Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most frequently dispensed nonprescription drugs in community pharmacies. However, inappropriate use of NSAIDs by consumers has been associated with development of gastrointestinal (GI) injuries and renal injuries. Community pharmacists’ education of consumers on proper use of NSAIDs and their associated adverse effects has been shown to reduce the GI and renal injuries. In Qatar, no studies have been done to assess the community pharmacists’ knowledge, attitude, and practices related to renal and GI adverse effects of NSAIDs. Therefore, this study aimed to assess Qatar community pharmacists’ knowledge, attitude, and practices on the safe use of nonprescription NSAIDs to reduce the risk of kidney and GI injuries.A cross-sectional web-based survey was conducted among community pharmacists in Qatar. A pre-tested 28-item questionnaire that was developed through a multi-phase iterative process was administered to a convenient sample of community pharmacists in Qatar. Data were analyzed using descriptive and inferential statistics with statistical significance set at p < 0.05.Overall, 114 community pharmacists responded to the online questionnaire (response rate 15.2%). Approximately 90% of the community pharmacists demonstrated from good to excellent knowledge on the renal and GI adverse effects of NSAIDs, with none of their sociodemographic and professional characteristics having a significant effect on their knowledge scores. More than half of the pharmacists reported that they always or usually educated patients on the dosage (98.6%), administration (95.8%), side effects and precautions (78%), and contraindications (71.2%) of NSAIDs during their routine practices. The majority of the pharmacists had positive attitude towards educating patients about adverse effects of NSAIDs, as well as identifying high-risk patients who should avoid nonprescription NSAIDs. However, 45.7% of the pharmacists strongly agreed or agreed that educating patients about NSAIDs can be time consuming.Community pharmacists in Qatar demonstrated good knowledge of the renal and GI adverse effects of NSAIDs with some obvious areas of improvement, and this can be reinforced through continuing professional development. They also showed positive attitudes towards protecting patients against the renal and GI adverse effects of NSAID. However, a significant proportion of the pharmacists admitted that educating patients on NSAIDs was time consuming, which is a cause of concern warranting further investigation. Community pharmacy managers should provide community pharmacists adequate time and support to educate individuals at risk of renal and GI injuries who obtain NSAIDs from their pharmacies. Also, the Ministry of Public Health of Qatar should consider making counseling on high-risk medications  by community pharmacists mandatory so that measures can be put in place in the pharmacies to free the pharmacist for education and counseling. For example, in the United States of America (USA), 39.7% of households purchased ibuprofen as an OTC NSAID in 2017 . HoweverIn the presence of underlying risk factors for GI and renal injuries, the risk of NSAIDs-related injuries is elevated further. These risk factors include advanced age (>60 years), previous history of ulcer, concomitant use of an oral anticoagulant or corticosteroid, use of more than one NSAID, and presence of comorbid conditions such as renal and hepatic failure . In manyThe community pharmacy setting is a safety net for patients or customers who self-medicate with OTC NSAIDs. The community pharmacist is often the first port of call and the most accessible healthcare professional who provides free consultation services and professional advice . TherefoIn Qatar, anyone can obtain a wide range of NSAIDs from community pharmacies without a prescription. Yet, the prevalence of NSAIDs use is poorly documented in Qatar even though it is widely dispensed in community pharmacies. A significant proportion of the population of Qatar are laborers engaged in manual labor who regularly buy NSAIDs from community pharmacies as a result of occupation-related musculoskeletal pain. Similarly, sedentary workers are also likely to utilize NSAIDs regularly for chronic and lower back pain. A large cross-sectional study conducted in the primary care setting on a representative sample of non-manual workers and unemployed patients reported high prevalence of low back pain of 59.2% in Qatar .It is a professional obligation for the community pharmacist to ensure NSAIDs dispensed to customers will not cause harm or fatal organ injury. Educating patients in the community pharmacy on NSAIDs avoidance have been shown to be an effective practice in reducing acute kidney injuries in high-risk groups . Similar22.1This was a cross-sectional questionnaire-based study that was conducted between October 2016 and May 2017 in community pharmacies in Qatar. Licensed community pharmacists were e-mailed the link to the survey via SurveyMonkey®. In addition, the pharmacy managers of the three largest pharmacy chains in Qatar  and one independent pharmacy  were contacted to disseminate the survey link to their pharmacists.2.2A minimum sample size of 255 was calculated with the Raosoft® online calculator using the following parameters: 5% margin of error, 95% confidence interval, estimated number of community pharmacists of 750, and estimated response distribution of 20% . Qatar i2.3The 28-item questionnaire was created and validated in English only. The three composites of the questionnaire assessed the current knowledge of the community pharmacists on NSAIDs-related kidney and GI injuries; current practices on proper use of NSAIDs and; attitudes towards NSAIDs use in Qatar. The items of the questionnaire were adapted and modified from two previous studies . The devThe final version of the questionnaire contained 28 items, divided into three composites (22 items) and a section on demographics (6 items). The knowledge composite contained nine multiple-choice questions and one true or false question. Therefore, the maximum obtainable score for this composite was 10 points. The current practice composite comprised nine questions related to patient education on safe use of NSAIDs, as well as the community pharmacist’s professional development endeavor related to NSAIDs. Lastly, the composite of attitudes towards NSAIDs use comprised three items measured on a 5-point Likert scale focusing on the attitudes of community pharmacists regarding counseling patients and customers on NSAIDs-related kidney and GI side effects as well as the importance of the community pharmacist’s role in today’s practice.2.4SurveyMonkey®, an online survey tool was used to administer the anonymous survey. The survey link was emailed to 750 valid email addresses of licensed community pharmacists in QCHP database, as well as through the managers of the largest chain community pharmacies in Qatar. Reminders were sent out to the community pharmacists by email and through pharmacy managers at least every two weeks to encourage more participation during the six-month period the survey link remained active.2.5Data collected via the SurveyMonkey® responses were exported into IBM’s Statistical Package for Social Sciences (SPSS) version 23 software . Both descriptive and inferential statistical analyses were applied. Total knowledge score was calculated for each respondent and categorized as poor (0–4 points), good (5–6 points), very good (7–8 points), and excellent (9–10 points). Since the possible knowledge score ranges from 0 to 10 points, the scores below the 50th percentile (0–4) were categorized as poor, and scores equal to or greater than the 50th percentile were categorized as good, very good or excellent. Mann-Whitney U and Kruskal-Wallis tests were used to analyze respondents’ knowledge score based on their demographic and professional characteristics. In addition, the Chi-square test was used to determine the influence of demographic and professional characteristics on the knowledge score categories . A p-value of < 0.05 was used to determine statistical significance.2.6The study protocol, questionnaire, and informed consent form were reviewed and approved by the Qatar University Institutional Review Board .33.1Over a six-month period, a total of 114 community pharmacists responded to the online questionnaire, achieving a response rate of 15.2% (114/750), of the population of community pharmacists practicing in Qatar who received the online survey link through email. Of the 114 that responded, 34 completed only the socio-demographic and professional characteristics without completing the remaining parts of the questionnaire. Therefore the usuable rate was 70.2% (80/114)., from North Africa , and East Asia . More than half of the respondents  got their license to work as community pharmacists in Qatar less than five years prior to completing the questionnaire. The majority of the community pharmacists  last updated their knowledge on NSAIDs within one year prior to completing the questionnaire. The highest degree obtained by most of the community pharmacists practicing in Qatar  was bachelor’s (BSc) degree.Details regarding the community pharmacists’ socio-demographic and professional characteristics are provided in 3.23.3U test and Kruskal-Wallis test) showed that none of the characteristics  had a significant effect on the knowledge score . In terms of age, those in the age ranges of 30–39 years and 40–50 years age groups seemed to be more knowledgeable with median (IQR) scores of 8 (2) and 9 (2) respectively. Respondents from a Middle Eastern country had a higher median (IQR) score of 9 (3) in comparison to those from a North African country, 8 (3) or an Asian country, 7.5 (3). The median (IQR) score for pharmacists who obtained their licenses more than 10 years prior to the questionnaire administration, 9 (3) was higher than those who obtained their licenses between 5 and 10 years, 8 (3) and less than 5 years before completing the questionnaire, 7.5 (3). In addition, the median (IQR) score for pharmacists whose highest professional degree in pharmacy is BSc, 8 (2) was higher than those with master’s degree, 7 (4).Further analysis of the results were done to assess the influence of the pharmacists’ characteristics on their knowledge score. The inferential analyses  stated that they had updated their knowledge on NSAIDs within a year prior to completing the questionnaire, while 22.8% (18/79) updated their knowledge on NSAIDs one to five years prior. Of those who updated their knowledge, 68.8% (55/80) used online resources, 35% (28/80) used drug information books, 30% (24/80) used research articles, 15% (12/80) through another community pharmacist and 26.3 % (21/80) attended a continuing education program to update their knowledge on NSAIDs . Only seThe frequency with which community pharmacists provided education to patients/customers on the instructions of taking the medicine, dosage, side effects/precautions, and contraindications are summarized in Regarding the sources where community pharmacists obtained information on NSAIDs, most of the community pharmacists working in Qatar  stated that they usually use internet sources for information on drugs, followed by the drug company’s package insert , and research articles  respectively. Finally, there was one practice-related question regarding the least important information on NSAIDs that pharmacists should educate patients on during patient counseling. Most of the pharmacists  selected nonprescription status of pain relievers, followed by counseling the patient to ask a doctor or pharmacist about the safety of adding a new medication while using NSAIDs . Only 6.9% (5/72) of the pharmacists selected the item related to counseling patient that NSAIDs may be harmful in those at risk of kidney injury because of its harmful effect of lowering blood supply to the kidney.3.5Of the respondents, 74.6% (53/71) believed that their current knowledge on NSAIDs is sufficient to allow them to advice patients on its safe use. Conversely, 14.1% (10/71) did not believe that their current knowledge suffices for them to educate patients on the safe use of NSAIDs, while 11.3% (8/71) do not know if their knowledge on NSAIDs is adequate.Approximately 94.4% (67/71) of the respondents believed that community pharmacists have a critical role to play in preventing NSAIDs users from the associated kidney and GI adverse effects of this class of agents. More than two-thirds  of the respondents, believed that today’s practice in Qatar in relation to counselling patients on the appropriate use of NSAIDs has to undergo some changes to ensure the prevention of the kidney and GI adverse effects. Whereas, 18.3% (13/71) of the community pharmacists who responded to this question did not believe that any changes should be implemented in current practice, and 14.1% (10/71) did not know whether current practice needs to undergo changes or not.Those who believed that current practice has to change were asked to answer an open-ended question that asked them to mention the changes they want to see in practice. They responded with the answers that revolved around the following themes: offering education, CPPD workshops, free conferences, free access to published articles, and free online sessions to community pharmacists on NSAIDs appropriate usage at least two times per year for updating. In addition to the need to change the legislation in that, not all NSAIDs concentrations shall be nonprescription. Moreover, community pharmacists shall conduct prior interview with the patient to identify risk groups and a proper counselling shall be done after the dispensary of NSAID when appropriate. Additionally, the re-evaluation of community pharmacists’ role by the Ministry of Health, and objective structured clinical examination (OSCE) training is demanded for community pharmacists.4This cross-sectional questionnaire-based study to explore Qatar community pharmacists’ knowledge and attitudes on the GI and renal adverse effects of OTC NSAIDs showed that approximately 90% of the community pharmacists have at least good knowledge on the adverse effects of the medication class. In addition, more than half of the pharmacists educated patients on the dosage, administration, side effects, precautions and contraindications of NSAIDs during their routine practices. Similarly, the majority of the pharmacists have positive attitudes towards educating patients about adverse effects of NSAIDs and identifying high-risk patients who should avoid OTC NSAIDs. However, almost half (45.7%) of the pharmacists strongly agreed or agreed that educating patients about NSAIDs can be time consuming. The community pharmacists indicated the need for continuing education programs at least twice a year to update their knowledge on NSAIDs and the changes in legislation to classify some NSAIDs as prescription only medicines.A large proportion of the community pharmacists in Qatar demonstrated they are knowledgeable about the adverse effects of NSAIDs. This result is important for the safety of patients and the public in Qatar that utilize the services of community pharmacists, because a knowledgeable pharmacist will properly educate patients on steps to prevent or minimize adverse effects that could sometimes be fatal. To our knowledge, there is no other study conducted to evaluate the knowledge and practices of community pharmacists about the adverse effects of NSAIDs. A study among physiotherapists in Australia reported a similarly high percentage of physiotherapists with good knowledge on NSAIDs’ adverse effects . NeverthSince most NSAIDs can be obtained from the community pharmacy in Qatar without a prescription or pharmacist’s consultation, it is important for pharmacists to take proactive role and the initiative to educate patients/customers who obtain these medications from them. Therefore, it is positive that more than half of the pharmacists surveyed reported routinely educating patients about NSAIDs use, despite admitting that it is a time consuming exercise. More importantly, almost all the pharmacists (94.4%) believed that the community pharmacist plays a critical role in protecting NSAIDs users from renal and GI adverse effects. This finding is similar to a cross-sectional study of community pharmacists in Thailand which found that at least 65% of them conducted screening for history of GI adverse effects or renal impairment prior to dispensing NSAIDs, as well as 36.9–63.2% of pharmacists communicating ADR information occasionally or regularly when they dispense NSAIDs . In addin = 114) was lower than the estimated sample size (n = 255), despite the prolonged data collection period and several reminders sent to eligible participants to complete the survey. The low participation by community pharmacists was most likely related to their fatigue in filling several questionnaires from different research groups in Qatar. Moreover, not all the three composites of the survey were completed by all the respondents. There is a possibility of type 2 error in the inferential analyses where none of the demographic and clinical characteristics of the pharmacists significantly influenced the knowledge score, because the minimum sample size was not reached. Nevertheless, it is assuring that both the overall mean and median scores fell in the range of good knowledge. There is also the possibility that the community pharmacists who completed the survey have particular interest in the research topic. Therefore, readers should interpret the results with caution as the responding community pharmacists may not be representative of the knowledge of all community pharmacists in Qatar. Another limitation of the study is the use of a convenient sample of pharmacists. Even though the initial intention was to reach out to all community pharmacists in a government database, pharmacy managers of the three largest chain pharmacies in Qatar were contacted to send the questionnaire link directly to pharmacists in their respective organizations after the initial emailing of the link to community pharmacists in order to boost up the numbers of respondents. Furthermore, it is possible that the study participants accessed drug information resources when completing knowledge-based questions of the survey, which may have led to the high knowledge score. In an ideal community pharmacy practice setting, pharmacists should be encouraged to consult appropriate drug information resources in order to prevent avoidable harm to patients. Therefore, if it is part of the routine practice of Qatar community pharmacists to access appropriate drug information resources during patient care, then it is a good practice to improve patient safety. Finally, social desirability bias cannot be ruled out especially for the composites of the survey related to current practices on proper use of NSAIDs and attitudes towards NSAIDs use. However, administering the survey online and assuring respondents that their responses will be kept anonymous hopefully minimized this bias in the study. Therefore, readers should take the possibility of social desirability bias into consideration when interpreting the results of the study.This study has several limitations, some of which are inherent to all cross-sectional survey type of studies. First, the number of community pharmacists who responded to the questionnaire  by community pharmacists mandatory so that measures can be put in place in the pharmacies to free the pharmacist for education and counselling.This study was funded by Qatar University under the Student Grant number QUST-CPH-SPR\2017-14.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Accurate measurement of trajectories in longitudinal studies, considered the gold standard method for tracking functional growth during adolescence, decline in aging, and change after head injury, is subject to confounding by testing experience.learning  on accuracy and after speed domain scores. Then developmental effects were  determined for each domain for accuracy and speed having removed the measured learning effects.We measured change in cognitive and motor abilities over four test sessions  in 154 male and 165 female participants (baseline age 12–21 years) from the National Consortium on Alcohol and NeuroDevelopment in Adolescence (NCANDA) study. At each of the four test sessions, these participants were given a test battery using computerized administration and traditional pencil and paper tests that yielded accuracy and speed measures for multiple component cognitive  and motor (Ataxia and Speed) functions. The analysis aim was to dissociate neurodevelopment from testing experience by using an adaptation of the twice-minus-once tested method, which calculated the difference between longitudinal change  and practice-free initial cross-sectional performance for each consecutive pairs of test sessions. Accordingly, the first set of analyses quantified the effects of The greatest gains in performance occurred between the first and second sessions, especially in younger participants, regardless of sex, but practice gains continued to accrue thereafter for several functions. For all 8 accuracy composite scores, the developmental effect after accounting for learning was significant across age and was adequately described by linear fits. The learning-adjusted developmental effects for speed were adequately described by linear fits for Abstraction, Emotion, Episodic Memory, General Ability, and Motor scores, although a nonlinear fit was better for Attention, Working Memory, and Average Speed scores.Thus, what appeared as accelerated cognitive and motor development was, in most cases, attributable to learning. Recognition of the substantial influence of prior testing experience is critical for accurate characterization of normal development and for developing norms for clinical neuropsychological investigations of conditions affecting the brain. Age was allowed to be a nonlinear smooth effect, implemented via thin plate splines [s(age)] with 3 internal knots [The primary analysis tools were the General Additive Mixed Model (GAMM) and Likelihood Ratio Tests (LRT) using the al knots , herein al knots .To determine the learning effect from visit to visit, three data sets were constructed: 1) all subjects’ visit 1 plus visit 2; 2) all subjects’ visit 2 plus visit 3; 3) all subjects’ visit 3 plus visit 4. For each dataset, a regression analysis fitting the data with age (GAMM with smoothed age) was performed without and with visit as a factor. The results of the two models were compared with an LRT; significant improvement by adding visit to the model indicated significant learning from visit to the subsequent visit. The improvement in the amount of variance explained (R-squared) is reported here as an index of the amount of learning between visits.To test for age effects on learning, for each test visit pair, GAMMs with and without an age-by-visit interaction were compared using LRTs. A significantly better fit with age-by-visit interaction indicated significant age effects on learning.To test for sex effects on learning, for each visit pair, GAMMs with age-by-visit plus sex-by-visit were performed and examined for learning-by-sex interactions.To quantify learning across the four visits, a sequence of model fits was performed  that allcross-sectional fit of dependent variable y vs. age was computed across all participants for each visit separately producing: fit1 (based on only 1st visits), fit2 (based on only 2ndvisits), fit3 (based on only 3rd visits), fit4 (based on only 4th visits).The age-related learning effect from the previous visit was estimated by computing the difference between the predicted values from the cross-sectional fit at the current visit minus the predicted value when applying the fit from the previous visit to the ages at the current visit. This procedure was done cumulatively across visits 2, 3, and 4, producing learning-adjusted  values. Because visit 1 had no learning relevant to these test sessions, visit 1 values were not adjusted for experience effects. This adjustment is a direct extension of the “once vs. twice tested” method to more than two testing occasions.For visits 2, 3, and 4, the fit2 applied to subject visit 2 ages minus the predicted values of cross-sectional fit1 applied to the same visit 2 ages:The estimated age-dependent learning at visit 2 was the difference between the predicted values of cross-sectional This is the simple case of baseline with one follow-up test session as used in Sullivan et al.  and is tfit3 applied to subject ages at visit 3 minus the predicted values of cross-sectional fit2 again applied to subject ages at visit 3. This quantity was then added to the estimate from visit 1 to visit 2 to obtain the cumulative learning effect from baseline to visit 3:For subsequent visits the learning effect required testing of additional learning from visit to visit, calculated as follows: The estimate of learning at visit 3 was the difference between the predicted values of cross-sectional fit4 applied to subject ages at visit 4 minus the predicted values of cross-sectional fit3 applied to the same subject ages at visit 4, which was then added to the estimate from visit 1 to visit 2 and from visit 2 to visit 3:The estimate of cumulative learning at visit 4 was the difference between the predicted values of cross-sectional To examine the effect of age on performance, GAMMs examining composite scores as a function of smoothed age were performed before and after adjusting for learning. ANOVAs were computed to allow comparison of the GAMM with smoothed age to the GAMM with linear age to determine whether the developmental trajectory of that test composite was better describes as smoothed or linear.To test for sex effects on development across all visits, GAMMs examining learning-adjusted values as a function of smoothed and linear age with and without sex as a factor were compared with ANOVA.p-values ≤ 0.00625 (two-tailed) to be considered significant.To account for the multiple comparisons made, family-wise Bonferroni correction was determined for 8 test session pairs for each metric (accuracy and speed) with α = 0.05 required The first set of results quantifies the learning effects for each performance metric (accuracy and speed) of each composite score by age and sex. The second set quantifies the developmental effects with the measured learning  effects removed.p-values. The additional variance explained by age plus visit in the model is indicative of learning and is depicted in red in the second bar of each visit pair in Fig. over age are presented in Figs. For each composite score, learning was quantified by computing the difference in the variance explained by age between pairs of two GAMM models with and without visit as a factor; these statistics are presented in Table Overall, smooth age fits were better than linear age fits in describing the unadjusted data, which comprised both learning and development   fits (p = 0.999) for Abstraction, Emotion, Episodic Memory, General Ability, and Motor scores. The smooth fit was better than the linear fit for Attention, Working Memory, Average Speed, and Efficiency . FurtheThe original, cross-sectional analysis of the WebCNP composite scores noted that sex differences had smaller effect sizes than age but were evident, with female participants outperforming their male counterparts on attention, word and face memory, reasoning speed, and all social cognition tests, whereas male participants outperformed their female counterparts in spatial processing and sensorimotor and motor speed . ComportCognition, which is based on the Web CNP, Basner and colleagues [To quantify practice effects associated with subtests of the computer-based test battery lleagues  varied tlearning ranged from 14% for Emotion to more than 100% for the two Memory composites between test sessions 1 and 2  design are poised to do so. Had our method not dissociated the effects of learning from development, the course of developmental changes over the adolescent years would have been interpreted as following an accelerating increase, notable in younger ages. By contrast, removing the learning effects revealed a linear developmental trajectory for all accuracy composite scores for all cognitive functions examined. Recognition of the substantial influence of prior testing experience, which does not necessarily rely on repetition and memory for specific test items  and can


There are few treatments shown to slow disability progression in progressive multiple sclerosis (PMS). One challenge has been efficiently testing the pipeline of candidate therapies from preclinical studies in clinical trials. Multi-arm multistage (MAMS) platform trials may accelerate evaluation of new therapies compared to traditional sequential clinical trials. We describe a MAMS design in PMS focusing on selection of interim and final outcome measures, sample size, and statistical considerations. The UK MS Society Expert Consortium for Progression in MS Clinical Trials reviewed recent phase II and III PMS trials to inform interim and final outcome selection and design measures. Simulations were performed to evaluate trial operating characteristics under different treatment effect, recruitment rate, and sample size assumptions. People with MS formed a patient and public involvement group and contributed to the trial design, ensuring it would meet the needs of the MS community. The proposed design evaluates 3 experimental arms compared to a common standard of care arm in 2 stages. Stage 1 (interim) outcome will be whole brain atrophy on MRI at 18 months, assessed for 123 participants per arm. Treatments with sufficient evidence for slowing brain atrophy will continue to the second stage. The stage 2  outcome will be time to 6-month confirmed disability progression, based on a composite clinical score comprising the Expanded Disability Status Scale, Timed 25-Foot Walk test, and 9-Hole Peg Test. To detect a hazard ratio of 0.75 for this primary final outcome with 90% power, 600 participants per arm are required. Assuming one treatment progresses to stage 2, the trial will recruit ≈1,900 participants and last ≈6 years. This is approximately two-thirds the size and half the time of separate 2-arm phase II and III trials. The proposed MAMS trial design will substantially reduce duration and sample size compared to traditional clinical trials, accelerating discovery of effective treatments for PMS. The design was well-received by people with multiple sclerosis. The practical and statistical principles of MAMS trial design may be applicable to other neurodegenerative conditions to facilitate efficient testing of new therapies. Compared to the predominantly inflammatory pathology in relapsing multiple sclerosis (MS) targeted by current treatments, the neurodegenerative processes driving progression in PMS are complex and less well-defined.6 There is a pipeline of candidate therapies from preclinical studies, but the challenge is testing them efficiently in clinical trials with appropriate outcome measures to determine whether they can successfully slow disability progression.Progressive multiple sclerosis (PMS) is a significant health problem worldwide7 and infectious diseases.8 They are also increasingly being considered for neurologic conditions such as Alzheimer disease,9 Parkinson disease10 and motor neuron disease.12 These neurodegenerative conditions share commonalities with PMS, where there is a marked translational gap between the relative abundance of early phase trials stemming from increased understanding of disease pathobiology and lack of positive phase III trials leading to disease-modifying treatments.One potential avenue is improving efficiency of trials by incorporating adaptive elements in a multi-arm multistage (MAMS) platform design. MAMS trials aim to evaluate multiple experimental arms and seamlessly integrate traditional phase II and III evaluations into a single trial. They have been successful in accelerating evaluation of therapies and changing practice in other disease settings, such as cancer13  group throughout the development process. The work of the treatment selection working group on identifying and shortlisting candidate treatments, focusing on licensed drugs that can be repurposed, has been reported elsewhere.This article describes the work of the trial design and outcome measures working groups. We discuss key elements of the MAMS trial design based on evaluation of 3 candidate treatments against standard of care in 2 analysis stages, including selection of the primary interim and final outcomes, sample size, and other statistical considerations.The outcome measures working group comprised individuals with expertise in MS trials, imaging, and biomarkers, as well as people with MS with lived experience of the condition. The group reviewed the literature to determine outcome measures relevant to a MAMS trial evaluating predominantly neuroprotective treatments. Individual members submitted proposed outcome measures based on their expertise with final prioritization of outcomes determined in consensus meetings.15The MAMS design allows distinct interim (stage 1) and final (stage 2) outcomes. The final outcome should be clinically derived and relevant to patients and regulators. The interim outcome serves as an early indicator of whether a treatment is likely to be effective and hence should be continued into the second stage of the trial while minimizing the likelihood of ceasing truly effective treatments. It should reflect the underlying association between the treatment and the clinical outcome. The absence of effect on the interim outcome should be indicative of the absence of effect on the final outcome, although the converse may not necessarily hold.The trial design working group, comprising experts in design and implementation of MAMS trials, statisticians, and MS clinicians, was tasked with generating design options for running an efficient, scalable, and flexible clinical trial by exploring different scenarios to determine the best design type. The group reviewed data from phase II and III randomized controlled PMS trials from January 1, 2009, to January 1, 2019, to inform key design measures for both stage 1 and 2 analyses, such as effect size, and the relationship between the interim and final outcomes.links.lww.com/WNL/B900). To support the design of the stage 1 analysis, we analyzed brain atrophy data from the MS-STAT116 and ASCEND17 clinical trials . Modeling was conducted in Stata version 15 (StataCorp) and Microsoft Excel 2016.To assess the statistical operating characteristics of the trial , we simulated multiple trials with different correlation structures for the treatment effects on stage 1 and stage 2 outcomes, under different treatment effect assumptions. We also modeled the expected trial progress over time based on different assumptions (such as recruitment rates) and design parameters (such as sample size and treatment stopping rules) to the second stage. Further details of the simulation methods are reported in eAppendix 1 and 2  and the MS Society Public Involvement Officer. They contributed to discussions as the project developed and focused on ensuring that the research would meet the needs of the MS community. Additional workshops attended by a total of 34 people with MS held in 3 UK locations brought in further expertise of people with MS on topics including relevance, feasibility, and acceptability of all aspects of the trial design as well as recruitment and engagement strategies.Data not published in this article will be made available by request from any qualified investigator.Our review identified 15 eligible phase II (n = 8) and phase III (n = 7) randomized trials in PMS . The medWe propose the design of a 2-stage MAMS trial in PMS: 1 interim analysis to examine early evidence of treatment effect (stage 1) and 1 final confirmatory analysis of efficacy (stage 2). The trial would include 4 arms in stage 1: 1 standard of care (control) arm and 3 experimental arms. Any treatment that is sufficiently promising at the interim analysis will progress to stage 2, which will continue until the required number of events is reached, as represented in It is expected that in the initial phase of the trial the standard of care arm for most participants will comprise best supportive care. Whereas ocrelizumab and siponimod have been approved for PMS, these treatments are not currently available to or suitable for all patients, in particular nonambulatory patients, who would be eligible for this proposed trial. If an efficacious therapy is subsequently found, this would then become the standard of care for future participants entering the platform.14 Participants will be randomized with an equal probability between each of the 4 arms (1:1:1:1 ratio). In a standard multi-arm trial with n experimental arms, the optimal allocation ratio would be 18 Unequal allocation would also make the trial less attractive to people with MS, as it results in a lower likelihood of being randomized to an experimental arm.The number of experimental arms was informed by feasibility constraints and the treatment selection group's work on number of repurposed therapies ready for clinical testing.19 used to determine the time to disability progression. Its strengths and limitations are well-documented20 and numerous attempts have been made to evolve it, including using a composite measure based on progression in 1 or more of 3 endpoints: (1) increase in EDSS (of ≥1 point if baseline EDSS was <5.5 or ≥0.5 points if baseline EDSS was ≥5.5), (2) ≥20% increase in 9-Hole Peg Test (9HPT), or (3) ≥20% increase in Timed 25-Foot Walk (T25FW) (if ambulant).21The classic measurement tool and regulatory standard has been the EDSS,links.lww.com/WNL/B900), which can reduce trial duration and sample size. For example, in the INFORMS phase III trial of fingolimod in PPMS, >70% of participants had reached progression on the 3-month confirmed disability composite outcome by 3 years, as opposed to 50% based on EDSS alone.22 Inclusion of a measure of upper limb function also addresses the PPI group's interest in expanding the traditionally narrow EDSS inclusion criteria to include patients with higher levels of disability, to whom arm function is critical and measures of ambulation less relevant.23Composite measures achieve higher event rates than single measures , we expect the rate of 6-month confirmed composite disability progression to be around 50% at 3 years.Based on these considerations, we selected time to 6-month confirmed composite disability progression as the primary outcome for the final (stage 2) analysis. The composite outcome will be measured at baseline and every 6 months until the end of the follow-up. The time to progression will be from randomization until date of the initial disability progression (if subsequently confirmed). Based on earlier trials  trials.24Whole brain atrophy on MRI, measured as annualized percentage of brain volume change (PBVC), was selected as the primary interim outcome, based on the initial candidate drugs having primarily neuroprotective mechanisms of action. Brain atrophy reflects underlying neuroaxonal loss, which contributes to accrual of disability in PMS, and has been successfully used as a primary outcome in phase II trials, including MS-STAT1,26 Some therapies, particularly those with anti-inflammatory effects, can excessively reduce brain volume in the first months (pseudoatrophy),27 so it was recommended to assess PBVC also after at least 6 months on treatment.Methods of measuring PBVC include registration-based techniques  or brain parenchymal fraction, which quantify the amount of brain tissue contained within a contour surrounding the entire brain including CSF.28 neurite indices derived from diffusion MRI, which reflect the microstructural changes of axons and dendrites, and magnetization transfer imaging, which reflects demyelination and axonal loss.19 However, technical challenges limit widespread implementation and standardization across multiple centers.27 Although biofluid markers such as neurofilament light chain are associated in high concentrations with disability and brain atrophy,29 there are mixed findings on whether they are sensitive to treatment and they are not ready to be used as primary outcome measures until a validated, standardized, and widely accessible assay is available, with normative values of neurofilaments across age groups. Moreover, there is divergence of their utility in relapsing MS compared to PMS.31We considered other imaging-based measures, including spinal cord atrophy, which contributes to MS disability progression and occurs at a faster rate than brain atrophy,links.lww.com/WNL/B900). Brain atrophy rate varied between 0.4%/year and 0.7%/year in control arms. There was no clear pattern of differences between trials in PPMS or SPMS or by follow-up length. The SD for atrophy rate decreased with increasing follow-up length, ranging from 0.59%/year to 0.78%/year over 1 year, and 0.37%/year to 0.60%/year over 2 years.Nine of the reviewed trials reported a direct measure of change in whole brain volume  to the data from MS-STAT1 and ASCEND are shown in The predicted SDs based on applying our statistical model . The SD at this point is predicted to be around 0.55%/year.Timing of the interim analysis is an important consideration of adaptive designs. It should occur after accruing sufficient participant data to make a reliable decision on continuing or dropping treatment arms, but early enough relative to total trial recruitment to have value in informing adaptation of the trial design.links.lww.com/WNL/B900) and summarized in 26A key criterion for the stage 1 outcome is the ability to identify treatments expected to be ineffective and also potentially effective in terms of the final (stage 2) outcome. We reviewed trials reporting treatment effect on both brain atrophy rate and clinical progression. Trial results are reported in eAppendix 2 .Our trial targets a treatment effect of 25% relative reduction in the 6-month confirmed disability progression rate, that is, a hazard ratio of 0.75. This is a clinically important effect in slowing progression in ambulation, upper limb function, or disability, which has been achieved in previous trials.Based on the review of previous trials, we assumed an effective treatment would reduce the rate of whole brain atrophy by around 0.15%/year, from 0.55%/year to 0.40%/year.15 but differs from the 5% commonly used in confirmatory analysis, as the objectives here are different. Assuming an SD of 0.55%/year (see above), 111 observations per arm will allow 95% power to detect a 0.15%/year difference at a 1-sided significance level  of 35%. Allowing for 10% drop-out, 123 participants are needed per arm.The sample size for stage 1 analysis was based on pairwise comparisons of whole brain atrophy rate at 18 months between each intervention arm and standard of care. A 1-sided test is used for the interim analysis, with a treatment continuing to the second stage if there is evidence in favor of a lower atrophy rate compared to standard of care. We chose 95% power because a priority of the interim analysis is to minimize the chance of stopping an arm when the treatment is genuinely active in slowing brain atrophy . Stage 1 alpha (type I error rate) captures the probability of an ineffective treatment to continue to the second stage. It should be chosen to balance minimizing this risk while ensuring the timeliness of the interim analysis. Designs were considered with stage 1 alpha between 20% and 50% with the final choice of 35% representing an achievable sample size and timely interim analysis . This is in line with other MAMS trials,p value is below 0.35, then the treatment arm is continued into stage 2.Therefore, we recommended that stage 1 analysis be conducted once 18 months' brain atrophy data are available for 111 participants per arm, with pairwise comparison for each experimental arm compared to the control arm. If the 1-sided The sample size for the stage 2 analysis was based on comparing the time to confirmed disability progression between each intervention arm to standard of care. For each pairwise comparison, to have 90% stage 2 power to detect a hazard ratio of 0.75 at the 2-sided stage 2 significance level of 5% , 281 progression events are required in the control arm and 600 participants per arm are needed.18 We aimed to select drugs with different mechanisms, which might be viewed as independent evaluations, similar to multiple trials being conducted,34 and therefore did not apply any correction for multiple comparisons. If drugs of similar action are selected , an appropriate correction  should probably be applied. The statistical power in a time-to-event analysis is determined by the number of events. Recruiting 600 participants per arm should be sufficient to observe the required 281 progression events in the control arm in a timely manner. This number of events is anticipated to occur around 18 months after the last participant has been enrolled, assuming a 10% drop-out rate and 50% disability progression rate by 3 years and recruitment rate, as described in eAppendix 4 (links.lww.com/WNL/B900) .The stage 2 significance level was set at a 2-sided 5% level, as in standard confirmatory trials, corresponding to a 1-sided level of 0.025. The question of multiplicity (adjusting significance level due to multiple comparisons) has been discussed before in MAMS.links.lww.com/WNL/B900, for Methods and full Results). We conducted simulations to assess the operating characteristics of the proposed trial design under different scenarios  describes the assumptions made and how the timeline was modeled. Results are summarized in An important consideration in adaptive trials is to anticipate the possible dynamics of the trial over time, including the relative timing of the interim and final analyses. eAppendix 4 .The proposed MAMS design leads to efficiencies in both sample size and trial duration compared to traditional separate phase II and III trials of single treatments. A single control arm is used to assess multiple experimental arms and participants recruited in stage 1 seamlessly continue to be included in the stage 2 analysis without additional setup time in between. This trial is expected to last 6–7 years with 1,900 participants, encompassing the stage 1 and 2 evaluations of 3 initial candidate treatments (16 and phase III (MS-STAT2)36 trials. Recruitment to MS-STAT started in 2011 and MS-STAT2 is expected to be completed by 2025, which corresponds to 14 years overall.In comparison, under the traditional approach, around 630 patients would be required in each of 3 phase II studies to have 90% power with 5% type I error under the same assumptions. If one of these treatments was found to be effective and proceeded to phase III, 1,200 additional participants would be required, totaling 3,090 participants. Separate phase II and III trials of a single treatment would be expected to take more than 10 years, with 3–5 years for phase II, 5–7 years for phase III, and additional setup time between the two . For exaPlanning and setting up a MAMS adaptive platform trial is considerably more complex than standard phase II and III trials and may take up to 12 to 18 months. In particular, statistical simulations examining different design options, scenarios, and parameters are essential to optimize efficiency and select appropriate trial operating characteristics while preserving the overall integrity of the trial. The initial modest investment in time and resources will be further offset by shorter subsequent setup times for further treatments added to the platform.7 and infectious diseases8 and our experience in these other disease areas suggests that regulators are becoming more open to, and knowledgeable and welcoming about, such designs.As adaptive platform designs are relatively novel in neurodegenerative diseases, there is a perception that regulatory agencies may not immediately accept them as equal to more conventional phase III studies. However, a precedent has been set for regulatory approval of MAMS platform trials in settings such as oncologyThe PPI group actively participated in the entire trial design process, as well as treatment and outcome measure selection, to ensure the needs of people with MS were being met. For example, it was important, particularly for nonambulatory people with MS, to include an assessment of upper limb function in the primary efficacy endpoint and proposed secondary outcomes included patient-reported outcome measures of key symptoms such as fatigue. Feedback indicated the trial design was well-received and acceptable, despite being more complex. Perceived advantages included the ability to evaluate multiple candidate treatments and the relatively lower likelihood of randomization to placebo. If a participants' treatment arm is discontinued after interim analysis, there is the potential opportunity to re-enter the trial in a continuing arm or future trials in the platform. Some participants expressed concern about the total trial duration, but this was offset by the favorable consensus overall regarding potential acceleration of treatment discovery.37 A program grant proposal based on activity of all working groups of the UK MS Society's Expert Consortium for Progression in MS was submitted to the UK MS Society in November 2019 and received favorable international peer and lay review. Funding has been awarded to develop the protocol and deliver the first active arms plus standard of care in the MAMS trial platform, with recruitment expected to commence in 2022. Whereas this article focuses on evaluation of only the first 3 candidate therapies, the adaptive MAMS platform will allow addition of new treatment arms7 and re-randomization of participants from discontinued arms in the future.38 Drugs with predominantly remyelinating potential will likely require additional and alternative endpoints at the interim analysis stage. Further aspects of the trial protocol, for example secondary and exploratory outcomes and recruitment infrastructure, are beyond the scope of this article.This article is based on work conducted by the trial design working group and in many senses is an evolution from our work carried out a decade ago.39 However, these conditions face similar challenges of efficiently translating candidate drugs into effective treatments with many disappointing phase III clinical trial results to date. Various reasons for this have been proposed, including the need to improve trial design.40Like PMS, conditions such as Parkinson disease, Alzheimer disease, and motor neuron disease are increasing in prevalence, have significant impact on patients, carers, and health care systems, and have no or few therapies that slow or prevent progression. An improved understanding of disease pathophysiology in recent years has led to a growing pipeline of potential therapeutics. For example, a 2020 review identified 121 agents in 136 phase I to III clinical trials for Alzheimer disease, with an increasing number of disease modification treatment candidates over the past 5 years.11 The Dominantly Inherited Alzheimer's Network Trials Unit (DIAN-TU) platform trial established in 2012 was a multi-arm trial of 2 anti-amyloid monoclonal antibodies. Although neither drug met the primary cognitive endpoint,41 lessons learned, including refinements in participant and outcome measure selection and trial duration, have led to several emerging platform trials, such as the AHEAD study evaluating different doses of an anti-Aβ monoclonal antibody in 2 phase III clinical trials that respectively use amyloid PET and cognitive testing as the primary outcome measures.9MAMS designs are particularly relevant when there are multiple candidate therapies to be trialed and when a reliable early marker of clinical efficacy is available. MAMS adaptive platform trials have been planned and initiated to accelerate successful drug discovery in these disorders. The Motor Neuron Disease–Systematic Multi-arm Adaptive Randomization Trial (MND-SMART) will initially test 2 repurposed drugs against a common placebo.The principles of designing a PMS MAMS trial as outlined in this article are relevant to other neurodegenerative conditions, but each condition will present unique considerations and challenges, including selection of biologically and clinically relevant, sensitive, and timely interim and final outcome measures, determination of the most appropriate patient population for inclusion, and trial duration required to detect a meaningful effect.We propose a design for a MAMS trial in PMS for evaluation of 3 repurposed neuroprotective drugs compared to standard of care. Although more complex in design, efficiencies in participant numbers and trial duration, as well as the ability to incorporate adaptive elements and continually test newly identified treatments through an ongoing platform, make this approach more likely to succeed in finding effective therapies that target disability progression in PMS in a timely manner.


Neurospora crassa, we show that molecular timekeeping is robust even under severe limitation of carbon sources, however, stoichiometry, phosphorylation and subcellular distribution of the key clock components display drastic alterations. Protein kinase A, protein phosphatase 2 A and glycogen synthase kinase are involved in the molecular reorganization of the clock. RNA-seq analysis reveals that the transcriptomic response of metabolism to starvation is highly dependent on the positive clock component WC-1. Moreover, our molecular and phenotypic data indicate that a functional clock facilitates recovery from starvation. We suggest that the molecular clock is a flexible network that allows the organism to maintain rhythmic physiology and preserve fitness even under long-term nutritional stress.The circadian clock governs rhythmic cellular functions by driving the expression of a substantial fraction of the genome and thereby significantly contributes to the adaptation to changing environmental conditions. Using the circadian model organism  Circadian clocks rely on transcriptional-translational feedback loops (TTFL) in which positive elements of the machinery activate the expression of oscillator proteins which in turn negatively feed back on their own transcription. Circadian clocks are closely linked to metabolism. On the one hand, the clock rhythmically modulates many metabolic pathways  composed of the GATA-type transcription factors WC-1 and WC-2, and Frequency (FRQ) represent the core components of the circadian clock. The WCC supports expression of FRQ which then interacts with an RNA helicase (FRH) and the casein kinase 1a (CK1a)  glucose deprivation triggers compensatory mechanisms at the transcriptional and posttranscriptional levels that maintain expression levels of the core clock proteins, thereby keeping period length constant  . The FRQa (CK1a) . The negq levels . The negq levels . Similare genome . The WCChe clock . In Neurconstant .wt and the clock-less mutant Δwc-1 and found that the WCC has a striking impact on nutrient-dependent expression of a large set of genes, including enzymes and regulators of carbohydrate, amino acid, and fatty acid metabolism. We show that molecular timekeeping is robust even under severe limitation of carbon sources. Moreover, our data provide evidence that the TTFL is able to function in a wide range of stoichiometric conditions of its key elements, dependent on glucose availability. Our results show that Neurospora recovers faster from starvation in the presence of a functioning clock, suggesting a significant impact of the circadian clock on organismal fitness.Aim of this study was to characterize how chronic glucose deprivation affects the molecular clock and what role the circadian clock plays in the adaptation to starvation. We analyzed the transcriptome response to long-term glucose starvation in wt Neurospora grown at 2% glucose (standard medium) were transferred to a starvation medium with 0.01% glucose for 40 hr. Cultures were kept in constant light (LL). In LL, both the negative and the positive feedback persist, however, the circadian oscillation stops and both RNA and protein levels of the core clock components are at steady state , when transcription of frq is repressed, and therefore changes in frq levels reflect RNA degradation. frq RNA levels after LD transition were similar under both culture conditions, suggesting that changes in RNA stability do not contribute to the maintenance of frq levels during starvation , an unknown mechanism must recalibrate the central clockwork to keep frq transcript levels and oscillation glucose-compensated despite the decline in WCC levels. To examine clock output function, we measured RNA levels of two clock-controlled genes, ccg-2 and fluffy  transfer under both nutrient conditions. The light-induced initial increase of RNA levels was lower in starved than non-starved cultures, whereas the steady state expression levels after light adaptation were similar under both conditions  in the two strains upon glucose withdrawal. The different behavior of frq promoter activity in frq9 and wt in response to glucose suggests that FRQ-mediated processes contribute to the compensation of frq levels at different glucose availability. Amount of WC-1 did not change significantly in frq9, whereas WC-2 levels were moderately reduced in response to glucose depletion   and FCD2impaired . Upon glNeurospora and yeast when glucose is limited -inducible qa-2 promoter  is an important factor of the starvation response in many organisms including yeast  and was a crassa . As GSK promoter . We perffindings , in the  . Under these conditions, the clock is entrained in wt, and at ZT 12 an adapted state for light-dependent genes can be expected as it was shown for frq RNA  enrichment tool , we founrocesses . Furtherarvation . In analarvation –6. Howeved to wt . Among co wt , the conserved mitochondrial gene tca-3 (NCU02366), encoding a putative aconitase that catalyzes the citrate-isocitrate transition, and the gene of choline dehydrogenase  promoting the production of glycin-betain from choline, and thereby participating in amino acid metabolism.To validate RNA-seq data, we performed qRT-PCR measurements for genes involved in the metabolism of carbohydrates or amino acids as well as for genes important for conidiation, the well-characterized nutrient-dependent process in fungi . All teswt and ∆wc-1 in the adaptation to glucose limitation, we hypothesized that starved wt might be better prepared to growth regeneration and could respond more effectively to the resupply of glucose than clock deficient strains. wt and the clock mutants 10frq and ∆wc-1 were incubated in light-dark cycles and following the 40 hr starvation the medium was supplemented with 2% glucose  to synchronize the clock with the changing environment .Plasmids and strains generated in this study are available upon request from the lead contact.Neurospora crassa strains wt (FGSC #2489), wt,bd (FGSC #1858), 10bd;frq (FGSC #7490), 9bd;frq (FGSC #7779), rgb-1 (FGSC #8380) and mcb (FGSC #7094) were obtained from the Fungal Genetics Stock Center .Extraction of rn blots  and subcrn blots  was perfRace tubes containing standard race tube medium  with 2% wt grown in 2% glucose containing standard medium in each experiment unless indicated otherwise. As levels of previously used housekeeping gene actin decreased under starvation conditions, additional genes were selected based on literature data  isolation reagent and transcript levels were quantified as described earlier . Values ure data . We founC spectrophotometer, the Qubit 4.0 Fluorometer (Invitrogen) and the Agilent TapeStation 4150 System.Liquid cultures were grown under standard and glucose starving conditions in 12–12 hr light-dark cycles for 48 hr and samples were harvested at ZT12 (n=4 for each group). RNA was purified using the TriReagent (Sigma Aldrich #93289) isolation reagent. Following DNase treatment RNA quality was controlled by the Nanodrop OneN.c. genome from Ensembl (Neurospora_crassa.NC12.48) (https://doi.org/10.5061/dryad.t4b8gtj4p).Library preparation and sequencing were performed by BGI Genomics, China, using PE-100 library. Sequencing quality check was performed with FastQC . MappingNC12.48) , and theNC12.48) . Read coNC12.48) . DiffereNC12.48)  package NC12.48) .  according to the manufacturer’s instructions. Each reaction contained 10 μg protein and was performed for 20 min at room temperature. Absorbance was measured at 600 nm.For statistical analysis, the Statistica 13  software was used. Error bars indicate ± SEM. Results were considered to be statistically significant when p value was<0.05.  Further statistical details can be found in the figure legends. N represents the number of independent biological samples. frq are affected by starvation, indicating a mechanism that recalibrates the central clockwork. Furthermore, Neurospora recovers faster from starvation in the presence of a functioning clock, adding further evidence for the importance of the circadian clock for organismal fitness.This manuscript is of interest to researchers working in the areas of chronobiology, metabolism, or environmental adaptation mechanisms. The authors show that starvation decreases the abundance of the fungal circadian clock component white collar complex (WCC). However, neither phase nor the amplitude of the RNA oscillation of the critical circadian clock gene  public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:Neurospora crassa" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Jonathan Cooper as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Adaptation to starvation requires a flexible circadian clockwork in The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.As you can also see from the individual reviews below, our opinion is that the conclusions that could be drawn from the experiments differ substantially from the conclusions currently presented in the manuscript.In our opinion the key messages are:a) WC-1,2 are required for the adaptation to starvation in Neurospora and provide a potential link between metabolism, the circadian clock, and light. However, which of the different roles of the WCC is required for this remains unclear. It could be the light receptor, circadian clock, or a light/clock independent transcriptional regulation function.Here the authors should also reconcile the somewhat contradictory findings that starvation leads to strong decreases in WCC levels, but the transcriptional and growth response to starvation requires WCC.b) The severe downregulation of WC-1 and WC-2 protein levels and WCC transcriptional activity by starvation, but the simultaneous lack of effects on frq RNA and protein levels is not explainable by the conventional TTL model of the Neurospora circadian clock.eLife's "Ideas and Speculation" subsection to hypothesize how this could work.) scientifically interesting enough to potentially merit publication in As essential revisions, we thus ask the authors to carefully re-inspect their results and avoid biased interpretations or overinterpretations. Consider another title that is more representative of the results presented in the manuscript.Besides the re-interpretation of the data, another essential revision is that the data in Figure 2 must be analyzed quantitatively, as those are critical for the manuscript's conclusions.Please also carefully consider the major comments made below. As the requested revisions are substantial the revised manuscript will be sent for re-review.Reviewer #1 (Recommendations for the authors):The text is well-written, with very few typos or grammatical errors.Page 4 Line 10: The reference to Adhvaryu does not seem appropriate to support the statement it is attached to.P5 L1-2: Is there a source for the claim that constant light results in steady-state levels of frq RNA and protein?P7 L 4 and Figure S2: The fluffy data are not convincing. There is only one cycle assayed and the levels simply decline. At least 2 cycles are needed for a convincing rhythm.P7 L17 and Figure 3A: The finding of no change in WC-1 levels in frq9 on starvation is not convincing. Levels of WC-1 are already very low in frq9 on high glucose and the finding that they do not drop further on starvation may simply indicate that a constitutive background level of WC-1 expression is all that is present in frq9 and it can't drop lower on starvation.P12 L5: Low growth rate of frq10 on a solid medium is not the usual phenotype – frq10 grows as fast as, or faster than, wild type. I would be very suspicious of the genotype of this strain if it does not grow well on normal solid agar. If it grew well on a normal medium but did not grow after starvation, that is an observation worth reporting.P16 L2-10: Correct nomenclature for multiple mutant strains should be used, with mutations in order of linkage group and semicolons between linkage groups .P16 L7-9: Is it necessary to thank an author of the paper for providing strains?P17 L9-10: How did you "find gna-3 to be the least variable"? Did you test this yourself or use the published data?P32 L19 and 21: Left panel and Right panel should be Upper and Lower panels?P36 Figure 5C: This experiment is missing an important control. What would the growth of these strains be without any starvation? Without this control, we don't know if the "recovery" growth is the same as growth in high glucose without prior starvation.P36 Figure 5D: The race tube method is unclear. The agar medium is not specified. How many days of growth are we looking at on the race tubes? The Y axes of the graphs should use the same scale, starting at zero and ending at the same value.P37 L16: Are the race tubes really 0.2% glucose and not 2%?P41 L3: Does this journal have STAR methods?P48 and P52 and P54, Suppl Table 2 and 3 and 4: There doesn't seem to be a supplemental table 1.Reviewer #2 (Recommendations for the authors):More clarity could be achieved by describing the logic for the different experimental protocols and conditions. I think much of my confusion came from the order of the Figures, might not Figure 2 be better first, describing the effect of starvation on clock function, before Figure 1 which I a fairly detailed investigation of the effect on light induction?Page 9 "However, while starvation did not alter frq levels in wt, it decreased the amount of frq9 RNA . This suggests that FRQ contributes to both WCC depletion and FRQ level compensation." I really did not understand this, do you mean that because the RNA did not change it must be something to do with the protein? Is not an alternative explanation that FRQ is not involved in the starvation response and the changes in WCC are due to another system? This needs to be clarified. My interpretation of the data is that FRQ is not involved in the changes in WCC in response to starvation.Page 9 "As shown in Figure 3A, absence of a 17 functional FRQ protein attenuated the effects of starvation, i.e. amount of WC-1 did not change in response to glucose deprivation, whereas WC-2 levels were moderately reduced." Isn't likely that a reduction in WC1 and WC2 levels could not be measured in response to starvation because they are both already so low in frq9 mutant? Again I feel that the evidence that FRQ participates in the starvation of WC abundance is not compelling.Please provide a more detailed analysis of the transcriptional responses providing an indication of the regulatory pathway, ie is it direct from WCC to the regulation of the transcripts, or are there intermediatory steps?I think the Discussion would be improved by a comparison of the effects of glucose on the Neurospora clock with the effects of glucose found in mice and Arabidopsis.As far as I understand there are three main conclusions, starvation reduces WCC, the clock is able to run despite the uncoupling of WCC from FRQ, and that WCC is required for the full starvation response. However, the Discussion section lacks an explanation of the apparent contradiction of sucrose decreasing WCC, but WCC is needed for the transcriptional response. Please can you address this? Reviewer #1 (Recommendations for the authors):The text is well-written, with very few typos or grammatical errors.Page 4 Line 10: The reference to Adhvaryu does not seem appropriate to support the statement it is attached to.We thank you for the correction and removed the reference.P5 L1-2: Is there a source for the claim that constant light results in steady-state levels of frq RNA and protein?We added the following references to the text: Crosthwaite et al., 1995, Cell; Elvin et al., 2005, GD. Crosthwaite et al. showed that frq RNA stops oscillating in constant light. In Elvin et al. both RNA and protein levels were analyzed for 48 hours in LL following a dark-light transfer. frq RNA did not display further changes after 1-4 hours in light, whereas FRQ protein levels gradually increased during the first 8-12 hours and remained high for the rest of the detection period.P7 L 4 and Figure S2: The fluffy data are not convincing. There is only one cycle assayed and the levels simply decline. At least 2 cycles are needed for a convincing rhythm.In the revised version of the manuscript fluffy expression is shown for the same period as the expression of frq and ccg-2. We added a reference  regarding the expected time points of peak and trough of fluffy expression.P7 L17 and Figure 3A: The finding of no change in WC-1 levels in frq9 on starvation is not convincing. Levels of WC-1 are already very low in frq9 on high glucose and the finding that they do not drop further on starvation may simply indicate that a constitutive background level of WC-1 expression is all that is present in frq9 and it can't drop lower on starvation.9 (even in the high glucose medium) makes it difficult to compare the glucose dependence of WCC expression in frq9 and wt. The conclusion has been modified and the new version of the text focuses mainly on the strain-dependent difference in the changes of frq RNA expression .We agree with the reviewer that the low expression of the WCC in frqP12 L5: Low growth rate of frq10 on a solid medium is not the usual phenotype – frq10 grows as fast as, or faster than, wild type. I would be very suspicious of the genotype of this strain if it does not grow well on normal solid agar. If it grew well on a normal medium but did not grow after starvation, that is an observation worth reporting.10 was largely variable on solid medium in our first experiments, especially at the beginning of the race tubes, therefore we disregarded their evaluation. For the revised version we repeated the experiment with a larger sample size (n=12) and focused on shorter (6-hour) periods at the beginning of the tube. Results of this experiment are now shown in .We regret the inaccurate formulation in the former version of the manuscript. Growth rate of frq10 inoculated from non-starved cultures and found it similar to that of wt .We thank you for the comment and corrected the nomenclature in the Materials and methods.P16 L7-9: Is it necessary to thank an author of the paper for providing strains?We appreciate the comment and corrected the section in the Materials and methods accordingly.P17 L9-10: How did you "find gna-3 to be the least variable"? Did you test this yourself or use the published data?As starvation resulted in a decrease of actin expression even in wt, i.e. cycle times in real-time PCR changed characteristically, we looked for other possible housekeeping genes in the literature which were described as non-oscillating and least variable under different metabolic conditions . Tfc1, SarA and gna-3 were tested as possible reference genes. gna-3 was selected, as its levels remained practically constant despite changes in the glucose content of the medium ).t values are presented in Figure 1 – Source data 14. For sequences of oligonucleotides and hydrolysis probes see Key resources table.We completed this section in the Materials and methods with this information: "As levels of previously used housekeeping gene actin decreased under starvation conditions, additional genes were selected based on literature data . We found gna-3 to be the least variable under the applied experimental conditions. CP32 L19 and 21: Left panel and Right panel should be Upper and Lower panels?We thank you for the correction and changed the text accordingly.P36 Figure 5C: This experiment is missing an important control. What would the growth of these strains be without any starvation? Without this control, we don't know if the "recovery" growth is the same as growth in high glucose without prior starvation.We agree with the criticism and in the revised version of the manuscript we show the result of this control experiment: Non-starved control samples did not show strain-specific differences in the acceleration of growth after the transfer into fresh glucose-rich medium .P36 Figure 5D: The race tube method is unclear. The agar medium is not specified. How many days of growth are we looking at on the race tubes? The Y axes of the graphs should use the same scale, starting at zero and ending at the same value.10 was also included. The new diagram shows growth rates normalized to those of the non-starved samples (inoculated from non-starved cultures onto the race tubes and indicated with dashed line) of the same strain during the same period. In addition, a more detailed description of the experiment was added to "Figure 5 —figure supplement 1" and a subsection was added to Materials and methods.For the revised version of the manuscript we repeated the race tube measurements in a modified form allowing the analysis of growth rate for shorter (6 and 12-hour-) periods. The sample size was enlarged and frqP37 L16: Are the race tubes really 0.2% glucose and not 2%?The race tube medium in the experiments shown in the previous version of the manuscript contained 0.2 % glucose. This is the usual glucose concentration when the conidiation rhythm is analyzed. However, to achieve a more characteristic increase in the glucose level, in the experiments shown in the revised version we used race tubes with 2 % glucose.P41 L3: Does this journal have STAR methods?eLife.Thank you for the feedback, we amended the wording to ‘Materials and methods section, which is the correct title for this section in P48 and P52 and P54, Suppl Table 2 and 3 and 4: There doesn't seem to be a supplemental table 1.Because of the size limitation of the journal, Figure 4 – Source data 2 (in the previous version of the manuscript entitled as Supplementary Table 1) was uploaded as an excel file and shows data from the Gene Ontology enrichment analysis.Reviewer #2 (Recommendations for the authors):More clarity could be achieved by describing the logic for the different experimental protocols and conditions. I think much of my confusion came from the order of the Figures, might not Figure 2 be better first, describing the effect of starvation on clock function, before Figure 1 which I a fairly detailed investigation of the effect on light induction?To better justify the choice of different protocols , we integrated more explanation into the description of the experimental procedures in the Results section (e.g. P5 L7-L10). Based on the suggestion of the reviewer we also changed the order of Figure panels. In the revised version Figure 1 shows expression of clock proteins and RNAs in both LL and DD, while Figure 2 summarizes the data concerning the light induction of frq, wc-1 and al-2 transcription and the subcellular localization of the clock components.Page 9 "However, while starvation did not alter frq levels in wt, it decreased the amount of frq9 RNA . This suggests that FRQ contributes to both WCC depletion and FRQ level compensation." I really did not understand this, do you mean that because the RNA did not change it must be something to do with the protein? Is not an alternative explanation that FRQ is not involved in the starvation response and the changes in WCC are due to another system? This needs to be clarified. My interpretation of the data is that FRQ is not involved in the changes in WCC in response to starvation.9 makes it difficult to compare the glucose-dependence of WCC expression in frq9 and wt. We modified the conclusion by adding this information and now mainly focus on the strain-dependent difference in the changes of frq RNA expression. (P7 L22-P8 L14)After rereading the criticized section, we admit that the text was not well structured and we carried out several modifications. We intended to emphasize that upon drastic changes of the glucose availability frq RNA levels remained compensated in wt, but this compensation was affected when functional FRQ was not present. We agree with the reviewer's opinion that the low expression of the WCC in frqPage 9 "As shown in Figure 3A, absence of a functional FRQ protein attenuated the effects of starvation, i.e. amount of WC-1 did not change in response to glucose deprivation, whereas WC-2 levels were moderately reduced." Isn't likely that a reduction in WC1 and WC2 levels could not be measured in response to starvation because they are both already so low in frq9 mutant? Again I feel that the evidence that FRQ participates in the starvation of WC abundance is not compelling.9.As mentioned in the previous answer, we agree with the concerns of the reviewer and in the criticized section we modified the conclusion (P8 L11-L14). We emphasize in the new version of this section that changes in frq RNA expression (frq promoter activity) are mainly different in wt and frqPlease provide a more detailed analysis of the transcriptional responses providing an indication of the regulatory pathway, ie is it direct from WCC to the regulation of the transcripts, or are there intermediatory steps?We now show the list of genes  that changed in a strain-specific manner in response to glucose starvation and, based on ChIP-seq results, were earlier described as direct targets of the WCC . Based on the literature data showing that the WCC affects the expression of several other transcription factors and controls basic cellular functions which might affect the expression of further genes, it was not surprising that only 90 out of the 1377 genes were reported to be direct targets of the WCC.I think the Discussion would be improved by a comparison of the effects of glucose on the Neurospora clock with the effects of glucose found in mice and Arabidopsis.We included a short section into the Discussion which gives a short overview about known interactions between glucose availability and circadian timekeeping in different organisms (P15 L18-P16 L7).As far as I understand there are three main conclusions, starvation reduces WCC, the clock is able to run despite the uncoupling of WCC from FRQ, and that WCC is required for the full starvation response. However, the Discussion section lacks an explanation of the apparent contradiction of sucrose decreasing WCC, but WCC is needed for the transcriptional response. Please can you address this?We agree with the reviewer that the problem of how low levels of WCC could sufficiently support the transcription of frq and different output genes under starvation conditions was not discussed properly. Our results suggest a model in which the maintained level of nuclear WCC and the weakened inhibition by both FRQ (the hyperphosphorylated form is less active in the negative feedback) and PKA (its activity lowered upon glucose depletion) together might ensure that transcriptional activity of the WCC is preserved upon glucose withdrawal in both DD and LL despite the decrease of the overall level of the complex. In the revised version these aspects are discussed more thoroughly (P16-18).


Pre-exposure prophylaxis (PrEP) is highly effective at preventing the acquisition of HIV. There is a substantial gap, however, between the number of people in the United States who have indications for PrEP and the number of them who are prescribed PrEP. Although Twitter content has been analyzed as a source of PrEP-related data , methods have not been developed to enable the use of Twitter as a platform for implementing PrEP-related interventions.Men who have sex with men (MSM) are the population most affected by HIV in the United States. Therefore, the objectives of this study were to (1) develop an automated natural language processing (NLP) pipeline for identifying men in the United States who have reported on Twitter that they are gay, bisexual, or MSM and (2) assess the extent to which they demographically represent MSM in the United States with new HIV diagnoses.Between September 2020 and January 2021, we used the Twitter Streaming Application Programming Interface (API) to collect more than 3 million tweets containing keywords that men may include in posts reporting that they are gay, bisexual, or MSM. We deployed handwritten, high-precision regular expressions—designed to filter out noise and identify actual self-reports—on the tweets and their user profile metadata. We identified 10,043 unique users geolocated in the United States and drew upon a validated NLP tool to automatically identify their ages.Ending the HIV Epidemic initiative. Furthermore, the age distribution of the users reflected that of MSM in the United States with new HIV diagnoses.By manually distinguishing true- and false-positive self-reports in the tweets or profiles of 1000 (10%) of the 10,043 users identified by our automated pipeline, we established that our pipeline has a precision of 0.85. Among the 8756 users for which a US state–level geolocation was detected, 5096 (58.2%) were in the 10 states with the highest numbers of new HIV diagnoses. Among the 6240 users for which a county-level geolocation was detected, 4252 (68.1%) were in counties or states considered priority jurisdictions by the Our automated NLP pipeline can be used to identify MSM in the United States who may be at risk of acquiring HIV, laying the groundwork for using Twitter on a large scale to directly target PrEP-related interventions at this population. Pre-exposure prophylaxis (PrEP) with antiretroviral drugs is highly effective at preventing the acquisition of HIV in men who have sex with men (MSM) . There iHannaford et al  found thThe Institutional Review Board of the University of Pennsylvania reviewed this study and deemed it exempt human subjects research under Category (4) of Paragraph (b) of the US Code of Federal Regulations Title 45 Section 46.101 for publicly available data sources (45 CFR §46.101(b)(4)).Between September 2020 and January 2021, we used the Twitter Streaming Application Programming Interface (API) to collect more than 3 million tweets containing keywords that men may include in posts reporting that they are gay, bisexual, or MSM. As a preliminary approach, we deployed handwritten, high-precision regular expressions—search patterns designed to automatically match text strings—on the 3 million tweets to filter out noise and identify actual self-reports . After aIn addition to tweet-based regular expressions, we also deployed handwritten regular expressions on the user profile metadata of the 3 million tweets collected from the Twitter Streaming API . The regTrue positives were defined as tweets or profiles in which the users reported that they are gay, bisexual, or MSM. Overall interannotator agreement (Cohen κ) based on independent, dual annotations for all 1000 users was 0.81, which is deemed to be “almost perfect agreement” [precision = true positives / . True positives and false positives were manually distinguished by 2 annotators in a random sample of 1000 (10%) of the 10,043 users that were identified by our automated pipeline, consisting of 500 matching tweets and 500 matching profiles. reement” . More spTo assess the utility of our automated pipeline for identifying MSM in the United States who may be particularly at risk of acquiring HIV, we analyzed their state- and county-level geolocations and ages. We detected a US state–level geolocation for 8756 (87.6%) of the 10,043 users identified by our automated pipeline, including users from all 50 states and the District of Columbia. As Ending the HIV Epidemic initiative [Our study demonstrates that gay men, bisexual men, or MSM in the United States publicly report their sexual orientation on Twitter and that these users can be accurately identified on a large scale. Moreover, among the 8756 users for which our automated pipeline detected a US state–level geolocation, 5096 (58.2%) were in the 10 states with the highest numbers of new HIV diagnoses [itiative . FurtherThis paper presented an automated NLP pipeline that can be used to identify MSM in the United States who may be at risk of acquiring HIV, laying the groundwork for using Twitter on a large scale to directly target PrEP-related interventions at this population.