Tear break up time and Schirmer I test were not correlated with OSDI. Significant albeit weak correlations were found between TFT and TFBUT  as well as Schirmer I . Subgroup analysis revealed that the correlation was stronger in the subjects with abnormal Schirmer I . Conclusions: The present study demonstrates an objective measurement of TFT using a novel OCT approach for DED that correlates with symptoms and signs of DED. Our data are consistent with the idea that TFT represents the aqueous-deficient component of DED.Purpose: To determine the association between tear film thickness (TFT) as measured with ultra-high resolution optical coherence tomography (UHR-OCT) and signs and symptoms of dry eye disease (DED). Methods: A total of 450 eyes from 225 patients with DED from six different randomized clinical trials were included in this pooled analysis. In all subjects, TFT was measured with a custom-built UHR-OCT system. Symptoms of DED were quantified using a standardized Ocular Surface Disease Index (OSD)I questionnaire and clinical signs including tear film break up time (TFBUT) and Schirmer I test were assessed. Associations of the average TFT with OSDI, TFBUT, and Schirmer I test were calculated using a linear regression analysis. Results: The average TFT of the included sample  was 4.2 ± 0.5 µm and the OSDI 36.2 ± 10.4. A significant negative correlation was found between TFT and OSDI (r = −0.36 to −0.31;  Dry eye disease (DED) is a highly prevalent ocular condition that is characterized by alterations in the homoeostasis of the precorneal tear film and the ocular surface ,2,3,4. AGiven that the total thickness of the human tear film is thin, ranging between 3 and 5 micrometers over the central cornea, quantification of tear film thickness (TFT) and its components requires highly sophisticated technical approaches . These iMore recently, optical coherence tomography (OCT) has been proposed to study TFT and tear film dynamics in vivo ,18,19. HUsing this system, we recently reported in a small cross-sectional study a correlation between TFT and symptoms of DED using the Ocular Surface Disease Index (OSDI) as well signs of DED such as tear film break-up-time (TFBUT) and tear osmolarity . These pThe aim of the present study was to extend these findings and to examine the relation between TFT with symptoms and signs of DED in a larger study population. We hypothesize that TFT, an objective measurement will be more correlated with symptoms of DED than current clinical tests. We also hypothesized that inter-eye differences between different signs of DED may show a better correlation with TFT than inter-subject differences.The present analysis includes pooled data from subjects with DED from six randomized clinical trials performed at the Department of Clinical Pharmacology in Vienna. The study protocols were approved by the Ethics Committee of the Medical University of Vienna  and were performed in adherence to the Declaration of Helsinki and the Good Clinical Practice (GCP) guidelines of the European Union. All subjects gave written informed consent before inclusion. In all these trials, the outcome was the effect of eye drops on TFT. Five of the studies have already been published ,26,27,28Main inclusion criteria were age over 18 years and signed and dated written informed consent. Patients had to have a history of the disease for at least 3 months, a TFBUT ≤10 s or Schirmer I test ≤10 mm/5 min and an OSDI score ≥13 points. Patients suffering from Sjögren’s syndrome or Stevens Johnson syndrome as well as patients not on stable concomitant medication that could have an influence on DED were excluded. Morever, intake of immune-suppressants was not allowed. All participating subjects did undergo a pre-study screening, which was scheduled in the 14 days before the baseline study day. This pre-study visit consisted of medical history, pregnancy test in women with childbearing potential, assessment of symptoms of DED using OSDI questionnaire, an ophthalmic examination including slit-lamp biomicroscopy and indirect funduscopy, visual acuity using the ETDRS acuity charts, measurement of TFBUT and Schirmer I test. All study participants had to abstain from administration of topical lubricants in the 24 h before the study day to avoid any undesirable influence on TFT.The OSDI  is a well-accepted questionnaire to assess the symptoms of DED . The OSD3  containing 512 × 256 × 1024 voxels. The recording of each volume takes 1 s resulting in a total measurements time of 3 s. The central TFT was calculated as the mean of the 15 horizontal frames above the central specular reflex that occurs at the apex of the cornea. The first volume was not used for data analysis, because TFT immediately after blinking shows considerable variability (unpublished observation). As such only volumes 2 and 3 were analyzed and the mean value obtained 1–3 s after blinking was used to calculate TFT. A typical ultra-high resolution OCT image is shown in A custom-built OCT system was used for TFT measurements that we have described in detail previously . As a liTear film break up time (TFBUT) testing is a clinical test used to assess for evaporative dry eye disease. We followed the guidelines published in the Report of the International Dry Eye Work Shop . For thiSchirmer I test without anesthesia was performed according to the protocol published in the Report of the International Dry Eye Work Shop  to evalup = 0.05. Statistical analysis was carried out using CSS Statistica for Windows® .For statistical analysis, both eyes were treated separately. Mean and standard deviation (SD) was calculated for each outcome parameter. The difference in TFT, TFBUT, and Schirmer was calculated between right and left eyes. The eyes were divided into those having normal TFBUT versus those having abnormal TFBUT (<10 s) as well as in those having normal Schirmer I versus those having abnormal Schirmer I (<15 mm). The association between the outcome variables was performed using linear regression analysis. All tests were performed at a significance level of n = 146; 65%). The severity of DED ranged from mild to moderate with an average OSDI of 36.2 ± 10.4. The mean age of the 225 patients with DED included in the present study was 45.0 ± 13.3 years with mean duration of DED of 6.3 ± 4.8 years. Majority of patients were female , a significant association was found only in the left eyes and not in right eyes. The correlation line was steeper for the entire population than for the subgroups with either normal or abnormal TFBUT  and abnormal (<15 mm) values, a relatively strong correlation was found between TFT and Schirmer I for eyes with abnormal Schirmer I test. However, in eyes with normal Schirmer I, no significant correlation was found. The regression line was steepest for the entire study population .The present study shows a significant, but relatively weak association between TFT and OSDI as well as TFT and TFBUT, which is in line with our previous results . Our preIn addition, we observed a relatively strong association between inter-eye differences for TFT and TFBUT as well as for TFT and Schirmer. These results indicate that the weak correlation between these parameters in the overall population is to a large degree related to hitherto unidentified factors. Factors that could potentially explain the weak correlation are composition of tears and surface tension. Based on our data, one may hypothesize that the latter factors show relatively little inter-eye variability but high inter-individual variability, which may explain at least partially the results of the present study. Further experiments are, however, required to test this possibility.In the present study, the association between clinical signs and reported symptoms of DED patients was weak, with TFT being the only variable significantly correlating with OSDI. This finding is in keeping with data from our own group as well as from previous other investigators ,8,23,30 Additionally, it needs to be considered that different clinical signs of DED also reflect different phenotypes of DED . ObviousSeveral non-invasive techniques have been realized to measure tear dynamics, tear clearance and TFBUT non-invasively ,39,40,41The present study has strengths and weaknesses that require further discussion: The major strengths of the current study include the relatively large sample size, the availability of OCT images from both eyes and the standardized protocol for OCT tear film imaging. A limitation of the study relates to the selection of the study population: As the current analysis is based on a pooled analysis of baseline data from several clinical trial, the sample does not represent a randomly selected cross-sectional population but rather DED patients who participated in different clinical trials. As such neither signs nor symptoms of DED were associated with known risk factors of the disease such as age and gender ,46. As tAnother limitation relates to the absolute values of TFT. It is important to understand that the measured TFT values are not only dependent on the imaging system but also on the estimator used for thickness extraction from the images. Indeed different approaches have been proposed that will result in slightly different estimates of TFT ,42,43,47In conclusion, our data show that TFT as measured with high resolution OCT is associated with symptoms of DED, although the correlation is not very strong. Pre-corneal TFT is also weakly correlated to TFBUT and Schirmer I. In patients with abnormal Schirmer I, the correlation is, however, more pronounced indicating that these signs rather reflect the aqueous-deficient component of DED. Given that OCT can also be used to estimate evaporation rates and quantify lipid layer thickness it may be an interesting approach in studying signs of DED.


Various levels of lockdown due to COVID-19 limit people’s habitual physical activity. Individuals addicted to exercise, health-oriented, and team-exercisers could be the most affected. We examined the COVID-19-related changes in exercise volume in 1079 exercisers from eight Spanish-speaking nations based on exercise addiction categories, primary reasons for exercise, and forms of exercise. The COVID-19-related decrease in exercise volume was 49.24% in the sample. The proportion of the risk of exercise addiction was 15.2%. Most (81.7%) of the participants exercised for a health-related reason. These exercisers reported lesser decrease in their exercise volume than those exercising for social reasons. The risk of exercise addiction was inversely related to changes in exercise volume, but after controlling for passion and perfectionism the relationship vanished. The reported effect of COVID-19 on training did not differ between the exercise addiction groups. The findings also confirm that exercise addiction research should control for passion and perfectionism. A physically active lifestyle has physical  the prevalence of the risk of exercise addiction in the sample; (2) the distribution of the primary exercise motive ) health-, (b) skill-, and (c) social) in three exercise addiction risk groups ) those at risk of exercise addiction [strong symptoms], (b) symptomatic [mild symptoms], and (c) asymptomatic [no symptoms]); (3) the change in exercise volume, in response to the COVID-19 pandemic in the three exercise addiction risk groups by considering their primary reason for exercise; (4) the relationship between the change in exercise volume during the COVID-19 pandemic and exercise addiction risk scores; (5) the change in exercise volume in response to the COVID-19 pandemic in three exercise addiction groups in the context of team or individual exercises; and (6) the perceived effect of the COVID-19 situation on the habitual exercise training regimen in the three exercise addiction risk groups.Apart from the first question that was exploratory, we posed five hypotheses: (1) the primary motives for exercise will differ between the three exercise addiction risk groups; (2) those at high risk of exercise addiction having a primary therapeutic motive for exercise will exhibit less change in exercise volume due to the COVID-19 situation than those who are at lower risk and exercise for other than health reasons; (3) a negative relationship will emerge between the exercise addiction risk scores and the change in exercise volume during the COVID-19 pandemic; (4) team exercisers in three exercise addiction risk categories will demonstrate greater change (decrease) in exercise volume than individual exercisers during the COVID-19 pandemic (due to lack of teammates and closed facilities); and (5) those at risk of exercise addiction will report greater negative impact of the COVID-19 pandemic in their training schedules than symptomatic or asymptomatic exercisers. Due to the very novelty of the COVID-19 literature, little or no background literature is available on the connection between the pandemic and the dependent measures. Therefore, the current hypotheses were generated primarily by relying on “common sense” or rational expectations. However, to investigate the claim of Szabo and Kovacsik et al.  that then = 102), Chile (n = 95), Costa Rica (n = 93), Ecuador (n = 99), Honduras (n = 77), Mexico (n = 96), Spain (n = 422), and Uruguay (n = 95). The total number of eligible participants was 1079 (52% men). Their mean age was 32.88 (± SD = 11.73) years, ranging from 18 to 75 years. More than half (53.1%) of the sample was involved in team/group exercises while the rest exercised on an individual basis. The majority (81.7%) exercised primarily for a health-related reason, 11.7% for a skill-related reason, and 6.6% for a social reason. While most participants were involved in one form of exercise, 24% of them reported practicing more than one exercise.Participants were recruited via social media from eight Spanish speaking countries: Argentina , the weekly amount of exercise (hours per week) before and during the COVID-19 pandemic, and form of exercise . A single item 11-point Likert scale was used for assessing the perceived impact of COVID-19 on respondents’ training regimen. Additionally, three psychometric instruments were employed for measuring the risk of exercise addiction, passion, and perfectionism.α) 0.70  reported for the EAI in a cross-cultural investigation  and HP   of the PS in the current work was 0.89, which is a higher value than that (0.81) reported by Chamarro et al. . Prior to being able to see and answer the questionnaires, participants gave consent to participation by marking with a mouse- click an “agree” button at the end of the consent form. This form contained information about the study as well as criteria for participation. Data were collected between the 6th and 20th of April 2020 and were saved in an Excel file. Subsequently, this file was imported in the SPSS statistical software (version 26) which was used for data verification and analyses.After obtaining ethical clearance from the Autonomous University of Madrid, demographic questions and the three psychometric instruments were published on Google Forms survey platform. Volunteers were eligible to take part in the study if they were aged 18 years or over and exercised for at least 1 hour per week. The gathered data were checked and the answers of those who did not meet the criteria for participation were deleted from the dataset (χ2) was used to examine the frequency of health, skill, and socially motivated exercise among the three exercise addiction risk groups. A bivariate and a partial correlation  were calculated to examine the relationship between exercise addiction risk scores and changes in exercise volume. Analysis of covariances (ANCOVA) was used in examining the change in exercise volume, as a result of the COVID-19 pandemic, in individuals at risk of exercise addiction having different primary motives for exercising as well as in those exercising in team and individual exercises. Further, an ANCOVA was also employed in examining the perceived impact of the COVID-19 pandemic on the training regimen of the three exercise addiction risk groups. In addition to gender and age, in all these analyses, we also controlled for passion as suggested by Szabo and Kovacsik et al. , and a score between 6 and 12 is indicative of low risk (asymptomatic)  hours/week before and 4.54 (± SD = 3.76) hours/week during the COVID-19 pandemic . The proportion of those at risk of exercise addiction was 15.2%, those showing some symptoms amounted to 79.6%, and 5.2% were asymptomatic. The χ2 test yielded statistically no significant difference between the eight nations. However, a statistically significant distribution emerged in the observed frequency of the three primary exercise motives  in the three exercise addiction risk groups . As illustrated in Table The overall decrease in habitual exercise was nearly 50% (49.24%) in the whole sample. Participants exercised an average of 9.22 , we also controlled for perfectionism in the second model. The results of the first model yielded a main effect for exercise addiction risk groups  and for primary exercise reason , but no interaction between the two. At this early point, we disregarded the between-group differences, because we wanted to obtain the results of the second ANCOVA which also included passion and perfectionism in the model. This test was no longer statistically significant for the exercise addiction risk groups, (p > 0.05), but it was still significant for the primary reason for exercise , but neither of these groups differed from those who exercised primarily for a skill-related reason.To test the hypothesis that those at risk of exercise addiction, having a health-related primary motive for their exercise, exhibit the least change in exercise volume due to the COVID-19 pandemic, we calculated two ANCOVAs. The first only controlled for age and gender while the second also controlled for passion (both OP and HP). Further, because a statistically significant correlation emerged between EAI scores and perfectionism . However, when a partial correlation controlling for passion and perfectionism was applied, as suggested in the literature . The zero-order correlations showed that exercise addiction was positively and statistically significantly related to all control variables . The result of the partial correlation justified the consideration of the second model in ANCOVAs which controlled for passion and perfectionism in the context of the risk of exercise addiction.To test the relationship between the risk of exercise addiction and the change in weekly exercise volume, first we calculated a bivariate correlation, which was weak, but statistically significant , as well as when we added passion and perfectionism to the model . This result was due to a significantly larger decrease in asymptomatic team exercisers than asymptomatic individual exercisers  the COVID-19 pandemic forces habitual exercisers to decrease their usual exercise volume to about half of the usual amount; (2) the majority of exercisers have a health-related primary reason for exercising; (3) if passion and perfectionism are not included in the model, there is a significant difference in the primary reason for exercise between the three exercise addiction risk groups, but when the model controls for these variables, the difference does not emerge anymore; (4) individuals exercising primarily for a health-related reason decrease less the volume of their exercise during the COVID-19 pandemic than those exercising for social reason; (5) a weak correlation between the decreased exercises volume during the COVID-19 pandemic and the risk of exercise addiction exists only if there is no control for passion; otherwise, the relationship is not significant; (6) only team exercisers in the asymptomatic exercise addiction risk group have decreased more their exercise volume than individual exercisers in the same risk group; (7) the three exercise addiction risk groups do not differ in their perceived impact of the COVID-19 pandemic on their habitual exercises.A decrease to about half in the usual exercise volume could be ascribed to the COVID-19 pandemic as revealed by the results of this international study. At this time, there is no report available in the literature to which these findings could be compared. However, the here observed overall change in the habitual exercise volume may be considered “reasonable” based on limited movement, distance keeping, and closure of many exercise facilities in many nations during the COVID-19 pandemic. The current results pave the road for research on the physical and mental health consequences of this major cut-back in physical activity in an already sedentary world.The decrease in exercise volume was initially statistically significant between the three exercise addiction risk groups, but when passion and perfectionism were added to the model, the three groups did not differ anymore. These findings support the strong connection between the concepts of risk of exercise addiction and passion  was lower than the values obtained for passion, indicating that perfectionism shares less common variance with exercise addiction than passion. Nevertheless, the connection between exercise addiction and perfectionism seems to affect the results related to exercise addiction.Our hypothesis that there will be a negative relationship between the risk of exercise addiction and change in exercise volume during COVID-19 was supported only until we did not control for passion and perfectionism. After controlling for these variables, the relationship vanished, and all zero-order correlations were significant, supporting the large commonality between these factors  explanations, emerge. The limitations of the present study are manifested in the volunteerism, which is a general problem in psychological studies. The internal reliability of the EAI was 0.65, in the current study which is at the lower end of the acceptable range and, hence, this is another limitation of this work. Further, these results cannot be generalized to non-Spanish speaking samples and despite all eight nations were affected by COVID-19, not all of them were affected equally. Indeed, the level of lockdown differed between these nations (BBC.com, April 6, Exercise volume decreases about 50% in regular exercisers 2 months into the COVID-19 crisis. The decrease and the primary exercise motive are unrelated to the risk of exercise addiction. Most exercisers report a health-related motive as the primary reason for their exercise and this motive is associated with lesser reduction of exercise volume than a social reason. During COVID-19 team exercisers decrease more their exercise volume than individual exercisers, but the difference is evident in the symptomatic exercise addiction risk group only. The findings confirm the call for controlling passion in exercise addiction research. They expand this call by showing that perfectionism could also moderate the results obtained on exercise addiction and, therefore, this variable should be also considered, along with passion, in future exercise addiction research.


Major depressive disorder (MDD) is highly prevalent and often managed by general practitioners (GPs). GPs mostly prescribe medication and show low referral rates to psychotherapy. Many patients remain untreated. Blended psychotherapy (bPT) combines internet-based interventions with face-to-face psychotherapy and could increase treatment access and availability. Effectively implementing bPT in routine care requires an understanding of professional users’ perspectives and behavior.This study aims to identify barriers and facilitators perceived by GPs in referring patients to bPT. Explanations for variations in referral rates were examined.Semistructured interviews were conducted with 12 of 110 GPs participating in a German randomized controlled trial (RCT) to investigate barriers to and facilitators for referrals to bPT for MDD . The interview guide was based on the theoretical domains framework. The interviews were audio recorded and transcribed verbatim, and the qualitative content was analyzed by 2 independent coders . A follow-up survey with 12 interviewed GPs enabled the validation of emergent themes. The differences in the barriers and facilitators identified between groups with different characteristics  were described. Correlations between referrals and characteristics, self-rated competences, and experiences managing depression of the RCT-GPs (n=76) were conducted.r(73)=−0.31, P<.001. The qualitative findings revealed a total of 19 barriers (B) and 29 facilitators (F), at the levels of GP (B=4 and F=11), patient (B=11 and F=9), GP practice (B=1 and F=3), and sociopolitical circumstances (B=3 and F=6). Key barriers stated by all interviewed GPs included “little knowledge about internet-based interventions” and “patients’ lack of familiarity with technology/internet/media” . Key facilitators were “perceived patient suitability, e.g. well-educated, young” (k=22) and “no conflict with GP’s role” (k=16). The follow-up survey showed a very high agreement rate of at least 75% for 71% (34/48) of the identified themes. Descriptive findings indicated differences between GPs with low and high referral rates in terms of which and how many barriers  and facilitators  they mentioned.GPs referred few patients to bPT, although varied in their referral rates, and interviewees referred more than twice as many patients as RCT-GPs . A negative correlation was found between GPs’ referrals and their self-rated pharmacotherapeutic competence, This study provides insights into factors influencing GPs’ referrals to bPT as gatekeepers to depression care. Barriers and facilitators should be considered when designing implementation strategies to enhance referral rates. The findings should be interpreted with care because of the small and self-selected sample and low response rates. Approximately 350 million people worldwide are affected by major depressive disorder (MDD) annually . It is tGeneral practitioners (GPs) are considered most important in the management of depression because they are often the first point of contact in the pathways to care ,8. AccorThere are several reasons for not adhering to clinical guidelines in primary care. Factors that may hinder GPs in adequately treating depression include low rates 47%) of correct recognition and diagnosis of depression , insuffi7% of corSome of the abovementioned barriers may be overcome by providing digital technologies as an additional treatment option. Digital approaches may help to improve access to specialists, decrease long waiting times, and reduce the high number of untreated patients ,24. InteDespite its potential, the uptake of evidence-based internet-delivered treatment remains limited ,28, and Given GPs’ central role as gatekeepers in the management of depression, research on their perspectives as gatekeepers seems crucial. A recent study on the implementation of internet-based therapy services in routine care, in guided, unguided, or blended formats, suggested that GPs’ low referral rates and unstable patient intake could be explained by GP skepticism and reservations and called for research on referrers’ attitudes . A studyThe abovementioned insights refer to GPs’ perspective on IMIs and show that research on bPT is scarce. To the best of our knowledge, no study has yet investigated GPs’ perspectives on barriers to and facilitators for referrals to blended treatments. GPs may be more willing to refer to an IMI when it is combined with face-to-face psychotherapy, which is well known and trustworthy. At the same time, referrals might be facilitated by improved treatment availability and access to treatment options. However, given low GPs’ referral rates to psychotherapy and the novelty of blended treatments, an assessment of factors determining GPs’ referrals to bPT is needed. From this, implementation strategies can be designed to address these and maximize the uptake of bPT. This mixed methods study aimed to identify barriers and facilitators that influence the referral behavior of GPs to a bPT for MDD.This study was part of a mixed methods German study arm of the European Project E-COMPARED, which was the first multicenter study that evaluated the clinical and cost-effectiveness of a blended depression treatment compared with treatment as usual across 8 countries. The objective of the German RCT was to evaluate the effectiveness of bPT based on cognitive behavioral therapy (CBT) for adults with a diagnosis of MDD compared with GP routine care. Further information on the RCT can be found in the study protocol .The trial was approved by the Ethics Committee of the German Society of Psychology and registered in the German Clinical Trials Register (DRKS00006866). All GPs provided informed consent to participate.Patients were recruited in GP offices in primary care and randomly assigned to the treatment conditions, bPT (n=86) and GP care (n=87). Professionals were not blinded. The bPT was conducted by psychologists in an outpatient university clinic. GP care consisted of (1) initial screening of MDD, informing about treatment options and referring depressed patients to the study; (2) conducting routine care for the control group; and (3) completing questionnaires about the diagnosis and treatment of patients at baseline and postassessment (13 weeks). All GPs received a print booklet with information about bPT and trial procedures as well as recruitment material  to inform and motivate their patients. Their effort was reimbursed with EUR 100 (US $117.6) per partaking patient. Further motivating activities were a kickoff meeting, newsletter, phone calls, and onsite visits.bPT was delivered as a short-term treatment (13 weeks) for MDD. It combined 6 internet-based CBT lessons , daily assessments of mood, and cognitive and behavior-related parameters on a mobile app , as well as 6 biweekly face-to-face sessions with a psychologist and CBT therapist in training . PatientBy the end of 2014, more than 1000 GPs were contacted through post, whose practices were located in the relevant catchment area for referrals to the university outpatient clinic (60 km). Overall, 137 GPs from 107 practices in Bavaria (Germany) agreed to refer to bPT. In the recruitment period from February 2015 to August 2016, 121 GPs referred to at least one patient, and 86 GPs had partaking patients in the RCT.Successful referrals were operationalized as the number of patients who completed screening for MDD after being informed of it in GP offices. Owing to the study design, it was not possible to document the intended referrals onsite. Higher referral rates can be assumed; since some patients may not have followed the recommendation of their GP, the GPs had reported a high number of patients treated daily , and not all patients could be assigned to a GP.The recruitment of GPs for this qualitative study took place shortly before the end of the study enrollment period (March to June 2016) by inviting 110 GPs from the trial population  by email or telephone to participate in interviews. In total, 12 GPs (11%) took part in semistructured interviews. Of 110 GPs, 39 refused to participate; some were interested but had time constraints, and others could not be reached by telephone and emails with 3 reminders.We used a qualitative method with a theory-based approach to gain insights into the experiences and perspectives of GPs regarding their referral behavior to bPT. This exploratory method is a recommended approach to identify barriers and facilitators for implementing interventions .The semistructured interview guide  was baseD01: KnowledgeWhat do you know about blended psychotherapy (bPT) and its effectiveness?D02: SkillsWhich skills and competencies do you consider as necessary to refer patients to bPT?D03: Social/professional role and identityDo you see referring suitable patients to bPT as part of your role? Is doing referrals to bPT incompatible or in conflict with professional standards or your identity?D04: Beliefs about capabilitiesHow difficult or easy is it to refer patients to bPT? How confident are you that you can overcome existing barriers?D05: OptimismBased on your experience, how confident are you that referrals to bPT will run optimally?D06: Beliefs about consequencesWhat do you think are the benefits of bPT?D07: ReinforcementDo you think the benefits of bPT for patients are sufficient to justify referral being part of your normal workflow?D08: IntentionsHow much of a priority is bPT in the care of patients with depression? How essential is your personal requirement to refer patients?D09: GoalsHow do you feel about the goal to implement bPT into the health care system in a way that you could refer to it in the future?D10: Memory, attention and decision processesIs referring to bPT something you usually do or remember to do? What helps or would help you to remember?D11: Environmental context and resourcesDo you think there are sufficient resources available if you start referring more patients to bPT? Which barriers outside your practice can prevent referrals?D12: Social influencesDid colleagues or patients/relatives ever prompt or encourage you to refer to bPT? Did they discourage your referral? What reactions do you expect?D13: EmotionDo you think bPT is something patients are willing to participate in? Does thinking about referrals to bPT evoke any concerns?D14: Behavioral regulationAre there procedures or ways of working that encourage referrals to bPT?The sample size was determined by the low response rates to interviews at the end of the RCT enrollment period. Hence, the sample size and composition could not be planned. In total, 3 interviewers conducted 4 interviews with GPs. IT trained 2 interviewers with a Bachelor of Science in Psychology in using the interview guide and gave feedback to their first interviews. The interviews were conducted through telephone between May 2016 and June 2016, whereas the GPs were at the workplace (n=9), at home (n=2), or in the car (n=1). The average duration of the interviews was 56 min . Interviews were audio recorded and transcribed verbatim, based on a transcription guide. Data were pseudonymized using code numbers.A qualitative content analysis was conducted, drawing on an inductive-deductive approach and using standardized methodical steps in qualitative research . Codes wFirst, a list of codes was developed using 50% (n=6) of interview material, which involved identifying emerging themes, discussing the possible meanings of text excerpts, and arranging themes into categories within consensus meetings. Consensual coding was applied to enhance the quality of the coding process [Second, a further text retrieval was done to (1) label text related to the first draft of the code list, (2) specify subthemes, (3) add new emerging themes to the code list, and (4) target a higher level of abstraction of the codes. A paragraph was coded if it contained themes from one or more categories. IT gave written feedback to the coders regarding all labeled text frames to enrich the data interpretation through clinical expertise. The list of codes was revised and completed with additional definitions and exemplary statements. A preliminary code system, based on 50% (6 interviews) of the material, was developed and discussed in a consensus meeting among IT, SS, and the coders. They reached final agreement on code definitions and excerpts, the structure of the code system, and coding rules. This iterative research process aimed to finalize the code list to fit the data and to optimize the content and number of identified categories.Third, all interview transcripts were independently coded by 2 coders in accordance with the code list. There was sufficiently moderate intercoder agreement with the coefficient kappa, k=0.71 . Data saFinally, to ensure the validity of the identified themes, the 12 GPs were questioned as to whether they agreed with the resultant barriers and facilitators via a survey. The themes were presented as a list. GPs agreed, on average, with 37 of 48 of all themes identified . The mean agreement rate per theme  was 78% . Thus, the identified barriers and facilitators yielded very good validation results.A comparison of all barriers and facilitators and the percentage of all 12 GPs who mentioned these in the interviews and in the follow-up validation survey was conducted. Agreement rates per GP were analyzed.d=1.96  or 1.85  in a Mann-Whitney U test, each with a power of 80% and an alpha level of 5% (two tailed).Secondary quantitative analyses were conducted to evaluate whether there were any group differences in the identified barriers and facilitators. The interviewees were divided into 2 groups according to the following characteristics: “referral rates: high (n=4) versus low (n=8)” , “experience years as licensed GP: high (n=6) versus low (n=6)” , and “training in psychotherapy: yes (n=4) versus no (n=8).” A frequency table and descriptive statistics with 95% CIs were used to present differences in the barriers and facilitators between the abovementioned groups. Inferential statistics were not applied because of small samples and limited power. Power sensitivity analyses indicated that both equal (6/6) and unequal (8/4) small sample sizes would have the sensitivity to detect only a large proportional difference of 0.75 in a Fisher exact test and large effect sizes of t test or Mann-Whitney U test for continuous variables. The results of the nonparametric Mann-Whitney U test were reported because of unequal sample sizes, if both assumptions  were not met. The test was applied for verification if the data were nonnormally distributed. All analyses were two sided, with an alpha level of 5%. Sensitivity power analysis for t tests, targeting unequal sample sizes (n=64 and n=12), an alpha level of 5% (two tailed), and 80% power, had yielded the sensitivity to detect an effect size of d=0.89.Independence tests of the interviewed sample (n=12) and the remaining RCT sample (n=64) were conducted using the Fisher exact test for categorical variables (because of cell frequencies <5) and using the r=0.31, with a power of 80%  in a bivariate correlation.Pearson correlations were conducted to determine the association between referrals and different continuous variables in the total RCT sample (n=76). The power analysis indicated that a sample size of 76 would have the sensitivity to detect an effect size of The tool MAXQDA 12  was used for the qualitative analysis and SPSS 25  for the quantitative analysis. Power analyses were conducted using G*Power, version 3.1.9.7 . A guideP≥.08 , with an average of 50.67 (SD 11.88) years and reported an average of 15.75 (SD 10.42) years of practice experience. Regarding their qualifications, 4 GPs had a license for psychotherapy and 9 had further education in psychosomatic basic care in addition to their university degree in medicine. GPs worked in both metropolitan  and rural  areas. Sociodemographic data for the remaining RCT sample (available for n=64) showed similar values, such as a proportion of 64% (41/64) being men, an average age of 52.14 (SD 8.73) years with 16.65 (SD 8.34) working years as a licensed GP. There were no statistically significant group differences in characteristics between the interviewed sample (n=12) and the RCT sample (n=64), all The patients treated with bPT (n=86) were aged on average 43.22 years , mostly women (61%), highly educated (56%), and employed (74%). Overall, 71% had no prior experience with psychotherapy.Of 698 successful referrals to bPT, 321 could be assigned to 121 GPs in the RCT. The majority of RCT-GPs  referred fewer than 3 patients to bPT during the 18-month study period, indicating a ceiling effect. However, there was a large variation between GPs in the number of referrals made , and interviewed GPs had higher referral rates than those in the RCT. r(73)=−0.31; P<.001), that is, GPs with higher self-confidence in delivering pharmacotherapy were less likely to make referrals to bPT. Correlations between referrals and demographics, medical experience, competence ratings, and depression management statement agreements were otherwise nonsignificant  the patient, (3) factors influencing the routine in the GP practice, and (4) factors relating to sociopolitical circumstances that influence the implementation of a bPT for MDD in Germany as well as the referral process. In total, 77% (37/48) of the identified themes were mentioned by at least five or more interviewed GPs (42%), whereas 44% (21/48) of the themes were mentioned by at least eight GPs (67%).Altogether, 19 barriers and 29 facilitators were identified and categorized into 4 main areas: (1) the Most emerging barriers  were assigned to the patient level. Five barriers  were named by at least nine GPs (75% of interviewees), whereas 2 barriers  were mentioned by all 12 GPs. These latter 2 key barriers are presented in the following section with a quotation illustrating the physicians’ experiences, whereas all 19 barriers are described with a definition and supporting quotations in All interviewed GPs reported feeling poorly informed about the content and the procedure of bPT and having little knowledge about the scientific evidence of the treatment, as one GP outlined:I know nothing about the effectiveness of bPT. I have not read anything about it in GP journals.GP098All respondents agreed that patients who are not familiar with computers or smartphones and have little experience with technology might struggle with the technology-based treatment:There are still people today who are not familiar with computers, especially in rural areas.GP053The analysis revealed more facilitating than hindering factors on the levels of the GP as a person, GP practice, and sociopolitical circumstances. Of the identified 29 facilitators, 16 (55%) were mentioned by at least 75% (n=8) of GPs and 2 (7%) by all interviewees. Below, these 2 key facilitators are presented with an outlining statement. All 29 facilitators are described with a definition and supporting quotations in All interviewed GPs perceived no role conflict between the referral to bPT and their professional identity and standards:I see the referral to bPT as part of my role. I see it as a welcome opportunity to be able to offer the patient a quick and uncomplicated means of assistance here in the practice.GP059Each GP identified patient groups for whom the bPT suits well. They characterized these patients as those who are young and highly educated, used to work with computers and smartphones, and ready to integrate them into their treatment:I think it is definitely suitable for younger patients and also for patients who are already at work and do not have that much time.GP083The follow-up assessment of the identified themes within the qualitative interviews showed a very high agreement rate between all 12 GPs of at least 75% for 34 (71%) of the identified barriers and facilitators. In total, 44 of 48 (92%) themes yielded an agreement rate of at least 58%, and 3 (6%) themes were agreed by all GPs. Only 2 (4%) themes had an agreement rate less than or equal to 25% of GPs.Low awareness of bPT as a therapeutic method was the barrier, and integration within guidelines as evidence-based treatment and short waiting time for internet-based intervention were the facilitators, which resulted in the highest agreement rates. Patients’ fear of stigmatization was the barrier, and GPs’ expectation of social reinforcement was the facilitator with the lowest agreement rates.All barriers, and the percentage of all 12 GPs who mentioned these in the interviews and in the follow-up validation survey of qualitative findings, are listed in All enabling factors and the percentage of all 12 GPs who mentioned the facilitator in the interview and the follow-up validation survey of qualitative findings are listed in Frequency tables indicated there were differences between GPs with different characteristics  in terms of whether they mentioned particular barriers and facilitators. Descriptive statistics and 95% CIs can be found in bridging the waiting time for traditional psychotherapy, little room for individualized treatment or personal issues, and limited therapeutic relationship or personal contact. Low referrers more frequently mentioned with a difference of 50% low awareness of bPT as a therapeutic method, personal contact with the therapists and provider, and patients’ motivation and willingness for treatment. Such meaningful differences were not found when comparing GPs with high or low experience and training or no training in psychotherapy.Barriers (10/19) and facilitators (13/29) with a discrepancy of at least 25% among GPs with high versus low referral rates are shown in The GPs with low referral rates mentioned fewer barriers  and facilitators  than those with high referral rates . Both GP groups mentioned, on average, more facilitators than barriers.This mixed methods study investigated facilitators and barriers for referrals to bPT for depression from the perspective of GPs. The results should be interpreted in light of the fact that the sample was limited, small, self-selected, and unlikely to represent the full range of all GPs’ experiences (because of low response rates). Although referral rates were quite low, they differed between GPs. The lower the RCT-GPs rated their pharmacotherapeutic skills, the more referrals they made. Interviewees referred more than double the number of patients than the RCT-GPs.All 29 facilitating and 19 hindering factors were identified on the levels of GP, patient, GP practice, and sociopolitical circumstances. The most frequently named barriers by the interviewed GPs concerned the use of new technologies within blended treatments, as most assumed that some patients would not be familiar with internet and technology or would be skeptical and uncertain as to whether an internet-based intervention could help them. These findings are in line with earlier research on barriers to the use of bPT by psychotherapists ,35,55,56The interviewed GPs reported not to be familiar with bPT, to have little knowledge of it, and to prefer more information and training. This is in accordance with the results of studies on bPT in GPs  and psycGPs had positive beliefs about the treatment success of bPT as well as high self-efficacy levels for patient referrals, contrasting previous findings on IMIs for depression in GPs . This miFurther facilitators for referrals mentioned by the interviewed GPs relate to the health care system  and are in line with research on IMIs with GPs ,42 and bGPs’ attitudes that patients are suitable for bPT if they are familiar with modern technologies or are affected by minor-to-moderate depression were in line with previous research on bPT with psychotherapists ,35,56 anThe finding that GPs perceived younger patients to be more suitable for bPT compared with older patients is in accordance with research on IMIs in clinicians  and GPs The findings can be interpreted in the context of prior systematic reviews on barriers and facilitators and help to develop implementation strategies. First, they align with the 7 stages of the implementation process of an intervention , which were identified using 256 publications to categorize barriers to optimal clinical practice in health care . These sThis study has noteworthy limitations, such as low response rates of GPs to interview invitations  and a limited sample size. This meant that further sampling to check the consistency of findings was not possible, and having a small sample size may have produced fewer themes and nuances in GPs’ views than the 20 to 30 interviews suggested by guidelines . Yet, suThe strengths of this work included the mixed methods approach, which enabled the research team to generate in-depth findings by using a theory-based interview guide, and validated qualitative findings with a survey. The consensus and iterative approach is used to develop codes, and independent coding with a moderate interrater agreement enhanced rigor in producing the results. Identifying GP subgroups and comparing differences between these provided an indication of whether barriers or facilitators differed in their importance to different GPs.Findings relating to barriers and facilitators could be used to design implementation strategies to support the integration of bPT as a referral option in clinical practice. The TDF is associated with the Behavior Change Wheel framework, which connects theoretical domains to 3 broad drivers of behavior: capability, opportunity, and motivation (COM-B model). It also connects these to specific intervention options ,71, descIn addition, future studies might explore barriers and facilitators in larger samples, invite GPs that have referred to bPT in routine care, and consider different delivery options of digital interventions . Furthermore, the roles of GPs as referrers to or users of blended treatments for different mental health disorders should be evaluated.This study provides insights into barriers and facilitators determining GPs’ referral behavior in relation to bPT for depression. The results indicate that GPs perceive bPT as an additional and valuable treatment delivery format. Having a central position in depression treatment, they experience positive consequences for their own professional group and for patient care when they are able to use bPT as a referral option. Thus, GPs appear to be ready as stakeholders to integrate digital interventions blended with face-to-face psychotherapy in their depression management. However, they experienced considerable barriers to their referrals, which might have led to their low referral rates in this study. Understanding and addressing their perceived barriers and facilitators might enhance their uptake of bPT as a referral option and therefore improve patients’ access to specialized care.On the basis of these findings, the following issues should be taken into account when developing an implementation strategy: (1) address the organizational, legal, and reimbursement requirements; (2) consider GPs’ suggestions for implementation, such as the integration of bPT as an additional care pillar in treatment guidelines and the development of measures to increase familiarity with bPT and its advantages ; (3) save GPs’ resources ; (4) ensure fit with GPs’ habits and routine activities ; and (5) address GPs’ need for information and training on bPT as well as personal contact and feedback in communication with therapists.


Published 10, February 2020We noticed mistakes in citations (omitted and wrong citations) in the original article. The following citations have been added or changed in the text and reference list:#1Original text:In addition, c-Maf may also directly contribute to RORγt expression in ILC3s, as it has been reported for T cells .Corrected text:In addition, c-Maf may also directly contribute to RORγt expression in ILC3s, as it has been reported for T cells . Our data supports and extends the findings of a very recent report that was released after completion of this manuscript .#2Original text:Flow cytometry was performed according to previously defined guidelines .Corrected text:Flow cytometry was performed according to previously defined guidelines .In connection with the changes above, the following citations have been added to the Reference list:Parker M.E et al., 2020. c-Maf Regulates the Plasticity of Group 3 Innate Lymphoid Cells by Restraining the Type 1 Program. J Exp Med, 217(1).Cossarizza, A., et al., 2019. Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). Eur J Immunol, 49(10): p. 1457-1973.We apologise for the omission and mistakes of these citations.In addition to the changes above, the following changes have also been made to the manuscript:#3Original text:All animal experiments were in accordance with the ethical standards of the institution or practice at which the studies were conducted and were reviewed and approved by the responsible ethics committees .Corrected text:All animal experiments were in accordance with the ethical standards of the institution or practice at which the studies were conducted and were reviewed and approved by the responsible ethics committees of Germany  and Russia.We also added the previously omitted funding information for Andrey Kruglov:Russian Science Foundation grant # 17-74-20059Andrey KruglovThe article has been corrected accordingly.


Pannexins are large-pore forming channels responsible for ATP release under a variety of physiological and pathological conditions. Although predicted to share similar membrane topology with other large-pore forming proteins such as connexins, innexins, and LRRC8, pannexins have minimal sequence similarity to these protein families. Here, we present the cryo-EM structure of a frog pannexin 1 (Panx1) channel at 3.0 Å. We find that Panx1 protomers harbor four transmembrane helices similar in arrangement to other large-pore forming proteins but assemble as a heptameric channel with a unique constriction formed by Trp74 in the first extracellular loop. Mutating Trp74 or the nearby Arg75 disrupt ion selectivity, whereas altering residues in the hydrophobic groove formed by the two extracellular loops abrogates channel inhibition by carbenoxolone. Our structural and functional study establishes the extracellular loops as important structural motifs for ion selectivity and channel inhibition in Panx1. Large-pore forming channels play important roles in cell-to-cell communication by responding to diverse stimuli and releasing signaling molecules like ATP and amino acids . PannexiTo identify a pannexin channel suitable for structure determination, we screened 34 pannexin orthologues using Fluorescence Size Exclusion Chromatography (FSEC). Frog PaThe frPanx1-ΔLC structure revealed a heptameric assembly, which is unique among the known eukaryotic channels . Other lSyrjanen et al., 2020The overall protomer structure of Panx1 resembles that of other large-pore forming channels including connexin, innexins, and LRRC8. Like other large-pore forming channels, each Panx1 protomer harbors four transmembrane helices (TM1-4), two extracellular loops (ECL1 and 2), two intracellular loops (ICL1 and 2), and an amino (N)-terminal loop . The traSyrjanen et al., 2020-), Erev shifted to +26 mV, suggesting the channel is more permeable to Cl- than to Gluc-. When exposed to the large cation, N-methyl-D-glucamine (NMDG+), Erev remained close to 0 mV, suggesting that Na+ and NMDG+ equally (or do not) permeate Panx1. These results are consistent with Panx1 being an anion-selective channel. The Arg75Lys mutant maintains the positive charge of this position, and displayed Erev values comparable to WT. Removing the positive charge at this position, as shown by the Arg75Ala mutant, diminished Cl- selectivity as the Erev in NaGluc remained near 0 mV. Interestingly, the Erev in NMDGCl shifted to −22 mV, suggesting the channel had lost anion selectivity and Na+ became more permeable than NMDG+. A charge reversal mutant, Arg75Glu, shifted the Erev in NaGluc to −16 mV and in NMDGCl to −45 mV, indicating that Gluc- became more permeable to Cl-. Overall, these results support the idea that the positively charged Arg75 plays a role in anion selectivity of Panx1.The Panx1 permeation pathway spans a length of 104 Å, with constrictions formed by the N-terminal loop, Ile58, and Trp74 . The nar+  diminished CBX-sensitivity . These dThe frPanx1-ΔLC structure uncovered a unique heptameric assembly of a large-pore channel that harbors an extracellular constriction formed by Trp74 and Arg75. These residues are located on ECL1 and face toward the central pore of the channel and thus, are situated to regulate channel function. Mutagenesis studies at these positions revealed that both residues play pivotal roles in ion selection. Unlike the LRRC8A anion channel, however, the positively charged Arg75 does not seem to form a canonical selectivity filter. Instead, the guanidino group of Arg75 likely mediates a cation-π interaction with Trp74 in the neighboring subunit, which seems to control ion selection. One possible ion selection mechanism is that this cation-π interaction stabilizes the inter-subunit interactions, which in turn creates an electrostatic environment that favors anion permeation. Another possibility is that tight inter-subunit interactions in the extracellular domain are necessary to form an ion selectivity filter in the missing region in our current model .Which functional state does our model represent? Based on the lack of channel activity at 0 mV , our curWe found that ECL1 and ECL2 interact to each other and form a potential CBX binding pocket. Both ECL1 and ECL2 may undergo movement based on conformational alterations of the TMDs and cytoplasmic domains. For example, it is conceivable that movement of the TMDs caused by membrane stretch or voltage, or changes in the cytoplasmic domain triggered by caspase cleavage may be coupled to conformational rearrangements in the extracellular domain. The major role of the extracellular domain in pannexin function is strongly supported by our experimental results demonstrating that mutating Trp74 and Arg75, as well as surrounding residues in ECL1 and ECL2, alter channel properties including ion selectivity. Furthermore, we previously demonstrated that application of CBX to mutants at Trp74  potentiates voltage-dependent channel activity , which iIn contrast to the extracellular domain, roles of the intracellular domain remain elusive. While the C-terminal domain has been demonstrated to play important roles in Panx1 channel gating , our stuIn conclusion, our frPanx1-ΔLC structure provides an important atomic blueprint for dissecting functional mechanisms of Panx1. While we did not observe a gate-like structure in the current cryo-EM map, the missing domains, especially the N-terminal loop and the C-terminal domain, may serve as a channel gate on the intracellular side of the channel. Further structure-based experiments such as cysteine accessibility and molecular dynamics simulations will facilitate our understanding of how this unique large-pore channel functions.HEK293 (CRL-1573) cell lines were purchased from the American Type Culture Collection , and therefore were not further authenticated. The mycoplasma contamination test was confirmed to be negative at ATCC.6 cells/ mL and cultured at 27°C for 48 hr. Cells were collected by centrifugation, washed once with PBS, and lysed by nitrogen cavitation  at 600 psi in PBS containing leupeptin (0.5 μg/mL), aprotinin (2 μg/mL), pepstatin A (0.5 μg/mL), and phenylmethylsulfonyl fluoride (0.5 mM). Broken cells were centrifuged at 12,000 x g for 10 min, and membranes were collected by ultracentrifugation at 185,000 x g for 40 min. Membranes were suspended and solubilized in PBS containing 1% C12E8 (Anatrace) for 40 min, followed by ultracentrifugation at 185,000 x g for 40 min. Solubilized material was incubated with StrepTactin Sepharose High-Performance resin  for 40 min in batch. Resin was collected onto a gravity column (Bio-Rad), washed with 10 column volumes of wash buffer , and eluted with five column volumes of wash buffer supplemented with 2.5 mM desthiobiotin. Eluted protein was concentrated and further purified on a Superose 6 10/300 Increase column  with 150 mM NaCl, 10 mM Tris pH 8.0, 0.5 mM DDM as the running buffer. Peak fractions were collected and pooled. All steps were performed at 4°C or on ice.frPanx1 (NP_001123728.1) was synthesized (Genscript) and cloned into the BamHI/XhoI sites of pCNG-FB7 vector containing a C-terminal Strep-tag II (WSHPQFEK). Amino acids from the IL1 and IL2 were removed by standard PCR strategies, and the BamHI site was also removed by quickchange mutagenesis. The full length frPanx1 and hPanx1  were also subcloned into pCNG-FB7 vectors by standard PCR. Sf9 cells were infected with high titer baculovirus (20–25 mL P2 virus/L cells) at a cell density of 2.5–3.0 × 10MSP2N2 apolipoprotein was expressed and purified as described previously , and the2/frame and a total exposure per micrograph of 57.3 e / Å2. Data were collected using EPU software (FEI). For full-length frPanx1 in nanodiscs, a total of 574 images were collected at 130 k magnification with a pixel size of 1.06 Å in electron counting mode. Each micrograph was composed of 50 frames collected over 10 s at a dose of 1.4 e / Å2/frame. The total exposure per micrograph was 70 e / Å2. Data were collected using SerialEM  were applied to glow-discharged lacey carbon-coated copper grids (Electron Microscopy Services). The grids were blotted for 4 s with blot force 7 at 85% humidity at 15°C, and plunge frozen into liquid ethane using a Vitrobot Mark IV (Thermo Fisher). All data were collected on a FEI Titan Krios (Thermo Fisher) operated at an acceleration voltage of 300 keV. For frPanx1-ΔLC, a total of 2034 images were collected at 130 k magnification with a pixel size of 1.07 Å in electron counting mode. Each micrograph was composed of 32 frames collected over 4 s at a dose of 1.79 e / ÅSerialEM . Data foWarp was used for aligning movies, estimating the CTF and particle picking for frPanx1-ΔLC and full-length hPanx1. For full-length frPanx1, movie alignment and CTF estimation were performed using the program Unblur and CTFFind, respectively, within the cisTEM package . 2D clas2, 1 MgCl2. A rapid solution exchange system  was used to perfuse cells with CBX or various salt solutions. Currents were recorded using an Axopatch 200B amplifier (Axon Instruments), filtered at 2 kHz (Frequency Devices), digitized with a Digidata 1440A (Axon Instruments) with a sampling frequency of 10 kHz, and analyzed with the pClamp 10.5 software (Axon Instruments). For voltage step recordings, Panx1-expressing cells were held at −60 mV and stepped to various voltage potentials for 1 s in 20 mV increments before returning to −60 mV. For ramp recordings, cells were held at −60 mV, and ramped between −100 mV and + 100 mV over 3 s duration.HEK293 cells were plated onto 12 mm glass coverslips (VWR) in wells of a six-well plate and transfected 24 hr later with 500–800 ng plasmid DNA using FUGENE 6 (Promega) according to the manufacturer’s instructions. Recordings were performed ~16–24 hr later using borosilicate glass micropipettes (Harvard Apparatus) pulled and polished to a final resistance of 2–5 MΩ. Pipettes were backfilled with (in mM) 147 NaCl, 10 EGTA, 10 HEPES pH 7.0 with NaOH. Patches were obtained in external buffer composed of (in mM) 147 NaCl, 10 HEPES pH 7.3 with NaOH, 13 glucose, 2 KCl, 2 CaCl In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:eLife, and the authors have done an excellent job of revising the manuscript to address the concerns of the reviewers.This is an important manuscript describing the first near atomic resolution structure of a pannexin channel, a family of large pore channels thought to be important for the movement of ions and small molecules such as ATP across cellular membranes. The mechanisms by which the activity of pannexin channels are regulated are poorly understood, yet these channels are thought to play important roles in innate immunity, neuronal signaling, apoptosis, cancer and ischemia. Having a structure of the channel will enable further mechanistic studies on how this protein plays important biological roles. By combining data from cryo-EM and electrophysiology, this manuscript describes the first structure of a pannexin channel at near-atomic resolution and it sheds light on the determinants of anion selectivity, gating and pharmacology. The data appears to be of high quality and the major conclusions of the manuscript are sound. The reviewers agreed that the work is a strong candidate for publication in Decision letter after peer review:eLife. Your article has been reviewed by Kenton Swartz as the Senior Editor, a Reviewing Editor, and three reviewers. The following individuals involved in review of your submission have agreed to reveal their identity: Raimund Dutzler (Reviewer #2); Ming Zhou (Reviewer #3).Thank you for submitting your article "The Cryo-EM Structure of a Pannexin 1 Channel Reveals an Extracellular Gating Mechanism" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:eLife. As is spelled out in the reviews below, all three reviewers have suggestions for improving the manuscript in revision. Most of the issues they raise can be readily addressed by careful revision of the text to openly discuss unresolved questions or to tone down some conclusions . We ask that you prepare a revised version that addresses the issues raised to the best of your ability.This is an important manuscript describing the first near atomic resolution structure of a pannexin channel, a family of large pore channels thought to be important for the movement of ions and small molecules (e.g. ATP) across cellular membranes. The reviewers thought this was an excellent manuscript that is appropriate for publication in Reviewer #1:This manuscript presents for the first time the structure of a pannexin channel. This is a timely and very important finding for the field that will serve as a basis for understanding and studying pannexin-1 channel function.I think it is important to note that in the current (and frenetic) race of structural biologists to determine channel structures, the main concern for physiologists is whether these structures really represent physiological conformations of the channel. I believe the authors prove, at least partially, that this is the case with the frog pannexin-1 channel. The structural interpretations that the extracellular loop 1, in particular, residues at positions W74 and R75 regulate ionic selectivity are nicely supported by their functional data. The functional data supporting inhibition by carbenoxolone at extracellular region are complementary, but less demonstrative.The major concern with this work is with respect to some forced interpretations that do not properly address the limitations of the pannexin-1 structure and require proper clarification. This refers mainly to the lack of reliable structural data related for the cytosolic domains N-terminus (NT), intracellular loops (ICL) and C-terminus (CT), which have been shown to be crucial in gating and permeation of these channels. This needs to be properly acknowledged in the manuscript.Another manuscript describing the pannexin-1 structures from frog and human was published a few days after the current manuscript in BioRxiv. They share similar structural results, and both lack structural information for the N-terminal and C-terminal domain.Essential revisions:1) As stated by the authors, the NT region has been shown to regulate gating and permeation in pannexin-1 channels. In this presented pannexin-1 structure the first 10 residues of the NT region are not resolved. The remaining residues are lining the pore as shown for other large pore channels including connexin, innexin and LRRC8 channels. Importantly, the intracellular loops in the aforementioned structures are critical for stabilizing the N-terminal region. This has been more recently demonstrated for connexin hemichannels. To solve the pannexin-1 structure, the authors had to remove 24 amino acids from the first ICL. How the deletion of this segment could alter the interactions between ICL1, ICL2, and NT, and the potential implications in channel gating or selectivity need to be discussed.2) The structure does not contain a significant part of the C-terminal region, which has been shown to play an important role in the gating of human, rat and mouse Pannexin 1 channels. The lack of the CT in pannexin-1 leads to constitutively open channels in these species, even at negative potential. How is therefore explained that the frog pannexin-1 structure lacking the CT region corresponds to a closed channel conformation as the authors propose?3) The authors suggest that the motif formed by the residues W74 and R75 is an extracellular gate in pannexin-1 channels. They referred to this motif in the text as a unique mechanism of gating, which gives the impression of a unique gate. If W74 is the only gate, one would predict that mutations in W74 (such as W74A) will disrupt the gate and produce a leaky channel, due to the substitution of an amino acid with a short side chain. This does not seem to be observed by the authors. If the mutations do not lead to a leak current, it is likely that there is a second gate for atomic ions.4) Pannexin channel currents for different constructs shown in Figure 1B and Figure 1—figure supplement 2F are not normalized by channel levels. This implies that currents of frPanx1, hPanx1, and frPanx1-ΔLC are not comparable.5) Figure 1—figure supplement 2F suggests that the channels used to determine the cryo-EM structure  are not functional. As noted, the authors needed to add a GS motif in the NT region to increase voltage-sensitivity. This reinforces the idea of a role for the NT in gating.6) The Discussion section is incomplete. I think that many of the concerns in this review need to be addressed by the authors in this section.7) The authors conclude that "our structural and functional study establishes the extracellular loops as the unique structural determinants for channel gating and inhibition in Panx 1…". Considering the above issues this sentence is incorrect. It must be also noted that the inhibition mechanism proposed by the authors is only ascribable to CBX. I think that modeling/docking of CBX with the pannexin-1 structure will reinforce the experimental data and provide a more solid conclusion about the mechanism of blockade by CBX.Reviewer #2:eLife.By combining data from cryo-EM and electrophysiology, the work describes the first structure of a Pannexin channel at high resolution and it sheds light on the determinants of anion selectivity. This is an excellent manuscript that provides important insight into an important ion channel. The data appears to be of high quality and the major conclusions of the manuscript are sound. I thus think that the work is a strong candidate for publication in Essential revisions:– The cryo-EM data is of high quality and it supports the structural claims made in the manuscript. The ring of aromatic residues at the extracellular constriction and the close-by arginine residues are particularly interesting, and the proposed cation-π electron interaction is supported by the data (considering the limited resolution of the map). Whereas the role of both residues for shaping the selectivity properties of the protein have been nicely demonstrated in patch clamp recordings, the evidence for the same region to act as gate is to some degree vague and speculative since it has not been demonstrated that the same residues would provide that largest energy-barrier that obstructs permeation in the closed conformation.– Given the proposal that different region contribute to anion selectivity it would be interesting to know more about the electrostatics within the pore region.– I wonder whether there is evidence that the described Arg residues contribute to the observed voltage dependence of currents. Although the authors have shown traces of recordings of the mutants to demonstrate shifts in the reversal potential, possible changes in the voltage dependence of gating could be better documented. I expect in their structure, the positively charged Arg would be located outside the electric field.– I wonder whether the authors have additional evidence to exclude a potential role of CBX as pore blocker. Is the effect of CBX reversible?– Are small currents after addition of CBX reflect incomplete occupancy of the blocker or residual conduction of the CBX-bound channel and are currents in mutants a consequence of decreased potency or increased conductance of the blocked channel (given the large amount of data presented in the study such experiments might be focus of a future study).– In the electrophysiology section, it would be important to show some comparative data from mock-transfected cells.– Was the change in the junction potential in asymmetric solutions in the selectivity experiments calculated to be significant and was it corrected?Reviewer #3:The manuscript from the Kawate lab titled "The cryo-EM structure of a pannexin 1 channel reveals an extracellular gating mechanism" reports the structure of a pannexin 1 from frog at about 3.0 Å resolution and functional studies that tested structure-based hypotheses on ion selectivity and inhibitor binding site. The work is significant in that this is the first pannexin structure, and that the structure revealed a heptameric assembly different from other large-pore forming channels such as connexins, innexins and LRRC8. The initial FSEC and excellent biochemical work led to identification of a stable construct suitable for structural studies. The procedures for cryo-EM seem straightforward, and the quality of the density map seems consistent with the claimed resolution.I feel that the experiments are all solid and interpretations are sound. I have two comments.First, the study on CBX is a very good follow up on a previous JGP paper by the same lab, and it provides further evidence that extracellular loop 1 harbors the inhibitor. However, short of a CBX-pannexin complex, one could argue that the inhibitor may bind elsewhere and induce a propagated effect that is mediated by the extracellular loop.Second, I see utility of using ion permeation as a read out for testing certain aspects of pannexin, but I do not quite understand the motivation of studying ion selectivity in pannexin. If the main function of a pannexin is to form a large pore to allow release of ATP, would it be more relevant to study permeation of ATP and its gating on a proteoliposome assay? Is there any indication that anion permeation is relevant in physiology? Summary:This is an important manuscript describing the first near atomic resolution structure of a pannexin channel, a family of large pore channels thought to be important for the movement of ions and small molecules (e.g. ATP) across cellular membranes. The mechanisms by which the activity of pannexin channels are regulated are unknown, yet these channels are thought to play important roles in innate immunity, neuronal signaling, apoptosis, cancer and ischemia. Having a structure of the channel will enable further mechanistic studies on how this protein plays important biological roles. The reviewers thought this was an excellent manuscript that is appropriate for publication in eLife. As is spelled out in the reviews below, all three reviewers have suggestions for improving the manuscript in revision. Most of the issues they raise can be readily addressed by careful revision of the text to openly discuss unresolved questions or to tone down some conclusions . We ask that you prepare a revised version that addresses the issues raised to the best of your ability.We thank the reviewers for the valuable comments. As suggested, we have toned down uncertain conclusions and added more discussion throughout the manuscript, especially on the limitations of our current study. We believe our revised and retitled manuscript addresses the concerns raised by the reviewers. Below we summarize our point-by-point answers/comments to the suggestions made by the reviewers.Reviewer #1:[…]Essential revisions:1) As stated by the authors, the NT region has been shown to regulate gating and permeation in pannexin-1 channels. In this presented pannexin-1 structure the first 10 residues of the NT region are not resolved. The remaining residues are lining the pore as shown for other large pore channels including connexin, innexin and LRRC8 channels. Importantly, the intracellular loops in the aforementioned structures are critical for stabilizing the N-terminal region. This has been more recently demonstrated for connexin hemichannels. To solve the pannexin-1 structure, the authors had to remove 24 amino acids from the first ICL. How the deletion of this segment could alter the interactions between ICL1, ICL2, and NT, and the potential implications in channel gating or selectivity need to be discussed.We created 23 different deletions in ICL1 between K155 and K194. Except for the construct that lacks this entire region, all other constructs showed voltage-dependent channel activity in our whole-cell patch clamp experiments. We also investigated these deletion constructs using FSEC and found that the functional constructs were properly assembled into heptamers. Based on these results, we think the deleted region in the loop has insignificant contribution in channel gating. Indeed, the EM density in this region was weak and we could not model the residues between K155 and K194. The NT region on the other hand does seem to play a crucial role in channel gating, as we previously demonstrated . It is possible that the unmodeled NT region may interact with the deleted region of the CTD. We included this information in the Discussion section.2) The structure does not contain a significant part of the C-terminal region, which has been shown to play an important role in the gating of human, rat and mouse Pannexin 1 channels. The lack of the CT in pannexin-1 leads to constitutively open channels in these species, even at negative potential. How is therefore explained that the frog pannexin-1 structure lacking the CT region corresponds to a closed channel conformation as the authors propose?In our hands, CT truncated Panx1 (1-355 in human) does not open constitutively. This is independent of species or intracellular loop deletion. Because frPanx1-ΔLC is closed at 0 mV , we initially thought that the current structure represents a closed state. However, the +GS version of frPanx1-ΔLC shows larger leak currents , leaving a possibility that the CT truncation does promote channel opening but lack of the NT modification somehow renders it closed. If the conformation of the NT in frPanx1-ΔLC is compromised during purification or reconstitution into nanodiscs, it is possible that our structure may actually look closer to the +GS version, which may then represent an open state. We included this discussion in our revised manuscript.3) The authors suggest that the motif formed by the residues W74 and R75 is an extracellular gate in pannexin-1 channels. They referred to this motif in the text as a unique mechanism of gating, which gives the impression of a unique gate. If W74 is the only gate, one would predict that mutations in W74 (such as W74A) will disrupt the gate and produce a leaky channel, due to the substitution of an amino acid with a short side chain. This does not seem to be observed by the authors. If the mutations do not lead to a leak current, it is likely that there is a second gate for atomic ions.We agree with the reviewer. Because we do not have sufficient data to confirm W74's role as a channel gate, we removed the phrase "extracellular gate" from our manuscript.4) Pannexin channel currents for different constructs shown in Figure 1B and Figure 1—figure supplement 2F are not normalized by channel levels. This implies that currents of frPanx1, hPanx1, and frPanx1-ΔLC are not comparable.That is true. We do not intend to compare those constructs quantitatively, but aim to qualitatively demonstrate that all three constructs present voltage-dependent channel activity when the N-termini are modified with the addition of Gly-Ser.5) Figure 1—figure supplement 2F suggests that the channels used to determine the cryo-EM structure  are not functional. As noted, the authors needed to add a GS motif in the NT region to increase voltage-sensitivity. This reinforces the idea of a role for the NT in gating.We agree that the NT region plays essential roles in pannexin channel gating. Unfortunately, our current EM structure falls short of proposing a possible mechanism, as the first 10 amino acids are not modelled. As suggested by the reviewers, we expanded the Discussion section to clarify the limitation of our studies.6) The Discussion section is incomplete. I think that many of the concerns in this review need to be addressed by the authors in this section.Thank you for the comments. Our revised Discussion section should address the concerns raised by the reviewers.7) The authors conclude that "our structural and functional study establishes the extracellular loops as the unique structural determinants for channel gating and inhibition in Panx 1…". Considering the above issues this sentence is incorrect. It must be also noted that the inhibition mechanism proposed by the authors is only ascribable to CBX. I think that modeling/docking of CBX with the pannexin-1 structure will reinforce the experimental data and provide a more solid conclusion about the mechanism of blockade by CBX.We used "unique" in a sense that it is unusual for an ion channel to have tryptophan residues surrounding the most constricted region of the pore. However, we agree with the reviewer that the sentence is misleading. We therefore toned down its potential role as a gating machinery throughout the manuscript. We also agree that further studies are necessary to clarify the action mechanism of CBX.Reviewer #2:[…]Essential revisions:– The cryo-EM data is of high quality and it supports the structural claims made in the manuscript. The ring of aromatic residues at the extracellular constriction and the close-by arginine residues are particularly interesting, and the proposed cation-π electron interaction is supported by the data (considering the limited resolution of the map). Whereas the role of both residues for shaping the selectivity properties of the protein have been nicely demonstrated in patch clamp recordings, the evidence for the same region to act as gate is to some degree vague and speculative since it has not been demonstrated that the same residues would provide that largest energy-barrier that obstructs permeation in the closed conformation.Thank you for the comments, which we agree. As described above, we have toned down the potential extracellular gating mechanism.– Given the proposal that different region contribute to anion selectivity it would be interesting to know more about the electrostatics within the pore region.Thank you for the suggestion. The electrostatic potential map revealed that the ion permeation pathway is mostly basic except for a highly acidic region near the constriction formed by W74. This charge distribution is consistent with an anion channel with a selectivity machinery located near W74. We now include an extra figure  and discussion in the text (subsection “Ion permeation pathway and selectivity”).– I wonder whether there is evidence that the described Arg residues contribute to the observed voltage dependence of currents. Although the authors have shown traces of recordings of the mutants to demonstrate shifts in the reversal potential, possible changes in the voltage dependence of gating could be better documented. I expect in their structure, the positively charged Arg would be located outside the electric field.We did observe various degrees of voltage dependence from different R75 mutants. However, it is challenging to assess the contribution of the positively-charged side chain in voltage gating, as the interaction with W74 also seems to affect voltage dependence. One clear thing is that the positive charge at this position is not necessary for voltage-dependent channel activity, as R75A or even R75E give rise to voltage-dependent currents.– I wonder whether the authors have additional evidence to exclude a potential role of CBX as pore blocker. Is the effect of CBX reversible?Yes, the effect of CBX is reversible. We previously demonstrated that voltage-dependent channel activity of W74A, W74I, or W74K is potentiated by CBX . In combination with our systematic mutagenesis studies, CBX is more likely to be an allosteric inhibitor than a channel blocker.– Are small currents after addition of CBX reflect incomplete occupancy of the blocker or residual conduction of the CBX-bound channel and are currents in mutants a consequence of decreased potency or increased conductance of the blocked channel (given the large amount of data presented in the study such experiments might be focus of a future study).This is an interesting point. Our previous single channel recordings suggest that CBX reduces the channel open probability without changing the conductance . This suggests that the residual currents after CBX application reflect incomplete occupancy. But we agree that further experiments are necessary to clarify this.– In the electrophysiology section, it would be important to show some comparative data from mock-transfected cells.We have already published several responses from mock-transfected cells that confirm the channel activities in our lab indeed come from Panx1 .– Was the change in the junction potential in asymmetric solutions in the selectivity experiments calculated to be significant and was it corrected?We corrected junction potentials experimentally using an agar bridge.Reviewer #3:[…]I feel that the experiments are all solid and interpretations are sound. I have two comments.First, the study on CBX is a very good follow up on a previous JGP paper by the same lab, and it provides further evidence that extracellular loop 1 harbors the inhibitor. However, short of a CBX-pannexin complex, one could argue that the inhibitor may bind elsewhere and induce a propagated effect that is mediated by the extracellular loop.We appreciate the comments. We agree with the reviewer that CBX may bind outside of the tested residues. And yes, CBX-pannexin complex structure will be very helpful.Second, I see utility of using ion permeation as a read out for testing certain aspects of pannexin, but I do not quite understand the motivation of studying ion selectivity in pannexin. If the main function of a pannexin is to form a large pore to allow release of ATP, would it be more relevant to study permeation of ATP and its gating on a proteoliposome assay? Is there any indication that anion permeation is relevant in physiology?While ATP release has been widely studied, anion permeation through pannexin channels has also been implicated in physiology . The suggested in vitro ATP-release assay would be interesting.


Chronic non-puerperal uterine inversion is rare and usually associated with uterine pathology with a diagnosis that is challenging. We present the case of a 47-year-old para 4 Caucasian woman with a history of polyfibromatous uterus who was admitted for severe vaginal bleeding for the past 48 hours associated with hemodynamical instability and was refusing any surgery. Pelvic MRI showed the uterus presenting an unusual appearance with a highly vascularized intracavitary leiomyoma protruding through the cervix. Upon deterioration of her status despite an optimal blood transfusion, resuscitation and anti-fibrinolytic treatment, she accepted total abdominal hysterectomy. The diagnosis of uterine inversion was made intraoperatively and confirmed on histopathologic examination. It revealed two side-by-side benign fundal leiomyomas which had collapsed the fundus and protruded partly from the cervix. Non-puerperal chronic inversion of the uterus is rare, and its diagnosis should be based on ultrasound, pelvic MRI and a high index of suspicion, allowing rapid diagnosis and treatment and thus decreasing patient morbidity and mortality. Uterine inversion is the folding of the fundus into the uterine cavity through the cervix. It is usually a serious postpartum complication leading to massive hemorrhage which generally occurs in the third stage of labor. Chronic uterine inversion is very rare and is observed at least four weeks after delivery . About one-sixth of all inversion cases are spontaneous, non-puerperal, and are called chronic or gynecological uterine inversions -3. In soA 47-year-old Caucasian female, gravida 4 para 4 , came to the Emergency Department for severe vaginal bleeding in the past 48 hours associated with syncope and hemodynamical instability. She had been complaining of intermittent menometrorrhagia over the past year that was attributed to a polyfibromatous uterus visualized on abdominal ultrasound, but the patient had declined all medical and surgical treatments. Two years prior to this event, she had a non-complicated hysteroscopic myomectomy for a 4 cm intracavitary myoma with no malignancy at the pathology.Upon admission, the patient appeared pale but responsive with a pulse of 105 bpm, blood pressure (BP) 130/80 mmHg, and a hemoglobin value of 5.6 g/dL. Clinical examination revealed an enlarged uterus with a 6-7 cm firm non-sensitive mass protruding from the cervix. Transfusion was initiated upon the patient's initial refusal of a surgical treatment along with anti-fibrinolytic treatment to stop the bleeding. A pelvic MRI enhanced with gadolinium was interpreted with caution as it showed a 12.8 x 10 x 9.5 cm uterus presenting an unusual bell shape with a highly vascularized 11 x 7.9 x 6.6 cm intracavitary lesion (most probably a leiomyoma) that was protruding through the cervix  and two fresh frozen plasma (FFP). She made a full uneventful recovery and was discharged home on day four.This case was submitted to our institution's ethics committee for approval and was registered as CEHDF-1137.Non-puerperal uterine inversion is extremely rare, representing 16% of all uterine inversions .Uterine inversion can be classified into four stages as described by Salomon et al.: Stage 1: the inverted uterus remains in the uterine cavity; Stage 2: complete inversion of the fundus through the cervix; Stage 3: the inverted fundus protrudes through vulva; and Stage 4: inversion of the uterus and vaginal wall through the vulva [The clinical diagnosis of chronic uterine inversion mostly depends on the finding of a mass protruding out of the cervix (for Stages 2-4) and the absence of a uterine body during bimanual examination. The clinical diagnosis of Stage 1 is difficult and requires a high index of suspicion and appropriate imaging. The prominent symptoms are vaginal discharge, irregular uterine bleeding, and pelvic discomfort [The etiology of the inversion is not clearly defined and it appears to be multifactorial. The following etiologies have been described in the literature: uterine leiomyoma, leiomyosarcoma, rhabdomyosarcoma, endometrial polyps, endometrial carcinoma, and total uterovaginal prolapse ,2,5. In Prolapse and extrusion of a leiomyoma, especially a submucous leiomyoma of the fundus, tends to be the most common factor inciting the inversion in 80-85% of cases, along with other etiologies such as hormone replacement and the presence of increased intra-abdominal pressure ,6. A 201Ultrasound and MRI are important imaging tools that play a major role in the diagnosis of this entity. Ultrasound findings include a depressed longitudinal groove extending from the uterus to the center of the inverted uterus . Lewin eChronic uterine inversion is difficult to treat conservatively and surgery is usually needed. The morbidity and mortality associated with uterine inversion correlate with the degree of hemorrhage and the rapidity of diagnosis and treatment . ChronicSpontaneous non-puerperal chronic uterine inversion is an exceptional and life-threatening disease that most gynecologists will rarely encounter in their career. It is usually associated with uterine pathology. Careful examination, imaging studies such as pelvic ultrasound and MRI coupled with a high index of suspicion will yield correct diagnosis and treatment that would help to decrease patient morbidity and mortality.