defecting round n. If partner opted out of round n + 1: 1 if player cooperated and 0 if player defected in round n + 2 after defecting round n.(CSV)Click here for additional data file.S2 Supporting InformationExperimental instructions, comprehension questions, demographic questions, analysis of players’ response to partner opting out, supplementary tables, supplementary figures & R code used to fit generalised linear mixed models.(DOC)Click here for additional data file.


Organoselenium compounds find applications due to their biological activity and useful synthetic properties  are usually prepared from alkyl halides and Seous NaOH , liquid ous NaOH , sodium ous NaOH , and sodous NaOH  were repous NaOH  or selenous NaOH , are les2) and 1,3-bis-2-selena propane (4).Herein we report improved methods for the preparation of pyrazole- and benzotriazole-containing selenoethers 1,5-bis-3-selena pentane  in aqueous NaOH  (5) was obtained in high yield (84%); therefore selenoether 2 and not some other impurity acted as a ligand and the structure of the complex can be used for the elucidation of compound 2 structure.In order to establish the structures of compounds 5 crystallizes in a monoclinic crystal system; crystallographic parameters and details of the diffraction experiment are given in  5 it is evident that compound 2 is indeed a selenoether and not a diselenide. The lengths of C–C and C–N bonds in pyrazole rings are within the usual range +), 203 ([M-PzCH2CH2]+), 109 ([PzCH2]+). Anal. Calc'd for C14H22N4Se (325.31): C, 51.59; H, 6.82; N, 17.22. Found: C, 51.97; H, 7.01; N, 17.70.A suspension of selenium , sodium formaldehydesulfoxylate dihydrate , and NaOH  in water (5 mL) was stirred at room temperature, until the initially formed red solution turned colorless and white precipitate of Na 4 was prepared similarly to compound 2 from 2.10 g (12.54 mmol) chloro-derivative 1, 0.50 g (6.27 mmol) of selenium, 2.31 g (15.0 mmol) of sodium formaldehydesulfoxylate dihydrate, and 1.38 g (34.5 mmol) of NaOH in 6 mL of water and 15 mL of acetonitrile. Yield 1.94 g (90%), colorless crystals, mp 182-183°C (DMF). IR  1612, 1496, 1453 (νBta), 754 (νC–Se). 1H NMR (DMSO-d6): 6.19 , 7.44 , 7.58 , 7.97 , 8.08 . 13C NMR (DMSO-d6): 42.5 (CH2), 111.1 (7-C-Bta), 119.3 (4-C-Bta), 124.4 (5-C-Bta), 127.5 (6-C-Bta), 131.9 (8-C-Bta), 145.4 (9-C-Bta). Anal. Calc'd for C14H12N6Se (343.25): C, 48.99; H, 3.52. Found: C, 49.30; H, 3.83.Selenoether 2  in acetone (0.2 mL), solution of Cu (NO3)2·3H2O  in acetone (0.2 mL) was added. After standing for 2 hours, deep-green crystals of the complex were formed, which were filtered, washed with acetone, and dried. The crystals were suitable for X-ray crystal structure determination. Yield 0.086 g (84%). IR  1556 (νPz), 1026 (Pz breating), 811 (νC–Se). Anal. Calc'd for C14H22CuN6O6Se (512.87): C, 32.79; H, 4.32; N, 16.39. Found: C, 33.04; H, 4.50; N, 15.96.To a solution of selenoether4 and 5.Supplementary Material contains crystallographic information files (CIF) for compounds


Parkinson's disease (PD) is a chronic and progressive neurodegenerative condition characterized by a progressive depletion of dopaminergic neurons in the substantia nigra pars compacta . It affeThe management of PD has traditionally centered on drug therapy with levodopa viewed as the “gold standard” treatment . HoweverIn this special issue of Parkinson's disease, we invited investigators to submit their contributions about rehabilitation procedures in the management of people with PD. We particularly focused on articles proposing some evidence for innovative rehabilitation protocols (including treatments based on motor-cognitive approaches) and comparing the effects of different rehabilitation therapies. Other articles explored rehabilitation strategies such as physical activity, physiotherapy, electromechanical and robot-assisted training, virtual reality, and telerehabilitation. Finally, we focused on the role of cognitive dysfunction in PD rehabilitation. This issue highlights the emerging and important role that rehabilitation plays in the management of motor and nonmotor symptoms of PD. The wide range of topics included in this issue demonstrates the complexity of the disease and the need for a multidisciplinary approach to rehabilitation for PD.Alessandro PicelliAlessandro PicelliTalia HermanTalia HermanSerene S. PaulSerene S. PaulLaurie A. KingLaurie A. King


Helianthus annuus) seedheads. This phenomenon has competing biomathematical explanations, and our core premise is that observation of both Fibonacci and non-Fibonacci structure is informative for challenging such models. We collected data on 657 sunflowers. In our most reliable data subset, we evaluated 768 clockwise or anticlockwise parastichy numbers of which 565 were Fibonacci numbers, and a further 67 had Fibonacci structure of a predefined type. We also found more complex Fibonacci structures not previously reported in sunflowers. This is the third, and largest, study in the literature, although the first with explicit and independently checkable inclusion and analysis criteria and fully accessible data. This study systematically reports for the first time, to the best of our knowledge, seedheads without Fibonacci structure. Some of these are approximately Fibonacci, and we found in particular that parastichy numbers equal to one less than a Fibonacci number were present significantly more often than those one more than a Fibonacci number. An unexpected further result of this study was the existence of quasi-regular heads, in which no parastichy number could be definitively assigned.This citizen science study evaluates the occurrence of Fibonacci structure in the spirals of sunflower ( This haet al. [Helianthus annuus and a sample of 33 of the related perennial H. tuberosus.These models are in some ways now very mathematically satisfying in that they can explain high Fibonacci numbers based on a small number of plausible assumptions, though they are not so satisfying to experimental scientists . Despiteet al.  studied F4 numbers defined below [Neither the occurrence of Fibonacci structure nor the developmental biology leading to it are at all unique to sunflowers. As common in other species, the previous sunflower studies found not only Fibonacci numbers, but also the occasional occurrence of the double Fibonacci numbers, Lucas numbers and ed below . It is wed below  that numNeither Schoute nor Weisse reported their precise technique for assigning parastichy numbers to their samples, and it is noteworthy that neither author reported any observation of non-Fibonacci structure. One of the objectives of this study was to rigorously define Fibonacci structure in advance and to ensure that the assignment method, though inevitably subjective, was carefully documented.This paper concentrates on the patterning of seeds towards the outer rim of sunflower seedheads. The number of ray florets  or the green bracts behind them tends to have a looser distribution around a Fibonacci number. In the only mass survey of these, Majumder & Chakravarti  counted Incorporation of irregularity into the mathematical models of phyllotaxis is relatively recent:  gave an 2.2.1.F4 sequence is similarly 1,4,5,9,14,23,37,60,97,…. The double Fibonacci sequence 2,4,6,10,16,26,42,68,110,… is double the Fibonacci sequence. We say that a parastichy number which is any of these numbers has Fibonacci structure. The sequences F5=1,5,6,11,17,28,45,73,… and F8=1,8,9,17,26,43,69,112… also arise from the same rule, but as they had not been previously observed in sunflowers we did not include these in the pre-planned definition of Fibonacci structure for parsimony. One example of adjacent pairs from each of these sequences was, in fact, observed but both examples are classified as non-Fibonacci below. A parastichy number which is any of 12,20,33,54,88,143 is also not classed as having Fibonacci structure but is distinguished as a Fibonacci number minus one in some of the analyses, and similarly 14,22,35,56,90,145 as Fibonacci plus one.The Fibonacci sequence is the sequence of integers 1,2,3,5,8,13,21,34,55,89,144… in which each member after the second is the sum of the two preceding. The Lucas sequence is the sequence of integers 1,3,4,7,11,18,29,47,76,123… obeying the same rule but with a different starting condition; the 2.2.When looking at a seedhead such as in While, in practice, it is only feasible to estimate these parastichy numbers through a subjective visual process, for which we developed a protocol given in the electronic supplementary material, this is informed by the mathematical theory of phyllotaxis . This thhypothesis of geometrical phyllotaxis, HGP) but could not prove [A central insight of Turing in the 1950s  was thaot prove , and whaot prove  did provReturning to the subjective analysis, there is often a choice of which parastichy families to count. In general, we preferred to count parastichies visible at the outer rim of the seedhead where possible. As well as wanting to explore how parastichy number varied with capitulum size, it was also because the outer rim of the seedhead is developmentally the oldest part. The experiments of Palmer and students ,26 showe2.3.adjacent Fibonacci pair if they are adjacent members of the Fibonacci sequence, for example , and an approximate Fibonacci pair if each of the pair is approximately equal to adjacent members. If the developmental process starts with a pair of parastichy numbers x0 and x1 and continues to obey the HGP, all successive pairs must be of the form , where xn+1=xn+xn−1. It is a mathematical fact [xn+1/xn is close to the golden ratio τ and departures from this ratio indicate either phyllotactic systems not obeying the HGP or errors in the counting process. Moreover, if one of the parastichy numbers does have Fibonacci structure, then the system only displays evidence of the HGP if the parastichy number in the other direction is an adjacent member of the relevant Fibonacci-type series. In combination with the evidence in this paper that approximate Fibonacci pairing is not uncommon, this means that cases which are not approximate Fibonacci  pairs would pose a particularly severe challenge to models of the Douady and Couder type.We say that a pair of parastichy numbers are an cal fact  that farUnder the counting protocol, it was possible and common for a sample to be represented by only one parastichy number.2.4.To mark the centenary of Alan Turing’s birth, the Museum of Science and Industry, Manchester, UK (MSI) ran a project in 2012 which invited members of the public to grow their own sunflower and either submit their data online or to bring it into MSI for counting. A guide to counting was provided, but not all submitters entered a photograph and both parastichy counts. This project was conceived from the start as a Citizen Science project (see –31 and tEach data submission which included a photo was reviewed by one of the authors (J.S.), and each of these photos was marked up with parastichy families based on the protocol in section (b) of appendix C of the electronic supplementary material. These photoreviewed parastichy numbers were counted without sight of the submitted counts.2.5.There were 745 parastichy counts which were reported by both the original submitter and the photoreviewer of which there was agreement on 479 and disagreement on 266. Disagreements were further reviewed as described in appendix C of the electronic supplementary material, and a small number of errors in the photoreviewed data were corrected. When not otherwise stated below only these photoreviewed data were used in the analyses.2.6.As described in the electronic supplementary material, each parastichy count was associated with an ambiguity measure by the photoreviewer. Broadly, we classified each count as unambiguous, ambiguous (in the sense that different reviewers might subjectively choose a different count), unclear  or uncountable. The presence of ambiguity and lack of clarity are closely related to pattern disorder, and we interpret these ambiguity scores as a proxy measure for disorder. It was not uncommon for a seedhead to have a very strongly ordered, low ambiguity, parastichy family in one direction, but a much harder to unambigously count family in the other.3.3.1.The distribution of observed parastichy numbers is given in In this study, nearly 20% of parastichy numbers did not have Fibonacci structure, in contrast to the studies of both Schoute and Weisse which reported no parastichy numbers of this kind at all. For those parastichy numbers which did have Fibonacci structure, the breakdown by subtype is given in p-value that one of two equiprobable outcomes would be seen more than 49 times in 66 trials is 3.3×10−5, so there was a statistically significant excess of Fibonacci minus one counts over Fibonacci plus one counts.Out of the 136 non-Fibonacci counts, 49 were Fibonacci−1 and 17 were Fibonacci+1. The binomial 3.2.3.3.F5 and F8 series, respectively: neither of these have been previously reported in the sunflower.One typical example of a Fibonacci pair is shown in 3.4.As we have seen in F5 sequence could alternatively have been classified as an approximate  Lucas pair in this way. A further handful of samples for which one of the two parastichy numbers was four or five away from Fibonacci were assigned as ‘approximately Fibonacci’. There is no objective way of picking the cut-off point for approximating Fibonacci, although it is notable that while the number of potential pairs classifiable in this way increases, in fact observed counts decrease. Although subjective, assigning samples as approximately Fibonacci class is useful in focusing attention on the remainder which cannot be easily so assigned. We do this in More generally, there were a number of samples in which both counts were within two or three of a Fibonacci pair. The numbers of these are given in 3.5.There are a number of samples in which there are overlapping families of parastichies, neither of which convincingly foliates the whole sunflower, or in which there are particularly awkward transitions. An example of this is in sample 667 . Figure Sometimes, as in There were four samples that we labelled as ‘similar counts’ in which the two parastichy counts were close to each other. In most of these samples, the seedhead contained relatively few seeds.Finally, fitting none of the categories well there are 3.6.As discussed above, there were a number of seedheads for which it was not possible to count two parastichy numbers. This was often because either the sample itself or the photograph was inadequate to the task, and sometimes because the seedhead had regions of such disorder that no count could be reliably assigned; this itself is not independent of image quality. However, in some cases, there was a well-defined parastichy in one direction and none in the other; it can be seen from the discussion of competing parastichies above how this might occur and an example is given in 4.F5 and F8 sequences but by themselves they do not add much to the evidence base. Our second core result, though, is a systematic survey of cases where Fibonacci structure, defined strictly or loosely, did not appear. Although not common, such cases do exist and should shed light on the underlying developmental mechanisms. This paper does not attempt to shed that light, but we highlight the observations that any convincing model should explain. First, the prevalence of Lucas numbers is higher than those of double Fibonacci numbers in all three large datasets in the literature, including ours, and there are sporadic appearances of F4, F5 and F8 sequences. Second, counts near to but not exactly equal to Fibonacci structure are also observable: we saw a parastichy count of 54 more often than the most common Lucas count of 47. Sometimes, ambiguity arises in the counting process as to whether an exact Fibonacci-structured number might be obtained instead, but there are sufficiently many unambiguous cases to be confident this is a genuine phenomenon. Third, among these approximately Fibonacci counts, those which are a Fibonacci number minus one are significantly more likely to be seen than a Fibonacci number plus one. Fourth, it is not uncommon for the parastichy families in a seedhead to have strong departures from rotational symmetry: this can have the effect of yielding parastichy numbers which have large departures from Fibonacci structure or which are completely uncountable. This is related to the appearance of competing parastichy families. Fifth, it is common for the parastichy count in one direction to be more orderly and less ambiguous than that in the other. Sixth, seedheads sometimes possess completely disordered regions which make the assignment of parastichy numbers impossible. Some of these observations are unsurprising, some can be challenged by different counting protocols, and some are likely to be easily explained by the mathematical properties of deformed lattices, but taken together they pose a challenge for further research.Our core results are twofold. First, and unsurprisingly, Fibonacci numbers, and Fibonacci structure more generally, are commonly found in the patterns in the seedheads of sunflowers. Given the extent to which Fibonacci patterns have attracted pseudo-scientific attention , this suIt is in the nature of this crowd-sourced experiment with multiple data sources that it is much easier to show variability than it is to find correlates of that variability. We tried a number of cofactor analyses that found no significant effect of geography, growing conditions or seed type but if they do influence Fibonacci structure, they are likely to be much easier to detect in a single-experimenter setting.We have been forced by our results to extend classifications of seedhead patterns beyond structured Fibonacci to approximate Fibonacci ones. Clearly, the more loose the definition of approximate Fibonacci, the easier it is to explain away departures from model predictions. Couder  found on


The androgen receptor (AR) is a transcription factor involved in prostate cell growth, homeostasis and transformation regulated by post-translational modifications, including ubiquitination. We have recently reported that AR is deubiquitinated and stabilised by Usp12 resulting in increased transcriptional activity. In this study we have investigated the relationship between Usp12, PHLPP and PHLPPL tumour suppressors in the regulation of AR transcriptional activity in prostate cancer (PC). PHLPP and PHLPPL are pro-apoptotic phosphatases that dephosphorylate and subsequently deactivate Akt. Phosphorylated Akt is reported to deactivate AR in PC by phosphorylation at Ser213 and Ser791 leading to ligand dissociation and AR degradation. In contrast, PHLPP- and PHLPPL-mediated dephosphorylation and inactivation of Akt elevates the levels of active AR. In this report we demonstrate that Usp12, in complex with Uaf-1 and WDR20, directly deubiquitinates and stabilises the Akt phosphatases PHLPP and PHLPPL resulting in decreased levels of active pAkt. Decreased pAkt in turn down-regulates AR Ser213 phosphorylation resulting in enhanced receptor stability and transcriptional activity. Additionally, we observe that depleting Usp12 sensitises PC cells to therapies aimed at Akt inhibition irrespectively of their sensitivity to androgen ablation therapy. We propose that Usp12 inhibition could offer a therapeutic alternative for castration resistant prostate cancer. PSA and AR gene amplification and mutations, thus new strategies targeting the AR signalling cascade, both directly and indirectly, are likely to be efficacious in this disease state , CH, CH5], CIn this study we focused on the relationship between the AR and Akt pathways. The Akt pathway plays a pro-survival and pro-proliferative role and is involved in prostate carcinogenesis , 15]. T. T15]. TThe effects of Akt on AR can be opposed by the Akt phosphatases Pleckstrin Homology domain leucine reach repeat protein phosphatases, PHLPP and PHLPPL . PHLPP dHere we report that Usp12, in complex with Uaf-1 and WDR20, interacts with PHLPP and PHLPPL in PC cells resulting in their deubiquitination and protein stabilisation. Consequently, Usp12 decreases the levels of active phospho-Akt (pAkt). As a result Usp12 regulates the cross-talk between the Akt and AR pathways. As such, overexpressing Usp12 inhibits AR S213 phosphorylation resulting in increased AR stability and transcriptional activity. Additionally, depleting Usp12 sensitises PC cells to Akt inhibition irrespectively of their AR status. Therefore, we have deciphered a novel regulatory pathway of the AR that may be translationally-relevant in CRPC that is characterised by elevated pAkt.A previous proteomics study predicted PHLPP and PHLPPL as potential interacting partners of Usp12 . To estaC48A mutant failed to elevate PHLPP and PHLPPL levels suggesting the importance of Usp12 enzymatic activity for phosphatase regulation . GW7647 caused an increase in pAkt levels and as a result increased AR S213 phosphorylation. We further confirmed this result by repeating this experiment in COS-7 cells where Usp12 had the same effect , LNCaP-AI (castration resistant) and PC3 (AR negative) PC cells using two separate assays. We report that depleting Usp12 significantly sensitised PC cells to Akt inhibition irrespectively of their castration sensitivity or AR status Figure  and 6A-CRegulation of AR protein levels and activity by post-translational modifications is known to regulate both normal and malignant prostate cells. Alterations in this control system have been reported to affect disease outcome and survival prognosis . This isUsp12 was previously predicted to interact with PHLPPs using proteomics tools , as a reWe demonstrate that Usp12 plays a crucial role in PC by firstly, directly deubiquitinating the AR and rescuing it from proteosomal degradation resulting in increased AR protein and activity . SecondlIt is not uncommon for the same DUB to regulate AR in both a direct and indirect manner. Usp10, another DUB reported to target AR, was also shown to enhance AR activity by combining both direct and indirect activity. Usp10 can bind to AR and positively regulate its transcriptional activity  however,Similarly to Akt, PIM-1S was demonstrated to phosphorylate AR at S213 resulting in AR degradation and inhibition of its transcriptional activity . PIM-1S in vitro mutagenesis , pFlag-Usp12 wild type and C48A mutant  wh whin vitA mutant  and pHA-2. LNCaP-AI variant cell line was derived in-house by culturing LNCaP cells in steroid-depleted media to allow for the development of androgen independence , WDR20 was silenced with siRNA CGAGAAAGAUCACAA GCGA[dTdT] and Uaf-1 with CAAAUUGGUUC UCAGUAGA[dTdT]. Routinely, we achieved >60%, >65% and >80% knockdown for Uaf-1, WDR20 and Usp12, respectively in qPCR validation (data not shown). LNCaP, LNCaP-AI and PC3 cells were reverse transfected with siRNA using RNAiMax (Invitrogen) according to manufacturer's instructions and incubated in culture media for 96h prior to cell lysis and analysis by Western blotting as described previously .For proliferation analysis, cells were transfected with siRNA and treated either with DMSO (controls) or with PI3K inhibitor GDC-0941 (0.5μM) or Akt inhibitors MK-2206 (1μM) or Perifosine (5μM). IncuCyte measurements of cellular occupation of the wells were taken every 4 hours and additionally in a separate set of experiments cell numbers were counted at 96h to assess cellular proliferation.5 cells per 90 mm dish and transfected with 1μg of each plasmid as indicated, incubated for 72h and lysed directly into lysis buffer ). Lysates were incubated with 1 μg of antibodies as indicated for 16h at 4°C, antibodies were pulled down using Protein G Sepharose beads. For denaturing IPs, cells were subjected to 20 μM of MG-132 proteosomal inhibitor treatment for the final 16h followed by collection into lysis buffer with an addition of 2% SDS and denatured at 100°C for 10 minutes [Cells were seeded at 5×10


Nlk rescues SBMA phenotypes in mice, including extending lifespan. We also investigated the molecular mechanisms by which NLK exerts its effects in SBMA. Specifically, we have found that NLK can phosphorylate the mutant polyglutamine-expanded AR, enhance its aggregation, and promote AR-dependent gene transcription by regulating AR-cofactor interactions. Furthermore, NLK modulates the toxicity of a mutant AR fragment via a mechanism that is independent of AR-mediated gene transcription. Our findings uncover a crucial role for NLK in controlling SBMA toxicity and reveal a novel avenue for therapy development in SBMA.Spinal and bulbar muscular atrophy (SBMA) is a progressive neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR) protein. Despite extensive research, the exact pathogenic mechanisms underlying SBMA remain elusive. In this study, we present evidence that Nemo-like kinase (NLK) promotes disease pathogenesis across multiple SBMA model systems. Most remarkably, loss of one copy of DOI:http://dx.doi.org/10.7554/eLife.08493.001 Spinal and bulbar muscular atrophy (SBMA) is an inherited disease that eventually leads to degeneration in motor neurons and weakness in muscles. It is caused by a specific genetic mutation in the gene that encodes the androgen receptor protein, which leads to the production of a mutant protein that is larger than normal. Similar mutations in other genes can lead to the development of other so-called ‘polyglutamine’ diseases such as Huntington's disease and spinocerebellar ataxia. However, the precise details of how these mutations lead to disease symptoms are not known, and there are currently no effective ways of treating these conditions.Previous research has shown that an enzyme called Nemo-like kinase (or NLK for short) regulates the normal androgen receptor in cancer cells. NLK has kinase activity, that is, it adds phosphate molecules to other proteins to regulate their activity. Todd et al. used human cells, fruit flies, and mice as model systems to investigate whether NLK is involved in the development of SBMA.The experiments show that NLK promotes the development of features associated with SBMA in all three models. The kinase activity of NLK is required for these features to develop. Todd et al. also found that NLK can bind to and add phosphate molecules to the mutant version of the androgen receptor protein. This causes the mutant androgen receptor proteins to accumulate and increases the ability of the mutant proteins to activate particular genes.Todd et al.'s findings suggest that NLK promotes the development of SBMA by interacting with the mutant androgen receptor. Previous studies have shown that NLK is able to modulate the development of spinocerebellar ataxia type 1, which suggests that NLK may also play an important role in other polyglutamine diseases. The next challenge will be to fully understand the role of NLK in these diseases, which may aid future efforts to develop new treatments.DOI:http://dx.doi.org/10.7554/eLife.08493.002 Spinal and bulbar muscular atrophy  is an X-linked progressive neuromuscular disease . PatientAndrogen Receptor (AR) gene (SBMA is caused by the expansion of a polymorphic CAG trinucleotide repeat located in the first exon of the AR) gene . In wildAR) gene . SBMA isAR) gene . NonetheAR is a well-studied steroid hormone receptor that also plays a crucial role in additional diseases including androgen insensitivity syndrome and prostate cancer . StudiesNlk (resulting in a 50% reduction in protein expression) is beneficial in mouse models of the polyQ disease SCA1  domain. This effect on AR activity could then modulate SBMA-related aberrant AR-dependent gene transcription. In addition, reduced NLK expression can rescue the toxic effects of an N-terminal fragment of AR, suggesting that NLK can regulate the mutant AR protein—even in the absence of DNA binding and AR-responsive gene transcription.Given the importance of androgens to SBMA pathogenesis, many approaches to SBMA therapeutics have focused upon depleting androgen levels in patients . Unfortuase SCA1 . NLK is ase SCA1 . In thisIt was previously reported that NLK could interact with the wild-type AR in prostate cancer cell lines . HoweverWe next wondered whether NLK could modulate SBMA disease phenotypes. PolyQ expansion results in the aggregation of the host protein, and inclusion formation is a pathological hallmark of polyQ and other neurodegenerative diseases . We therTo test whether NLK can also increase mutant AR aggregation in an in vivo motor neuron setting, we cultured mouse primary motor neurons from spinal cord and transfected with GFP-tagged polyQ-expanded mutant AR and either a control plasmid or FLAG-tagged NLK-WT in the presence or absence of DHT. Cells were then blindly scored for the presence or absence of aggregation. We found that NLK is able to robustly increase mutant AR aggregation in DHT-treated neurons, while it only modestly increased aggregation in the absence of hormone .10.7554/Drosophila. When a full-length AR transgene is expressed in the Drosophila eye via the Gal4/UAS system . To ensure that this was not due to a non-specific background effect, we utilized two independent nmo loss-of-function alleles (adk1 and adk2)  . Both alnd adk2) , althouge of DHT . Loss of2 allele . AlthougDrosophila SBMA model. To do this, we crossed SBMA flies with flies expressing either the human NLK or an EGFP control  show a 50% reduction in NLK expression, while mice homozygous for the gene trap alleles show an approximately 90% reduction in protein expression (AR and its endogenous regulatory elements (BAC fxAR121). These mice recapitulate key SBMA disease phenotypes, including motor neuron pathology, muscle atrophy, and early lethality. These phenotypes are only seen in male mice, as is consistent with the hormone specificity of this disease , we were able to detect aggregates in the skeletal muscle of BAC fxAR121 mice via multiple assays. First, aggregation could be detected via immunofluorescence with AR antibodies, resulting in punctate, nuclear staining that was absent from wild-type or Nlkgt/+ muscle   . At lateks) , and so we could not assess if NLK influences phosphorylation at this site in vivo via this approach. However, the phospho-AR-S81 antibody could detect the mutant AR protein, and we found that male mice lacking one copy of Nlk showed a reduction in the level of AR-S81 phosphorylation , and this positively correlates with its effects on SBMA phenotypes in vivo, we reasoned that our cell culture system could be reliably used as an initial read-out for NLK-mediated effects on mutant AR. In order to test the specific contribution of AR-S81 phosphorylation to SBMA-related phenotypes, we introduced a phospho-resistant mutation into the polyQ-expanded AR construct at S81 . We found that the AR-S81A mutant tended to show slightly less aggregation than AR-S81  in the Drosophila eye results in a robust retinal degeneration and depigmentation phenotype  that interacts with its own AF-2 domain with a greater affinity than other motifs   . As prev6A/F27A) , this co6A/F27A)  and was 6A/F27A) . It also6A/F27A) , as was 6A/F27A) . Nonethe6A/F27A) . NLK inc6A/F27A) . NLK was6A/F27A) , as wellOne important remaining question is how NLK can increase AR-meditated gene transcription while inhibiting its N/C interaction. AR regulates target gene transcription by interacting with several cofactors at both its AF-1 and AF-2 domains , and theBased on our mammalian two-hybrid data , we specDrosophila, and mouse models, we show that reduced expression of NLK suppresses, while increased expression exacerbates, mutant AR-associated SBMA pathology, including protein aggregation, cellular toxicity and degeneration, and animal lethality phenotypes. It is particularly intriguing that the effects of NLK on the mutant polyQ-expanded AR and SBMA are consistent across different model systems, as this suggests that the role of NLK in SBMA pathogenesis is fundamental. Furthermore, all of these effects are clearly dependent on the kinase activity of NLK. Our work therefore strongly suggests that a reduction in NLK expression or enzymatic activity could be beneficial for SBMA patients.SBMA is a devastating neuromuscular disease without any cure or effective therapy to date. In this study, we explored whether and how NLK could modulate the pathogenesis of SBMA. By utilizing a variety of model systems, we clearly show that NLK is a key regulatory factor capable of modulating AR activity and SBMA pathology. Using a combination of cell culture, BAC fxAR121 mice die before reaching this final time point, however, and so we cannot rule out the possibility that the small cohort of mice analyzed at 40 weeks represent an ‘escaper’ subset of SBMA mice that are slightly healthier than the average BAC fxAR121 mouse. The reasons for the variation in the SBMA phenotype in these mice are not known, but may be interesting to investigate in the future. It is also worth mentioning that we analyzed the mice on a C57/129 F1 hybrid genetic background. Although they can be considered to be on a pure background for the purpose of this study, we cannot rule out the possibility of mouse background effects. Future studies on a different pure genetic background and/or using NLK inhibitors would be useful in corroborating our findings.Of particular importance is our finding that a 50% reduction in NLK expression partially rescues the phenotypes of an SBMA mouse model . This imDrosophila models . MAGE-11 has been reported to bind AR specifically at the 2 domain . MAGE-112 domain , 2010. T23FQNLF27 motif reduces mutant AR aggregation and toxicity . As all the mice assayed did show a rescue in the degenerative phenotype, however, we concluded that another mechanism must be playing a role in these mice and therefore investigated the possibility of a direct interaction between NLK and AR. That direct mechanism is the focus of the current study. Nonetheless, we noted that a subset of about 30–40% of the mice did show a reduction in mutant AR protein levels with a reduction in NLK expression. We therefore speculate that NLK may also play a role in protein clearance pathways, and that this, in turn, may contribute to the ability of NLK to regulate mutant protein aggregation and toxicity in varying disease cases. We ultimately suspect that both direct and indirect regulation of mutant protein expression/aggregation and activity underlies the role of NLK in disease.Data presented here and in a previous publication  suggest Nlkgt/+ heterozygous mice are generally healthy and our study provides convincing evidence that a 50% reduction in NLK protects against SBMA pathogenesis in vivo , UAS-EGFP (Bloomington Stock Center), UAS-AR14Q (current study), UAS-AR61Q (current study), UAS-trAR112Q . After crossing with the GMR-Gal4 driver line, two independent UAS-AR lines of each Q length that showed roughly equal levels of transgene expression were used for the analysis. For the genetic interaction analyses, appropriate fly lines were intercrossed and their progeny were raised at 22°C, 25°C, or 30°C on fly food containing or lacking 100 nM DHT. All experiments were carried out multiple times.The following mutant and transgenic flies were used in this study: trAR112Q , UAS-NLKS-NLK-WT , UAS-NLKS-NLK-KN , nmoadk1 mouse lines were maintained on the pure 129S6/SvEv background  were used in subsequent analyses.The Yale University Institutional Animal Care and Use Committee approved all research and animal care procedures. Mice were maintained on a 12/12-hr light/dark cycle with standard mouse chow and water ad libitum. Two independent ckground . BAC fxAckground . To perfMice were monitored for their general health and the date of death was recorded. Occasionally, mice were euthanized for humane reasons at the very end stage of disease progression, and the date of euthanasia was used as the death date in the analysis. Survival curves were generated using Kaplan–Meier statistical analysis and the log rank test was used to compare individual curves. The assay was capped at 2 years of age.Mouse quadriceps were harvested and snap frozen in liquid nitrogen-chilled isopentane. Samples were sectioned on a cryostat at 12 μm and collected on superfrost slides. Sections were then either stained with hematoxylin (3 min) and eosin (1 min) or incubated with 0.4 mg/ml NADH (Roche) and 0.8 mg/ml 4-nitro blue tetrazolium chloride  for 15 min, 37°C. Sections were then dehydrated with ascending ethanol solutions and incubated in xylenes. Coverslips were mounted with Permount. Slides were imaged on a compound light microscope using an Olympus camera and CellSens software. Fiber area and Feret's diameter of cross-sectional muscle fibers and the mean gray value of NADH transferase activity staining images were analyzed using ImageJ software . The NADH transferase activity images were obtained on the same day using identical camera settings.Mouse vertebral columns were dissected whole from freshly sacrificed mice and post-fixed in 4% paraformaldehyde overnight, 4°C. Samples were kept at 4°C through subsequent steps until freezing. After fixing, samples were incubated in 0.5 M EDTA in PBS overnight. The following day, the 0.5 M EDTA was replaced with fresh solution three times, rocking, with the last incubation lasting overnight. The next day, samples were moved to 10% sucrose, then 20% sucrose, and finally left in 30% sucrose overnight. Spinal cord and bone were frozen in Optimal Cutting Temperature (OCT) medium and later sectioned on a cryostat at 18 μm and collected on Superfrost Plus slides. After sectioning, the L4–L5 region was identified based on location and morphology as compared to a mouse spinal cord atlas. Alternating sections were stained with Cresyl violet (4 min) and dehydrated in ascending ethanol solutions. Slides were incubated in xylenes, and coverslips were mounted with Permount. The entire L4–L5 region was imaged on a compound light microscope using an Olympus camera and CellSens software, and then random images periodically spaced throughout this region were used for the measurement of neuronal soma size using ImageJ. Over 100 neurons were scored per animal.Quadriceps extracts were generated as for immunoblot and prepared as 400 μl (1 μg/μl) samples. Samples were then divided into 2 equal halves and ran separately though the filter trap assay using a BioRad BioDot SF apparatus according to the manufacturer's instructions, with the exception that a 0.22-μm cellulose acetate (CA) membrane (Whatman) was placed atop the 0.45-μm nitrocellulose (NC) membrane. The CA membrane collects insoluble AR, while the NC membrane detects soluble protein. For one sample half, both the CA and NC membranes were blocked and immunoblotted with anti-AR H280 antibody . For the other sample half, the NC membrane was immunoblotted for the loading control using either rabbit anti-Actin  or mouse anti-Tubulin . The amount of AR collected by each membrane was quantified using ImageJ.AR constructs, the full-length human AR cDNAs were PCR-amplified from GFP-AR25Q or GFP-AR120Q plasmids and inserted into an HA vector using the XhoI and NotI sites. The 130 amino acid N-terminal fragment was also subcloned into HA and GFP vectors via restriction digest and Gateway cloning, respectively. All AR point mutations used in this study were introduced via site-directed mutagenesis using the Stratagene Quikchange Kit. The FLAG-tagged Nlk constructs were kindly provided by Dr Kunihiro Matsumoto  and Dr Tohru Ishitani . ARE-luciferase plasmids were kindly provided by Dr Nancy L Weigel  and Dr Zafar Nawaz . The mammalian two-hybrid constructs were kindly provided by Dr Diane E Merry .To generate HA-tagged A mammalian cell culture system was used for co-immunoprecipitation and biochemical analyses, immunofluorescence, and luciferase reporter assays. Standard cell culture and plasmid transfection were conducted as described . BrieflyTo generate cell culture extracts, NSC-34 or HeLa cells were transfected and treated with DHT as described above. 24 hr after DHT treatment, cells were lysed in 300 μl NP40 lysis buffer . Extracts were cleared by centrifugation for 10 min at 4°C, and soluble extract was either boiled with sample buffer to generate ‘input’ samples or incubated overnight with either anti-FLAG M2 Affinity Gel (Sigma) or glutathione-sepharose 4B beads  as indicated. IP samples were washed three times with lysis buffer and submitted to western blot analysis.Drosophila heads were collected and ground in 50 μl RIPA buffer and incubated on ice for 15 min. Samples were then spun for 10 min at 13,000 rpm. Supernatant was boiled with sample buffer for 5 min, ran on 8% SDS-PAGE gels, and blotted. Antibodies used include: mouse anti-HA , mouse anti-FLAG , mouse anti-GAPDH , mouse anti-Tubulin , rabbit anti-GST , rabbit anti-AR N20 , rabbit anti-AR H280 , rabbit anti-AR phospho-S81 , rabbit anti-AR phospho-S308 , mouse anti-polyQ 1C2 , and rabbit anti-NLK . Quantification of immunoblots was carried out using ImageJ using loading controls ran on the same SDS-PAGE gel as the samples. Multiple trials were averaged.For non-co-IP blots, cells were transfected and treated with DHT and cell extracts were generated as described using triple cell lysis buffer . Extracts were boiled with sample buffer and ran on 8 or 12% SDS-PAGE gels. Gels were transferred to NC membranes, blocked and incubated with primary antibodies overnight in non-fat milk at 4°C. Membranes were then washed and probed with horseradish peroxidase-conjugated secondary antibodies  and exposed to film. Mouse tissue samples were harvested and lysed in 1 ml RIPA buffer  by dounce homogenization and cleared by centrifugation for 10 min at 4°C. Total protein concentration was measured using a BCA assay and equivalent concentrations of protein were ran on SDS-PAGE gels and blotted. For phosho-AR-S81 blots of mouse tissue, SuperSignal Western Femto (Thermo Scientific) was used in order to detect the signal. Adult Cells were plated unto coverslips and transfected and DHT-treated. 24 hr after DHT treatment, cells were fixed in 4% paraformaldehyde, permeabilized, blocked, and incubated with primary antibodies . They were then washed, incubated with Alexa Fluor conjugated secondary antibodies , and mounted onto slides in Vectashield. Immunofluorescence was imaged using a Zeiss spinning disc confocal microscope using Volocity software. All images are composite z-stacks encompassing the entire cell. Cells were scored as containing aggregates or not based on the presence of punctate vs solely nuclear staining. The ratio of aggregate-containing cells out of total cells was recorded and averaged over at minimum three trials. Mouse quadriceps were harvested and snap frozen in liquid nitrogen-chilled isopentane. Samples were sectioned on a cryostat at 12 μm and collected on superfrost slides. Slides were blocked, incubated with rabbit anti-AR H280 , washed and incubated with Alexa Fluor 488 secondary antibody  and TOTO-3 . Coverslips were mounted with Vectashield. Slides were imaged on a Zeiss LSM710 confocal microscope and images are z-stack composites encompassing the entire section. Aggregation rate was determined using ImageJ software: ‘Particle Analysis’ was used to determine the number of TOTO-3-stained nuclei and the ‘Find Maxima’ tool was used to locate all AR aggregates. The ratio of nuclei containing aggregates out of total counted was recorded for several sections and averaged over individual mice by genotype.GFP-AR120Q and/or FLAG-Nlk-WT plasmids by the Calcium-phosphate method. On the next day, 10 μM DHT was added to the medium. At DIV9, cells were fixed and subjected to immunofluorescence. Primary antibodies used were mouse anti-FLAG antibody , rabbit anti-GFP antibody , and goat anti-ChAT antibody . Appropriate Alexa secondary antibodies (Invitrogen) were used to visualize the proteins. The number of aggregate-containing cells per total GFP-positive cells was counted manually.Primary motor neurons were prepared from embryonic day 13 (E13) mouse embryos as described previously with slight modification . BrieflyARE-luciferase reporter, a pRL-TK renilla luciferase reporter and any other indicated constructs using lipofectamine 2000 and treated with DHT. 24 hr after DHT treatment, cells were lysed and subjected to a dual-luciferase assay using a Promega kit according to the manufacturer's instructions. Luciferase activity was measured using a Promega GloMax 20/20 luminometer and associated software. The ratio of the luciferase activity values was recorded for each sample and normalized to control samples in each case. Each experimental trial was performed in triplicate, and ratios were averaged over multiple trials. Protein expression was confirmed by immunoblot.NSC-34 cells were transfected with an Gal4-luciferase reporter was used in place of the ARE-luciferase construct and cells were transfected with the VP16- and Gal4-DBD-fused protein constructs as indicated.Mammalian two-hybrid assays were carried out as described for the other dual-luciferase assays, except a t-test using a two-tailed distribution and assuming unequal variance. Statistical significance between multiple sample sets was determined by one-way ANOVA using Tukey's post-hoc HSD test to compare individual group differences. Statistics were calculated using Microsoft Excel and GraphPad Prism software.Unless otherwise noted, statistical significance between two sample sets was determined by the Student's  review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and Hank Paulson (peer reviewer).Todd and colleagues explore the role of Nemo like kinase (NLK) as a regulator of disease processes in the neurodegenerative disorder, spinal and bulbar muscular atrophy (SBMA). SBMA is a polyglutamine expansion disease that specifically affects motor neurons. Todd and colleagues use a wide range of models and assays to address the potential role of NLK in disease pathogenesis. Particular strengths of the study include genetic crosses between SBMA mice and mice haploinsufficient for NLK that strongly suggest it is indeed a modulator of disease processes and studies establishing that NLK can phosphorylate the disease protein in SBMA, the androgen receptor (AR), at specific sites. Taken together, results in the mouse crosses, fly models of disease and in cell-based assays of transcription, phosphorylation and protein-protein interaction, support the view that NLK directly phosphorylates the androgen receptor and directly binds the protein. While much of the data support the view that AR phosphorylation mediated by NLK is a principal driver of AR protein accumulation, aggregation and toxicity, additional evidence suggests that a direct interaction between AR and NLK independent of kinase activity inhibits AR intramolecular (N/C) interactions and alters AR association with transcription factors, separately adding to the toxicity of expanded AR. Overall the results are thorough and build a solid case for the role of NLK in this polyglutamine disease. While there are some inconsistencies in the data – for example, the variable extent of modulation in two separate fly models deficient in the NLK ortholog and an intriguing discrepancy between established factors contributing to toxicity and the effect of NLK on intramolecular interactions of mutant AR – these are not a significant issue and do not lessen the import of the study. Indeed, the authors do a good job of explaining their, sometimes conflicting, data and avoiding over-interpretation of key findings.Studies by the same group previously showed that NLK could modulate SCA1 disease pathogenesis, SCA1 being another polyglutamine disorder. While this convergence on NLK in two related diseases arguably increases the impact of the current findings, it is striking that two separate disease proteins with little functional connection show similar relationships to NLK. One wonders whether there are indirect neuroprotective effects of this kinase on cellular pathways, not yet been explored, that may explain aspects of disease regulation by this signaling protein. The studies are a significant advance in the field and provide insight into the broader area of pathogenic factors underlying polyglutamine disorders.Essential revisions:t-test in these multivariable instances.1) 2) A larger question that needs to be addressed is why a major change seen in the molecular data regarding aggregation and phosphorylation and a relatively minor change is seen phenotypically in the mouse .3) The first paragraph of the Discussion belongs in the Introduction.4) Third paragraph of Discussion: why is the 40-week old data not shown but discussed in the manuscript?5) In the Discussion section, a hypothesis is floated out to the reader, conformational change, multiple times. This needs to be simplified as no data are presented.6) What is NLK's role in normal AR function and what about the polyQ causes the degenerative phenotype? 1)ANOVA should be used instead of t-test in these multivariable instances.ANOVA using Tukey’s post-hoc analysis to compare samples. We also updated the figure legends and methods section to reflect these changes. Some of the more subtle effects we noted in our original manuscript are not significant by ANOVA, and we have altered the text to reflect this. All major conclusions concerning the role of NLK in SBMA remain the same and in some cases are of increased significance using this method.We appreciate the reviewers’ drawing our attention to this oversight. The statistics for the figures listed have been updated to 2) A larger question that needs to be addressed is why a major change seen in the molecular data regarding aggregation and phosphorylation and a relatively minor change is seen phenotypically in the mouse .We apologize that we did not directly address this question in our original manuscript. We agree that it is an important question and have included a discussion of our thoughts on this topic in the Discussion section of the revised manuscript.3) The first paragraph of the Discussion belongs in the Introduction.We agree with the reviewers. We have moved this paragraph from the Discussion to the Introduction and merged it into the existing text.4) Third paragraph of Discussion: why is the 40-week old data not shown but discussed in the manuscript?The 40-week data we referred to in the Discussion are included in the graphs in 5) In the Discussion section, a hypothesis is floated out to the reader, conformational change, multiple times. This needs to be simplified as no data are presented.We agree that our original manuscript included a more lengthy description of our hypotheses than was warranted given our current data. We have edited the Discussion section to focus less on this topic and more on the questions in Essential Revisions #2 and 6 listed in this letter.6) What is NLK's role in normal AR function and what about the polyQ causes the degenerative phenotype?We agree with the reviewers that this is an important question, and in our revised manuscript we have tried to better explain our thoughts on this topic in our Discussion. To summarize, we currently suspect, based on the data presented, that NLK normally interacts with and phosphorylates AR and regulates its interactions with its cofactors. This in turn influences the transactivation activity of AR. However, others and we have postulated that its activity and its interactions with its cofactors are likely altered, resulting in protein dysfunction and pathology. As NLK is still able to bind this mutant AR, we predict that its normal role in regulating AR activity now acts to enhance the dysfunction and toxicity of the receptor. At the same time, our data suggest that phosphorylation of the mutant (not wild-type) AR by NLK promotes its aggregation, enhancing the toxic effects of this aggregation process, as well.