Aim: The aim was to determine the role of aging and exercise training on endothelial mechanosensor proteins and the hyperemic response to shear stress by passive leg movement.Methods: We examined the expression of mechanosensor proteins and vascular function in young  and old  healthy male subjects with eight weeks of aerobic exercise training. Before and after training, the hyperaemic response to passive leg movement was determined and a thigh muscle biopsy was obtained before and after passive leg movement to assess the acute effect of increased shear stress. Biopsies were analyzed for protein amount and phosphorylation of mechanosensor proteins; Platelet endothelial cell adhesion molecule-1 (PECAM-1), Vascular endothelial cadherin, Vascular endothelial growth factor receptor-2 and endothelial nitric oxide synthase (eNOS).Results: Before training, the old group presented a lower hyperaemic response to passive leg movement and a 35% lower (P < 0.05) relative basal phosphorylation level of PECAM-1 whereas there was no difference for the other mechanosensor proteins. After training, the eNOS protein amount, the amount of PECAM-1 protein and the passive leg movement-induced phosphorylation of PECAM-1 were higher in both groups. The hyperaemic response to passive leg movement was higher after training in the young group only.Conclusion: Aged individuals have a lower hyperaemic response to passive leg movement and a lower relative basal phosphorylation of PECAM-1 than young. The higher PECAM-1 phosphorylation despite a similar hyperemic level in the aged observed after training, suggests that training improved shear stress responsiveness of this mechanotransduction protein. In vivo in humans, the vascular response to increased flow and thereby shear stress can be evaluated by examining the hyperaemic response to passive leg movement  the lower hyperemic response to shear stress in older men is associated with impairments in the endothelial mechanosensor complex and eNOS activation and (b) shear stress induced hyperemia is improved after a period of exercise training and paralleled by improved ability of the mechanosensor proteins to sense and respond to shear stress resulting in increased eNOS phosphorylation.The study was approved by the Ethics Committee of Copenhagen and Frederiksberg communities (H-6-2014-085) and was conducted in accordance with the latest guidelines of the Declaration of Helsinki. Written informed consent was obtained from all subjects before enrolment in the study.Fourteen young (20–28 years) and 14 older (66–80 years) healthy men participated in the study. Before enrolment in the study, all subjects were screened by electrocardiography and their medical history and level of daily physical activity were registered. All included subjects were non-smokers without any known diseases, and were not engaged in regular intense physical activity or training (<2 weekly sessions of moderate to intense exercise).Each subject completed 8 weeks combined training consisting of two weekly intense bicycle ergometer (spinning) sessions and 1 cross-training session . Briefly, spinning sessions consisted of repeated, intense  5-min intervals separated by 1–2-min active recovery periods. Training intensity and duration was progressively increased with three intervals during training week 1 and 2 and 5 intervals during week 3–8. Training intensity was monitored with an online heart rate monitoring system during each session . The heart rate response was subsequently analyzed (Polar ProTrainer 5 software) for determination of heart rate response and distribution during training. Both groups showed similar training compliance during the 8 week intervention, and training intensity was equally high in both groups (Table O2peak) and for familiarization with the passive leg movement on an one-leg knee-extensor model. Body composition was determined in the morning in a fasted state with whole-body dual-energy X-ray absorptiometry scanning . The passive leg movement was performed on a knee extensor ergometer  with an incremental exercise test on a mechanically braked cycle ergometer . The incremental exercise test consisted of a 4-min submaximal bout at 50 and 100 W for old and young, respectively. Subjects then rested for 2 min after which the incremental test began at the work load corresponding to the submaximal bout and increased by 25 W min−1 until volitional exhaustion. O2peak was calculated as the average of the three highest consecutive 15 s values. Recognition of true O2peak required three of the following five criteria to be met: Individual perception of exhaustion, respiratory exchange rate (RER) >1.15, VO2 curve plateau, heart rate approaching age-predicted maximum, or inability to maintain pedaling frequency above 80 RPM. Verbal encouragement was offered throughout the test. Heart rate was measured throughout the exercise protocol .Pulmonary −1, AstraZeneca, Sweden). After insertion of the probes, subjects rested for 120 min to allow the muscle to attain a resting state and to recover from insertion trauma. The microdialysis probes were perfused with Ringer acetate buffer  at a rate of 5 μl min−1 and to determine the relative exchange over the membrane, a small amount (2.7 nM) of [2–3H] labeled adenosine was added to the perfusate to allow for calculation of probe recovery. Microdialysate was collected during the final 20 min of the 120 min rest, excluding the first 2 min to account for delay in the probe perfusate. Immediately after collection, samples were weighed and triplicates of 5 μl of dialysate were allocated into 3 ml Ultimate Gold scintillation liquid . The [2-3H] adenosine activity of the dialysate was measured on a liquid scintillation counter . The remaining dialysate was immediately frozen at –80°C. Probe recovery (PR) was calculated as [PR = ], where dpm denotes disintegrations per minute  was inserted into the antecubital vein for blood sampling. Blood was drawn at rest and after 20 min of passive leg movement . Two micr minute . The pro−1) rejected noise caused by turbulence at the vascular wall. Doppler tracings and B-mode images were recorded continuously and Doppler tracings were averaged over 45 s. Resting femoral arterial blood flow was measured before passive leg movement. During the 5 min passive leg movement, femoral arterial blood flow was measured at 15 s intervals during the initial 3 min, and after 4 and 5 min. Peak blood flows and mean blood flow for the first 5 min are reported herin.Passive leg movement was conducted by the subject being seated in a knee extensor model and the lower leg being moved back and forth in a kicking movement by one of the investigators at a frequency of 60 cycles per minute . The sub−1, AstraZeneca, Sweden) using the percutaneous needle biopsy technique with suction . The lysates were diluted to appropriate protein concentrations in a concentrated sample buffer , and equal amount of total protein were loaded for each sample in different wells on precasted Tris–HCL gels . For comparisons, samples from the same subject were always loaded on the same gel. After gel electrophoresis, the proteins were transferred (semidry) to a polyvinylidene difluoride membrane , which was incubated with ∼10 ml of primary antibody over-night and then washed three times for 5 min in Tris-buffered saline-Tween before incubation with secondary antibody for 1 h. The membranes were incubated with the following primary antibodies: VEGF-R2 ; VEGF-R2Tyr1175 ; eNOS ; PECAM-1 ; PECAM-1Tyr713 ; VE-cadherin ; VE-cadherinTyr731 . Secondary antibodies used were goat anti-rabbit or rabbit anti-goat HRP-conjugated antibodies . Following detection and quantification , the protein content was expressed in arbitrary units. The blots were also analyzed for glyceraldehyde 3-phosphate dehydrogenase , actin  or alpha tubulin  depending on molecular weight of the protein that was studied to check for loading inconsistencies  two times for 30 s . After rotation end-over-end for 1 h, the samples were centrifuged for 30 min at 17,500 × s Figure . Due to s Figure  the specs Figure  and reprser1177 . Sections were then incubated with goat anti-mouse IgG1 546 (eNOS) and goat anti-rabbit IgG 633 (eNOSser1177) secondary antibodies , in combination with Ulex europaeus I lectin–FITC conjugate  as a marker of the endothelium. Coverslips were then applied using a glycerol and Mowiol 4-88 solution.The procedures for immunofluorescence staining and subsequent image analysis has been adapted from the original method described by Cocks et al. . TransveImages were acquired using an inverted confocal microscope  with a 40× oil immersion objective. FITC fluorescence was excited with a 488 nm multiline argon laser and detected with 493–557 nm emission. Alexa Fluor 546 and 633 fluorophores were excited with 543 and 633 nm lines of the helium–neon laser and 548–623 and 638–747 nm emission, respectively. The images were acquired at a resolution of 1,024 × 1,024 pixels and stored in 24-bit tagged image format file format. Identical settings were used for all image capture within each participant. The mean number of views assessed was 6 ± 2. The number of capillaries quantified was approx. 30.ser1177 images. Fluorescence intensity was then quantified within the endothelial specific area. The mean fluorescence intensity of Pre-training Pre-passive movement samples were normalized to a value of 1, and the relative intensity of subsequent samples was calculated. In addition, as eNOS and eNOSser1177 were dual stained it allowed for assessment of eNOS/eNOSser1177 ratio on an individual vessel basis to be calculated. Changes in eNOS/eNOSser1177 ratio from the immunohistochemical analysis were used to detect changes in phosphorylation status going from rest to passive leg movement. The immunofluorescence analysis employed allowed for assessment of eNOS phosphorylation specifically within the endothelium, and as such a more sensitive assessment of changes to the passive leg movement than would be allowed using western blotting on whole muscle homogenates  in plasma and microdialysate, were measured using a fluorometric EIA kit . Interstitial concentrations were estimated based on the relative recovery of [2-ompounds .a-level of P < 0.05 at a power level of 0.8. Data are means ± SD.Subject number is based on power calculations of selected primary outcome measures. These include average differences and variability in femoral arterial blood flow in response to passive leg movement and protein expression and phosphorylation of mechanosensors and eNOS in muscle homogenates from m. vastus lateralis . Signifipost hoc procedure.A linear mixed-model approach  was used to investigate differences within and between groups. Fixed factors were “group” (young or old), “intervention” (before or after training) and “time” (rest or passive leg movement). Subjects were specified as a repeated factor and identifier of random variation. The homogeneity of variance and normal distribution was confirmed through residual and Q–Q plots. Pairwise differences were identified using the unadjusted difference O2peak and O2peak kg−1, were higher (P < 0.05) in the young compared to the old group (Table P < 0.05) both variables in the young and the old group. Both groups showed similar compliance with the training intervention  in the young group but not in the old. The peak change in blood flow from rest to passive movement was also lower . Blood flow traces from both groups before and after training are presented in Figures Peak and mean femoral arterial blood flow in response to passive movement was 25% lower .Before training, protein expression and relative phosphorylation levels of VE-CadherinP = 0.008, Figure P < 0.001) in both the young and old group and the level was 58% higher in the old compared to the young group (P = 0.013). Absolute PECAM-1tyr713 phosphorylation was not different between the young and old before or after training .Before training, PECAM-1 protein levels were 23% higher in the old compared to the young group  whereas it remained similar in the young group.Before training, passive leg movement increased the relative phosphorylation of eNOStyr713, VE-Cadherintyr731 and VEGF-R2ser1175 was not changed by passive leg movement in either the young or the old group  before training, compared to the young group, the older group presented a lower hyperaemic response to increased shear stress induced by passive leg movement, which was accompanied by a lower basal relative phosphorylation status of the endothelial mechanosensor PECAM-1; (II) eight weeks of exercise training resulted in a higher PECAM-1 protein level and an enhanced passive leg movement induced phosphorylation of PECAM-1 in both the young and the older subjects; (III) exercise training resulted in a higher expression of eNOS and a greater total eNOS phosphorylation in both young and aged individuals; (IV) after training, the hyperemic response to passive leg movement was higher in the young subjects. Combined, the results provide novel evidence for that the mechanosensor protein PECAM-1 is plastic in that both the protein amount and the phosphorylation status appear to be sensitive to aging and the level of physical activity. Although the changes in PECAM-1 observed with training were not paralleled by an improved hyperemic response during passive limb movement, there was a greater increase in phosphorylation of PECAM-1 for the same level of hyperemia in the old group, suggesting improved shear stress responsiveness.There is convincing evidence for that reduced availability of NO plays a central role in the age induced impairment in vascular function  includinser1177 phosphorylation was increased in the old but not the young subjects with passive leg movement. Although our data support that eNOS can be phosphorylated by shear stress in older subjects, overall these findings only partially support in vitro studies showing activation of the mechanosensor complex and eNOS in response to shear stress  were assessed in plasma and interstitial fluid before and after the training period but there was no direct relationship to eNOS level and eNOS phosphorylation. This lack of relationship may be due to that NOx is a rather rough estimate of NO bioavailability and the levels are affected by diet which was not controlled for in the current study.tyr713 phosphorylation in both groups. PECAM-1 has several other additional physiological roles of this protein in the vasculature (in vivo, the fact that the hyperemic response to passive leg movement was similar in the old group after training compared to before, whereas the change in PECAM-1 phosphorylation was increased, suggest a similar degree of shear stress and thus an enhanced shear stress induced activation. Although we could not detect an association with eNOS phosphorylation, the evident sensitivity of PECAM-1 both to aging and training provides evidence in humans for that this protein may be an important molecule in the development of vascular disease as proposed based on numerous in vitro studies (Out of the three proteins in the mechanosensor complex, the PECAM-1 protein content was enhanced in both the young and the old group after the training period. This increased PECAM-1 protein expression was paralleled by an increase in the passive leg movement induced relative PECAM-1culature . PECAM-1culature  angiogenculature . Moreoveculature . The pre studies . The neeper se. Accordingly, they had good cardiovascular health and it is plausible that greater differences in the mechanosensor complex and in the hyperaemic response to passive movement would be observed in older individuals with poorer health status.Passive leg movement was used to induce shear stress without muscle metabolic signaling. However, passive leg movement only induced activation of PECAM-1 and not VE-CAD, or VEGF-R2 and it is possible that a stronger shear stress stimulus or added metabolic signaling by active exercise, would activate the mechanosensor complex to a greater extent. It is also plausible that other mechanosensors, e.g., luminal G protein receptors and integrins also plays a functional role in determining the hyperemic response to passive leg movement. The older subjects recruited to the current study were healthy as the purpose was to evaluate the effect of age in vivo model in humans the drawback is that the potential differences in the size and number of the arterioles in the microcirculation, as well as the perfusion pattern, in the muscle is unknown and thereby its potential influence on the actual shear stress. However, aging has been associated with a lower capillarization (It could be speculated that the level of capillarization explains the differences and changes in PECAM-1. Shear stress is dependent on blood flow, blood vessel diameter and viscosity of the blood. In using an rization  and, it in vivo evidence in humans for plasticity of PECAM-1 and improvements in mechanosensing; factors which are likely to contribute to the known beneficial effects of physical activity on vascular function. The lack of association between the hyperemic response to passive leg movement and eNOS activation nevertheless warrants further investigation.Our results demonstrate that the impaired shear stress induced hyperemia in aged is not immediately explained by a lower eNOS activation but there appears to be an association with the phosphorylation status of the important mechanosensor protein PECAM-1. Moreover, that the finding that exercise training leads to enhanced PECAM-1 phosphorylation in response to acute passive leg movement, despite a similar increase in blood flow, agrees with our hypothesis that the responsiveness of mechanosensory proteins can be improved by training. Overall, our study provides the first LG and YH conceived and designed the study. LG, NR, MC, and YH analyzed and interpreted the data. All authors collected the data, drafted the article or revised it critically for important intellectual content, approved the final version, and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Since 2010 the National Institute for Health and Care Excellence has recommended screening adults for excessive alcohol consumption to try and help prevent alcohol-use disorders. Little is known about the extent to which these recommendations are followed, and the resulting completeness and validity of alcohol-related data recording in primary care.To investigate the completeness and accuracy of recording of alcohol use within primary care records in the UK.Cross-sectional study in the Clinical Practice Research Datalink.st January 2018 with ≥1 year of follow-up.We included all adult patients registered on 1We calculated prevalence of alcohol consumption recording overall and within patient groups. We then validated alcohol consumption data against recommended screening tools (Alcohol Use Disorders Identification Test (AUDIT)) as the gold standard. We also calculated how prevalence of alcohol recording changed over the preceding decade.In 2018, among 1.8 million registered adult patients, just over half (51.9%) had a record for a code related to alcohol in the previous 5 years. Recording of alcohol consumption was more common among women, older people, ex-smokers and those from more deprived areas, who were overweight/obese, or with comorbidities. A quarter of patients had units per week recorded in the last 5 years, but <10% had an AUDIT or Fast Alcohol Screening Test (FAST) alcohol screening test score. The recorded alcohol measures corresponded to results from gold standard AUDIT scores. The distribution of consumption among current drinkers was similar to the Health Survey for England.Half of adults in UK primary care have no recorded alcohol consumption data. When consumption is recorded, we have demonstrated internal and external validity of the data, suggesting greater recording may help identify opportunities for interventions to reduce harms. This study investigated alcohol recording across nearly 1.8 million UK adults in primary care, providing the power to understand in detail how alcohol use is recorded in general practice.We demonstrated the validity of alcohol recording internally within general practice records, and externally compared with the Health Survey for England.Though we demonstrated levels of alcohol recording and screening, it does not mean that general practitioners have acted on the screening result, or that they have screened at an appropriate time.Hazardous alcohol use is a key behavioural risk factor affecting health. It is estimated that over 20% of adults in England are drinking at levels that are considered harmful,Identification of high-risk patients can usefully be undertaken in primary care, where appropriate interventions can be promoted. The Alcohol Use Disorders Identification Test (AUDIT)et alKhadjesari Using primary care data from the CPRD, this study assessed the utility of recorded alcohol data in primary care by assessing completeness of recording of alcohol status at different levels and by various patient level factors, and comparing measures of alcohol recorded in the CPRD internally and to external sources. Our study also offers insight into the completeness of alcohol data in primary care since the introduction of 2010 NICE guidance,st January 2018.We undertook a cross-sectional study with data from the Clinical Practice Research Datalink GOLD database, an ongoing primary care database containing the anonymised medical records from general practitioners in the UK using the Vision software to capture health record data with coverage of 15.4 million patients. Data from CPRD includes diagnoses, tests, clinical measurements, prescriptions and specialist referrals. CPRD uses Read morbidity coding to summarise each patient encounter with a code (or codes) that correspond to a standard set of clinical terminology. Roughly half of all practices in the CPRD have consented to data linkage and therefore deprivation data (based on quintiles of the Indices of Multiple Deprivation) is available for these practices. The study included all patients in CPRD GOLD who were 18 years or over, alive and registered, for at least 1 year, with a CPRD GOLD practice on 1We investigated a range of potential factors that may be associated with alcohol-use recording in primary care: age, sex, deprivation, geographical region, selected comorbidities related to alcohol use that could be captured in electronic health record data  or those where health function monitoring is likely to be more frequent/complete (diabetes mellitus), time since registration at the current practice (1 to 5 years vs ≥5 years), body mass index (BMI), smoking status and ethnicity.st January 2018). We used existing morbidity code lists and algorithms to define smoking status, BMI and ethnicity.We identified liver disease, hypertension and diabetes mellitus based on presence of a recorded diagnosis at any time prior to the study date. We identified depression and anxiety based on the presence of a diagnosis at any time within the 5 years prior to the study date  or FAST screening.Codes quantifying alcohol use, comprised of:drinking status ,level of drinking ,units per week,AUDIT, AUDIT-C or FAST scores . We have also provided further detail regarding variable definitions in the We have made all morbidity code lists used in this study available to download  as the gold standard for alcohol recording. The AUDIT score is a 10-item instrument with a maximum score of 40 designed specifically for screening for hazardous or harmful alcohol use in primary care.st January 2018 (each patient could have codes recorded in more than one category). We then described alcohol recording prevalence in strata of individual characteristics . We also described the frequency of consultations with a record of an alcohol code in the 5 years before the study date, and the number of days from a patient’s registration to their first record of an alcohol use code.To investigate the completeness of alcohol recording, we estimated the proportion of patients with a record for one or more morbidity codes relating to alcohol in the categories outlined above  Any code suggesting that alcohol was discussed in the consultation; (ii) Codes indicating AUDIT, AUDIT-C or FAST screening or (iii) Codes quantifying alcohol use) in the 5 years prior to 1same date and the same patient. Full AUDIT versus AUDIT-C screening were considered separately. We calculated median AUDIT and AUDIT-C scores for each category of level of alcohol use recording: (a) drinking status, (b) drinking level and (c) units per week.We validated codes quantifying the level of alcohol use ) against an AUDIT score from the Similarly, we also validated codes indicating drinking status, drinking level and AUDIT/AUDIT-C scores against the number of units per week. For each variable, we only included a patient’s most recent record (to the study date).We identified current alcohol-use status for each individual with a relevant primary care alcohol record by identifying the most recent record indicating alcohol intake; that is, either a morbidity code indicating alcohol-use status (classified as current or non-/ex-drinkers) or a record indicating the number of units of alcohol consumed per week. We described current alcohol-use status separately for men and women from CPRD, and compared the results to the Health Survey for England 2016.st January each year between 2009 and 2018. We estimated the number of patients who would be eligible for the study, using the same criteria as above , and calculated the proportion of patients with a record of alcohol consumption (based on a record of drinking status), and level of drinking  over the past 5 years.To understand how alcohol recording has been changing over time, we carried out a cross-sectional study on the 1In the main analysis, we restricted the validation of alcohol codes to cases where patients had an AUDIT/AUDIT-C score recorded on the same date as another alcohol code of interest. In sensitivity analyses, we included cases where any AUDIT/AUDIT-C score was recorded up to 30 days before or after the code of interest.In the main analysis, we recorded liver disease, hypertension and diabetes mellitus based on presence of a diagnosis at any time prior to the study date. We tested this definition by repeating the analysis limiting to diagnoses of these comorbidities within 5 years prior to the study date.In the main analysis, we recorded depression and anxiety based on presence of a diagnosis within 5 years prior to the study date. We tested this definition by repeating the analysis limiting to diagnoses of these comorbidities within 12 months prior to the study date.Data were analysed using Stata V.14 .Neither patients nor the public were involved in the design or analysis of the study. This work uses data provided by patients and collected by the UK National Health Service as part of their care and support.st January 2018, and therefore included in the study; 49.4% were male, and their mean age was 49.7 years. The mean duration of registration prior to the study date was 18.7 years (SD 14.5 years). One-fifth had hypertension, 2.0% had liver disease, 6.8% had diabetes, 6.7% had depression and 4.6% had anxiety.There were 1 768 651 adults aged 18 years or over, alive and registered, for at least 1 year, with a CPRD practice on 1Overall, 918 254 (51.9%) patients had a record indicating that alcohol was discussed within the last 5 years. This included diagnoses of alcohol use disorders, their management, harm due to alcohol consumption, in addition to the consumption of alcohol.Drinking status  was recorded in 862 330 (48.8%) of patients, though this was not consistent across patient groups. Recording of drinking status was higher in women compared with men (52.7% in women vs 44.7% in men), increased with age  and was more common among recently registered patients (in the last 1 to 5 years) than in those registered for 5 or more years . There were also differences in recording between geographical region , by deprivation (recording was more common in deprived areas) and by ethnicity (highest for white patients). Patients who had missing smoking or BMI data had particularly low levels of alcohol status recorded, but even in those with smoking status recorded, drinking status was only recorded in approximately 50% to 60% of patients. If smoking and BMI were recorded there were differences in the recording of alcohol status at different levels of smoking/BMI: for example, ex and current smokers were more likely to have alcohol status recorded than non-smokers, as were patients who were overweight. Patients were also more likely to have alcohol status recorded with each of the morbidities investigated . Patients with diabetes were the most likely to have alcohol levels recorded  .Overall, 862 642 (48.8%) of adult patients had some level of alcohol consumption (including non-drinkers) recorded in the previous 5 years, identified through recording of Read codes , units per week or formal alcohol screening score using AUDIT, AUDIT-C or FAST.Level of alcohol consumption data were recorded using Read coding for 31.6% of patients, in units per week for 24.4% of patients and via a record of formal alcohol screening for 11.5% of patients with a Read code indicating that they had completed an AUDIT, AUDIT-C or FAST questionnaire, but only 8.5% of patients had the actual score available.Recording of level of alcohol consumption by patient and practice characteristics are shown in Among patients with a recorded drinking status, 20.3% were non-drinkers, 3.7% ex-drinkers and 76.1% current drinkers. If we use all data  to derive drinking status based on recorded status, units per week and AUDIT scores, an additional 35 105 patients (2.0% of eligible patients) have a drinking status. This derived status shows that 20.8% are non-drinkers, 3.5% ex-drinkers and 75.7% are current drinkers.In our study population, there were 77 212 (4.4%) patients with at least one AUDIT score record available, 21 099 (1.2%) patients with at least one AUDIT-C score record available and 431 394 (24.4%) with units-per-week records. Current drinking (based on Read coding) was associated with a median AUDIT-C score of 3 (IQR 2 to 5), which is under the standard threshold defining risky drinking, and non-/ex-drinkers had a median AUDIT-C score of 0. For those with full AUDIT scores, the non- and ex-drinkers had median scores of 0, and current drinkers had a score of 3 (IQR 1 to 5). On average, current drinkers drank six units per week (IQR 2 to 14).Light and moderate drinkers had low AUDIT and AUDIT-C scores, while heavy drinkers had a median AUDIT-C score of 8 (IQR 6 to 9) and a median AUDIT score of 6 (IQR 5 to 9), which indicates alcohol education. Light drinkers drank three units per week (IQR 1 to 8), moderate drinkers six per week (IQR 2 to 14) and heavy drinkers 18 per week (IQR 9 to 30).Units per week increased with increasing AUDIT and AUDIT-C scores, but even the highest category of units per week (43+) only had a median AUDIT score of 11 (IQR 8 to 12), which indicates simple alcohol advice.The majority (92.5%) of patients had scores in the lowest category  consumed fewer units per week; median four units per week (IQR 2 to 8) compared with a median of 12 units per week (IQR 6 to 20) in the high risk category (score ≥5). Where there were records for both AUDIT and AUDIT-C, in those with high risk AUDIT-C scores (which would indicate the need for a full AUDIT) the median AUDIT score was 6 (IQR 5 to 7).For men and women, the proportions of current and non-drinkers identified using Read-coding in CPRD were close to those estimated in Health Survey for England (HSE) . Units per week were also broadly comparable between CPRD and HSE although prevalence of higher risk drinking in CPRD looked slightly lower . HoweverAmong patients with at least one alcohol record in the last 5 years, 48.9% had just one record, 21.8% had two records, 10.8% had three records and the remaining 48.5% had four or more records; 51.4% of patients with a record had their first alcohol record on the date of registration, 84.4% within the first month and 95.5% within the first year of registration.For patients with more than one record of alcohol consumption, the median duration between records was 339 days (IQR 175 to 452). This was shorter for patients with heavy consumption (237 days (IQR 70 to 432) compared with light drinkers (median 351 (195 to 459 days)) or non-drinkers (median 335 (179 to 438 days)).Internal validity where we required that AUDIT score and units per week were recorded within 30 days of another code (rather than on the same date) resulted in more records being validated, but the results were identical to the main analysis .Individuals with diabetes mellitus, hypertension and liver disease diagnosed in the 5 years prior to the study date had higher alcohol recording than shown in the main analysis  .Similarly, depression and anxiety diagnosed in the past year had higher levels of alcohol recording than when diagnosed in the previous 5 years .In 2018, information about alcohol consumption was available for roughly half of all adult patients registered with practices contributing to the CPRD. Large differences in recording were seen across patient characteristics, in particular increasing with age, deprivation and higher in patients with liver disease, hypertension, diabetes mellitus, depression and anxiety. Patients who were missing data on other risk factors such as smoking, BMI and ethnicity were also less likely to have any recording of alcohol use. Those with missing alcohol, smoking, BMI and ethnicity data may represent a group who have minimal contact with primary care. However, even among those with smoking recorded, general practitioners (GPs) only recorded drinking status in approximately 50% to 60% of patients. This suggests that GPs record alcohol use less frequently than smoking status; so even when there are opportunities to ask about health behaviours, alcohol use in not necessarily included. Alcohol was more likely to be recorded in those with higher levels of other health risks  also suggesting that GPs may be more likely to ask about alcohol in patients who they perceive as at higher risk. These patients may also visit the GP more frequently, increasing the opportunities to ask about alcohol use.The patterns of alcohol recording seen here show that UK GPs may be failing to record alcohol consumption at the most basic level of drinking status for half of their adult population. However, patients who are at greater risk from alcohol-related illness are being appropriately targeted  and have better recording. Importantly, 20% to 40% of these at-risk patients still lack data on alcohol consumption and the proportion of patients screened for alcohol consumption appropriately using a valid screening tool is even lower . Alcohol use should be discussed as part of the cardiovascular disease health check for all patients over 40,et alCompared to a 2013 study using CPRD data , and both measures corresponded to informal coding of alcohol consumption using Read codes  although the informal codes were less useful for discrimination between heavier drinkers (the median AUDIT score in those with a heavy drinker code was 6). AUDIT scores were used as the ‘gold standard’ because they are considered valid in comparison with other self-reported alcohol measures,The Health Survey for England found that, in 2016, 17% of men and 22% of women were teetotal. Using CPRD drinking status, 20.3% of patients were recorded as non-drinkers, so this is comparable and demonstrates external validity, although also self-reported. The internal and external validity demonstrated in this study give weight to any study using alcohol data from CPRD. While care must be taken to avoid introducing bias by restricting to only patients with alcohol recorded, the measures of consumption are likely to be useful in ranking participants in terms of their alcohol of consumption, with the caveats required by all self-reported alcohol research.This study investigated alcohol recording across nearly 1.8 million UK adults, providing the power to explore recording in different subgroups of patients, and to understand in detail how alcohol use is recorded in primary care.To validate, we used AUDIT scores recorded on the same date as the codes of interest. If an AUDIT questionnaire is completed, subsequent recording of alcohol consumption (in Read codes or units per week) might be more accurate than at a time when no scoring is done, and validity may be lower outside of these times. Additionally, GP practices that record both measures during the same consultation may be different to practices that only record one measure, and perhaps more likely to record alcohol use measures accurately. However, our sensitivity analyses using AUDIT scores recorded within 30 days before or after the date of the alcohol-related code of interest showed that records within a month of an AUDIT score or measure of units were internally consistent. For the external validation we used data from the Health Survey for England 2016, whereas the time frame for the prevalence estimates of alcohol use from CPRD were from 2013 to 2018 however although the time frames do not match perfectly 2016 is within the middle of the time frame and this seems an appropriate comparator.Evidence suggests alcohol use in young people is common.Although we have demonstrated that alcohol recording and screening are higher for patients with certain high-risk conditions, it does not mean that GPs have acted on the screening result, or that they have screened at an appropriate time. For example, patients with depression or anxiety may not have been screened at the time when depression was diagnosed. We showed that the majority of screening took place within 1 month of registration, so whether this is repeated during consultations for high-risk conditions is not clear from these data.et al,Between 2009 and 2018, the proportion of CPRD patients with alcohol recorded more than tripled from around 13% to nearly 49%. It is likely that this increase in primary-care alcohol recording was contributed to by the 2010 NICE guidelines recommending screening on registration with a GP, and studies demonstrating the efficacy and cost-effectiveness of alcohol screening and brief interventions for alcohol misuse.This study helps establish best practise in using primary care data to investigate alcohol use in research. Researchers using CPRD data should be aware of the limitations of alcohol data. Though the validity of the existing data has been described here, using only patients with complete alcohol use data may introduce selection bias into studies by preferentially including older, sicker and more deprived patients.This study provides insight into how well aligned routine primary care practice is with NICE guidelines for alcohol screening. Recording of alcohol in primary care is not complete, meaning that opportunities for intervention may not be being identified. While we found evidence that alcohol use recording is higher in the high-risk groups highlighted by NICE , and at key times of patient registration recommended by NICE (among newly registered patients), there are still large numbers of patients in whom we might expect risky drinking who are not having their alcohol use recorded, and who therefore may not be targeted for intervention. In particular, younger individuals, in whom alcohol is a major cause of morbidity, maybe being overlooked.


BackgroundX-ray imaging is a common procedure performed on a regular basis for diagnostic purposes all over the world. The use of X-rays is increasing rapidly with the introduction of new radiation-oriented therapeutic practices. Although it carries significant diagnostic benefits, extensive exposure to X-ray imaging has been shown to be associated with multiple dose-dependent health risks. Awareness and knowledge among patients regarding the effects of X-ray imaging, therefore, becomes important. Through this study, we aimed to assess the knowledge and awareness of the hazards of X-ray imaging among different groups of patients visiting two of the public sector tertiary hospitals in Karachi, Pakistan. We also aimed to evaluate the necessary safety measures undertaken during X-ray imaging in these hospitals, and the perception of patients regarding the importance of these measures.Materials and methodsA cross-sectional study was conducted in October and November 2018 at two well-known public sector tertiary care hospitals, Dr. Ruth KM Pfau Civil Hospital, Karachi and Jinnah Postgraduate Medical Centre, Karachi. A non-probability convenience sampling technique was adapted to recruit 200 participants for the study. A pretested questionnaire was used to assess the knowledge of radiation among patients and their perception regarding the necessary safety measures required to be undertaken during the X-ray imaging procedure. Data were entered and analyzed using the IBM Statistical Package for the Social Sciences 17.0 . Frequencies were calculated for individual variables. Chi-square test was employed to measure the relationship between categorical variables. A p-value of <0.05 was considered to be significant.ResultsOut of 200 participants, 58% knew what radiation was, 42% did not. The relationship between the level of education of patients and the awareness of the term ‘radiation’ was found to be statistically significant . Television was the most common source of information (65.5%). One participant (0.5%) thought that it was possible for X-ray imaging to cause cancer. Similarly, only one participant (0.5%) thought that it could cause decreased fertility, five participants (2.5%) thought it could cause burns, seven (3.5%) thought it could cause cataract, and 20 (10%) were of the view that anemia could be caused.The majority of the participants (80.5%) thought that a lead sheet was important during the X-ray procedure for safety and protection. Most participants (71.5%) said that they were never provided with any such lead sheets. When asked if the participants requested for a lead sheet if not provided, the majority (71%) denied requesting for it. On analyzing, we found that a higher percentage of uneducated participants denied requesting a sheet compared to the educated ones. The relationship between the level of education and the choice of requesting for a lead sheet was found to be statistically significant .ConclusionsThe patients visiting the public sector tertiary care hospitals of Karachi seem to lack the knowledge and awareness regarding the hazards of ionizing radiations and the necessary safety measures required to be undertaken during X-ray imaging. More awareness programs should be conducted to increase the level of patients' awareness to protect them from unnecessary health risks. Radiations are categorized as ionizing and nonionizing. Ionizing radiations, like X-rays, possess sufficient energy to separate an electron from an atom or molecule, producing free radicals in the process which are chemically unstable and highly reactive . The emeDue to the extensive use of X-ray imaging, its effects should be fully understood. The most important factor when discussing the effects of X-rays is not the amount at a point in the air (exposure) but the amount of energy absorbed by tissue (dose) . The dosWhile radiations are extremely useful diagnostically, a study conducted in the UK estimated that up to 20% of medical X-rays ordered are not beneficial and only add to the unnecessary exposure in patients, contributing to 100-250 cases of cancer each year in the region . AlthougOver a period of two months of October and November 2018, we conducted a cross-sectional study at two public sector hospitals, Dr. Ruth KM Pfau Civil Hospital, and Jinnah Postgraduate Medical Centre after receiving ethical approval from research committee of the respective hospitals. We recruited 200 participants through a non-probability convenience sampling technique. The study included patients who presented to the radiology department to undergo X-ray imaging and were willing to participate in the study. We excluded patients who refused to participate, had cognitive dysfunction or neurological disease (which made them unable to understand and answer our questions), did not have the capacity to give informed consent, and/or if they were unable to understand the communication language (English or Urdu).A questionnaire was designed comprising 15 questions to assess the experience and knowledge of patients regarding X-ray imaging, and their perception regarding the necessary safety measures required to be undertaken during the X-ray imaging procedure. A pilot study was conducted first on 10 participants before the start of the study to assess the questionnaire for any loopholes or difficulties in understanding the questions. Each questionnaire was then filled by individually interviewing each of the participants of the sample population after seeking informed consent. Participants were assured that collected data will not be misused and that every possible measure would be undertaken to preserve the confidentiality of the data.Data were entered and analyzed using the IBM Statistical Package for the Social Sciences 17.0 . Frequencies were calculated for individual variables and were represented as percentages in tables and figures. The chi-square test was used to measure the statistically significant relationship between the level of education and radiation knowledge as well as the choice of requesting for a lead sheet during the X-ray imaging procedure. A p-value of <0.05 was considered to be significant.Characteristics of the study populationA total of 200 patients participated in our study. Their mean age was 35.96 ± 13.62 years with a minimum age of 12 years and a maximum of 80 years. Table Knowledge and awareness of X-ray imagingAll of the 200 participants had undergone an X-ray imaging before. Out of 200, 164 participants (82%) had less than three X-rays before this one, while 36 (18%) had three or more X-rays before this one.Out of 200 participants, 116 (58%) knew what radiation was, while 84 (42%) did not. When analyzed on the basis of education, we found that the uneducated people were more likely to be ignorant about the term ‘radiation’ (59.61%) rather than being aware of it (40.38%). The relationship between the level of education and the awareness of the term ‘radiation’ was found to be statistically significant . The television was the most common source of information (65.5%) for radiation knowledge, followed by books (25%), the internet (6.9%) and the newspaper (2.6%).We then asked the participants about the effects of X-ray imaging. A total of 166 participants (83%) thought that it was beneficial, 18 (9%) were of the view that it was harmful, one (0.5%) thought that it was harmless, while 15 (7.5%) participants said they did not know. The most common benefit of X-ray was thought to be of that in diagnosis (78.5%). When given options about the population group to which the X-ray radiation was the most harmful , the majority participants (n=182) (91%) said that they did not know, 13 participants (6.5%) correctly identified the most susceptible population group, while five (2.5%) gave an incorrect answer.When asked about the possibility of the harms of X-ray to be increased on repeated exposure, 117 participants (58.5%) thought that it was possible, 44 thought that it was not possible (22%), 24 said that they did not know (12%), while 15 gave no answer to the question (7.5%).The radiation knowledge of the patients was further assessed by questions presented in Table Awareness and practice of necessary safety measures during X-ray imagingWhen asked if they thought a lead sheet was important during X-ray procedure for safety and protection, the majority of the participants (n= 161) thought that it was important (80.5%), 12 thought that it was not important (6%), while 27 said that they did not know (13.5%). Most participants (n= 143) (71.5%) said that they were never provided with any such lead sheets, 48 (24%) said that they were always given lead sheets during the procedure, while nine (4.5%) said that they were given lead sheets sometimes.When asked if the participants requested for a lead sheet if not provided one, the majority of the participants denied requesting for it (n=142) (71%). Slightly more than a quarter of the participants (n= 58) (29%) said that they do request for a lead sheet.We then analyzed this data again based on education. We found that a higher percentage of uneducated participants denied requesting a sheet as compared to the educated participants. The relationship between the level of education and the choice of requesting for a lead sheet was found to be statistically significant . Figure Through this study, we aimed to highlight the knowledge and awareness of radiation risks of X-ray imaging among the patients. Our results show that a high percentage of the study population 42%) was unaware of the term ‘radiation'. The study population demonstrated a poor level of knowledge regarding the procedure and the harmful effects of X-ray imaging altogether. These results are consistent with those that we found in the literature. A study conducted in Hong Kong reported that 87.9% of the local patients were unaware of the fact that plain X-rays contain radiations . Our stu% was unaAccording to our results, 42% of the patients did not know that the type of imaging techniques which were associated with higher doses of radiation. Other studies have reported similar results where patients seem to underrate the equivalent dose of radiations from CT scans compared to the number of chest X-rays , 12. MosWhile assessing the perception and practice of necessary safety measures during the X-ray imaging procedure, we found that 80.5% of the patients were of the view that a lead sheet was important for safety against radiations. However, the majority of the patients (71.5%) said that they were never provided with any such sheet. A study reported similar results where half of the participants were never provided with protective devices during radiation exposure . Apart fAmong the patients who were able to identify the risks associated with X-ray imaging, the primary source of information was television. As opposed to this, other studies report healthcare providers as being the primary source of information for the patients , 13. TheThe overall knowledge of the patients visiting tertiary care government hospitals of Karachi regarding radiation and its hazards is unsatisfactory. Safety protocols are less implemented in these hospitals, probably due to limited resources. To ensure the protection of patients from unnecessary repeated radiation exposure, educating patients as well as the health care providers may prove to be beneficial. Public awareness programs should be conducted on a regular basis, where electronic media could play a central role. Healthcare providers should be taught to make a justified decision of exposing their patient to radiation only when the benefit outweighs the risk. It has been suggested that patients’ exposure history must be maintained and updated after each exposure. Informed consent should be sought and a clear explanation of the imaging and its associated risks should be provided to each patient prior to the procedure.


Meloidogyne genus, have a worldwide distribution and cause severe economic damage to many life-sustaining crops. Because of their lack of specificity and danger to the environment, most chemical nematicides have been banned from use. Thus, there is a great need for new and safe compounds to control RKN. Such research involves identifying beforehand the nematode proteins essential to the invasion. Since G protein-coupled receptors GPCRs are the target of a large number of drugs, we have focused our research on the identification of putative nematode GPCRs such as those capable of controlling the movement of the parasite towards (or within) its host. A datamining procedure applied to the genome of Meloidogyne incognita allowed us to identify a GPCR, belonging to the neuropeptide GPCR family that can serve as a target to carry out a virtual screening campaign. We reconstructed a 3D model of this receptor by homology modeling and validated it through extensive molecular dynamics simulations. This model was used for large scale molecular dockings which produced a filtered limited set of putative antagonists for this GPCR. Preliminary experiments using these selected molecules allowed the identification of an active compound, namely C260-2124, from the ChemDiv provider, which can serve as a starting point for further investigations.Root-knot nematodes (RKN), from the  Root-knot nematodes (RKNs) seriously threaten global food production: these microscopic, soil-dwelling worms are highly destructive and cause up to 100% yield loss in important life-sustaining crops like soybean, rice, cotton, tomato, etc. ,3. TheirConventional control methods, including crop rotation, use of resistant cultivars and biological control have limited efficacy or are not available against RKNs that have a wide host range . TraditiIn recent years, and since the availability of several nematodes genomes , bioinfoMeloidogyne graminicola), the disruption of neuropeptides involving G protein-coupled receptors (GPCRs) signalling disturbs both behavior and migration abilities infection [Meloidogyne incognita which is amongst the most devastating plant parasite worldwide [M. incognita in order to help control this agricultural pest. GPCRs constitute a superfamily of transmembrane proteins acting as receptors that sense molecules and trigger transduction pathways in cells. Their 3D structure is highly conserved in opposition to their amino acid sequences that are poorly conserved. Most GPCRs have are 200–1000 amino acids long, show a 7 transmembrane helices structure and several additional domains related to their functional specificity and useful for their classification.In the rice RKN  and to the human neuropeptide Y Y1 receptor (UniProt ID P25929) corresponding to the PDB structure 5ZBH [The first step in homology modelling is to identify the most suitable templates to use to build the query 3D model. In our case, the best template should be a GPCR structure having sequence similarities with our query and belonging to the same GPCR category. In order to decide which one of the three possible GPCRs found above would be retained for performing the homology modelling step, we performed a phylogenetic analysis with several GPRCs with available 3D structures and others belonging to the neuropeptide receptors family. According to the phylogenetic tree obtained  it appeaure 5ZBH , while MConsequently, we selected the PDB template 5ZBH to build the 3D model of Minc3s00007g00462 and the two proteins sequences were aligned according to their similarities and the position of their transmembrane helices .The 3D model of Minc3s00007g00462 is presented in When observing the variation of the RMSD of the GPCR backbone during the MD (taking the first frame as the reference), the largest values (up to 8 Å) came from the loops connecting the TM helices . In contrast, the TM7 helix bundle and the small From the 200 ns MD simulations, according to the RMSD analysis, it appeared that the molecular system adopted five families of stable conformations a, c1 to The observation of the binding pockets for each of these conformers revealed that all the pocket shapes differ due to different side chains orientation of the residues lining the binding site despite roughly similar values obtained for their volumes (around 2000–2500 Å1300 Å2) . This woFrom the docking campaign using these five main protein conformers, we retained the top 100 molecules from the complete PLP score list for further analysis. From this list, it appeared that 85 compounds came from the ChemDiv provider’s library. Hence, for consistency, we kept only these 85 compounds in the rest of our analysis.After inspection of molecular weights, solubility , compound similarities and toxicity, 13 molecules were finally selected . For exaA dendrogram representing the chemical relationship between the 13 retained compounds is presented in Looking at the interactions of these 13 compounds within the protein binding pocket, 12 retained ligands are found mostly bound to the c3 or the c2 conformations (9/13 and 3/13 respectively). The only exception was the C260-2124 compound that gives its highest score when bound to the c5 protein conformation. Considering hydrogen binding interactions, a large variety of binding possibilities were found, but the only residues that bind the majority of retained compound through H-bonds (or -stacking) are Trp102 and Arg285. All other interactions were specific to one or two ligands. We illustrate in The 13 candidate molecules, retained from the virtual screening list above, were tested in nematode assays. Direct exposure of nematode infective juveniles (J2s) to the selected compounds at 1% did not affect their viability. Nevertheless, in preliminary qualitative tests in pluronic medium (supp figs), the observed RKNs movements seem to be altered, probably because of the drug interaction with the predicted nematode chemosensor receptor target. Even if reduction effects on the penetration of J2s in plant roots were observed with several compounds, reliable and consistent results of treated nematodes were only obtained with the C260-2124 compound in three independent experiments . We obseM. incognita [M. incognita GPCR models with the one obtained for our C260-2124 compound. Such comparison is shown in Searches for novel nematicides have been tackled starting mostly from profiled peptides belonging to the FMRF-amide or to the neuropeptide families. FMRF-amide like peptides represent the most widely investigated family. They are characterized by -RFamide motif and are known to be involved in motor and sensory function coordination with effects on nematodes infectivity . The neuncognita . The mosApart from peptide-related compounds, few small molecular weight organic compounds were also reported in the literature. Besides BIBP-3226, which was the first selective neuropeptide Y1 receptor (NPY Y1R) antagonist found in 1996 . Some ofDocking of all these compounds according to a process similar to the one followed in our virtual screening shows that only the UR-MK299 compound gave a score in the range of the ones obtained with our 13 selected molecules. When looking at the interactions stabilizing this ligand, it appears that, besides several H-bonds implying the backbone of Glu192 and Cys109, as well as the sidechain of Gln116, the two residues Arg285 and Trp102 found as stabilizing all our 13 compounds through H-bonds or The binding cavity obtained for our nematode GPRC receptor presented a quite similar 3D shape when compared to the ones obtained for the two 5ZBH and 5ZBQ X-ray structures : calculaThe two inhibitors (UR-MK299 and BMS-193885 respectively) complexed to the neuropeptide Y Y1 receptor have very different chemical structures when compared to our selected compounds with Tanimoto coefficients less than 0.3;In our model most of the 13 compounds interact through H-bonds with Arg285 (which is not conserved in the Y Y1 receptor) and with Trp102, Gln116 and Gln173 . In the Y Y1 3D structures, only interactions with Gln120 are similar to the ones found with our compounds. Gln120 is proposed to be crucial for receptor activation as being the interaction partner for the NPY peptide C-terminus.Additionally, interactions are also found in our model with residues Tyr46 and Trp102  are also stabilizing the interactions with some of our 13 molecules. It could be concluded from this analysis that, despite a quite similar binding site compared to the ones in human YY1 receptor, our compounds should be very specific to neuropeptide nematode GPCRs with few or no interactions with human neuropeptide receptors, so that no unwanted effects can be expected.Because we used the human neuropeptide Y Y1 receptor (NPY Y1R) as a template, the question of the specificity of the compounds identified in our study to the nematode and their possible side effects must be considered. To check that, it is interesting to compare the protein/ligand binding characteristics found in the Y Y1 neuropeptide receptor template versus our model. When looking at the ligands chemical structures and binding properties found in the 5ZBH and 5ZBQ PDB structures and comparing them to the 13 proposed compounds from our virtual screening, it appeared that:The in silico approach was composed of four main steps as illustrated in M. incognita recent genome annotation available V2 [The initial dataset used for GPCRs prediction comprises lable V2 . As the lable V2  in orderlable V2  was usedlable V2 .Although automatic methods for GPCR homology modelling were recently developed ,48, the For the template choice, selected GPCRs were aligned by PSI/TM-Coffee  with PDBTo build the model, the selected protein and the template were aligned with AlignMe , a dedicThe next steps using Molecular Dynamics (MD) simulations were required to refine the preliminary crude model and then analyse the stability of the GPCR within the membrane. For that, we used the NAMD molecular dynamics software . MD is nFor this purpose, we used molecular dynamics simulation on the receptor homology models that were embedded in a fully hydrated POPC bilayer . No ligaThe MD simulations were carried out in the isobaric-isothermal ensemble, maintaining the pressure and the temperature of 1.0 atm and 300.0 K, respectively, using Langevin dynamics and the Langevin piston approach. The MD program NAMD was employed in conjunction with the CHARMM27 force field  to descrEach system was energy minimized and then equilibrated before recording trajectories. All MD trajectory frames were recorded at 1 ps intervals, for a total of 10 ns simulation. Model stabilities were then checked by analysing secondary structure evolution during the MD simulation. If at least one transmembrane helix broke, then the model was not considered stable. For the stable models obtained, the simulations were extended to reach a 100 ns simulation time.Once the MD simulations finished, all the frames were aligned on the first frame to calculate RMSDs with VMD (“RMSD trajectory tool” plugin) . An RMSDIn addition to the RMSD map, the trajectory obtained after molecular dynamics was analyzed by the Self Organizing Maps (SOM) method . The metThe initial dataset of chemical libraries were the GPCR-centred libraries encompassing six compounds providers: Asinex , ChembriThe docking was performed by GOLD which has been recognised as one of the best docking softwares ,81. As sormation . The binormation ,85.In order to avoid possible toxicity, the candidate compounds were surveyed using predictors such as PAINS-remover  and ProTAs it was not presently possible to perform in vitro tests to directly measure the binding of our selected compounds to the GPCR receptor, we will present here only very preliminary in vivo experiments. Robust protocols for in vivo testing of products acting on root-knot nematodes are difficult to handle and are time consuming. Valuable experiments are not as easy as for other systems because of the particular surrounding conditions necessary for establishing nematode growth. Furthermore, as an obligatory endo-parasite of plant roots, the only life stage that can be used for testing attack inhibition is the infective juvenile stage .Meloidogyne incognita was obtained from pure cultures maintained in roots of tomato plants ). Tomato seeds were surface sterilized with 1.4% NaOCl for 5 min followed by 2 × 15 min rinse in sterile water. After 7-days germination in Germitest paper, tomato seedlings were transferred to a plastic pot filled with a sand/hydrogel mixture (3 seedlings in 5-mL substrate per pot). To perform the mobility test nematode second-stage juveniles (J2) were obtained from M. incognita egg masses extracted from previously infected plants. After approximately two reproductive cycles, the nematode eggs were bulk extracted from severely infected tomato roots with 0.05% (v/v) sodium hypochlorite (NaOCl) as previously described [escribed . The J2 l method . FreshlyCompounds (2–5 mg each) were suspended in DMSO solution to reach a 10 mg mLM. incognita J2s previously treated in vitro (eppendorf) with each selected compound diluted to 1% with distilled water (v/v stock compound). Worms treated with DMSO 1% were used as control. To verify the inoculum viability after 24 and 48 h of exposure direct exposure to the diluted compounds, the J2’s motility was observed over a microscope slide under visible light (Leica). The in vivo validation of the selected compounds was performed by measuring the nematode ability to penetrate tomato roots when treated with the compounds. This ability was tested with exposure to compounds at 1% concentration by counting the number of J2s in roots at 3 days after inoculation (dai). A total of at least 300 freshly hatched J2 were added to each pot. The whole experiments contained 30 replicates per treatment. The inoculated plants were cultured at room temperature with 16h/day natural light. The M. incognita J2 penetration rate was recorded after 3 dai by observation under a stereo-microscope (Leica) of fuchsin-stained roots [Tomato roots were inoculated with ed roots .M. incognita is one of the most aggressive and damaging plant-parasitic nematodes as it uses a sophisticated chemosensory apparatus to detect potential hosts and to infect them. Chemical nematicides have so far constituted the most efficient tools against these agricultural pests. Because of their toxicity for the environment and danger to human health, most nematicides have now been banned from use. Consequently, new and more specific means of control, which are safe for the environment and human health, are urgently needed to avoid worldwide proliferation of these devastating plant-parasites. Understanding the parasite machinery, aiming at identifying novel targets for the development of future control methods is an important issue that concentrate the efforts of many research groups. For that purpose, three main lines of action can be followed dedicated respectively to the detection of the hosts by the parasite, its penetration within the host roots, and the development of the giant cells within the roots. We focused our study on the role of GPCRs possibly involved in the mechanism of parasite detection and locomotion towards its host’s roots. Among all the putative GPCRs genes in the M. incognita genome, we found one GPCR belonging to the neuropeptide receptors family that appeared as an interesting target against M. incognita. A drug design strategy merging in silico and experimental bioassays was successfully undertaken in order to find new low molecular weight chemical compounds able to interfere with this GPCR.The root-knot nematode The data obtained in the present study show that some of the tested compounds affect mostly or only the penetration rates most probably due to muscle contraction/behavioral disruption ,97. ThesM. incognita chemoreception of environmental stimuli as one of the major sensory system. These findings may allow further research, innovation and development. Nevertheless this work is the first one proposing a non-peptide compound acting as an antagonist of M. incognita neuropeptide receptor, therefore validating our design strategy. C260-2124 can be considered as a good seed to further develop new leads and this work demonstrated the strength of combining in silico and experimental approaches.Our biological assays were preliminary and further investigations now have to be performed to definitively validate our results and investigate the specificity and safety of this compound for non-target organisms. These preliminary studies indicates the targeting of some of the selected compounds against the nematode neuromuscular system as previously pointed by Kumari et al. . HoweverMeloidogyne hapla, M. javanica and M. arenaria. This data suggest that C260-2124 could also be active on these others nematode species of high economic interest.Moreover, all the putatively C260-2124-binding amino acid residues of Minc3s00007g00462 are conserved in the homologous sequences of the other RKN of major concern C. elegans nematode [This work participates in the efforts of the research community to identify new classes of nematicides and we believe this approach will help to strengthen the present arsenal. Besides recent screening approaches mostly conducted on the nematode ,99, our


Saturating concentrations (10 µmol/L) of either FKBP12 or 12.6 significantly reduced the frequency, spread, amplitude and Ca2+ spark mass relative to control, while the tomograms revealed both proteins shifted the tetramers into a largely side-by-side configuration. Phosphorylation of immunophilin-saturated RyR2 resulted in structural and functional changes largely comparable to phosphorylation alone. dSTORM images of myocyte surfaces demonstrated that both FKBP12 and 12.6 significantly reduced RyR2 cluster sizes, while phosphorylation, even of immunophilin-saturated RyR2, increased them. We conclude that both RyR2 cluster size and the arrangement of tetramers within clusters is dynamic and respond to changes in the cellular environment. Further, these changes affect Ca2+ spark formation.The effects of the immunophilins, FKBP12 and FKBP12.6, and phosphorylation on type II ryanodine receptor (RyR2) arrangement and function were examined using correlation microscopy (line scan confocal imaging of Ca They arrcolemma . The smae (CICR) . This are (CICR) .2+ influx through Cav1.2, RyR2 open stochastically but with a very low probability , and rows indicating whether the cells were (bottom) or were not (top) exposed to the phosphorylation cocktail.The images in 2+ spark parameters were not normal, so they were displayed as box plots , possibly reflecting their differing effects on RyR2 gating (−4). The number of sparks analyzed, cells examined, and animals used are in The immunophilins significantly reduced the spark frequency and most of the Ca2 gating . The spa2+ sparks.Given these results, and those from our previous study, we hypothesized that changes in the tetramers’ distribution would accompany the functional changes and used correlation microscopy to examine dyads in the same cells from which we had recorded the CaRyR2 is a homotetramer resembling a square-capped mushroom about 27 nm wide and 12 nm deep . Its sizUsing Amira, a single plane from a dual-tilt tomogram is displayed in Measurement of the tetramers’ centre-to-centre nearest neighbor distances (NND), also derived from RyR_fit, is displayed in Tetramer arrangements recorded in each of the indicated experimental conditions using the RyR_fit program which is available in Comparable images of a myocyte treated with the phosphorylation cocktail are shown in Permeabilized myocytes treated with saturating concentrations of either FKBP12  or FKBP1Exposing immunophilin-treated tetramers to cell-wide phosphorylation partially reversed the proteins’ effects , but seeTo determine which, if any, of the tetramer distributions could have been derived from the same population, the histograms were expressed as cumulative distribution functions CDF;  to enablTomograms currently provide the highest possible resolution with which to view the tetramers, but to determine their position and orientation the images must be limited to a small area within a single dyadic cleft. To determine whether the results were truly representative of wide-spread changes in the cells we imaged the distribution of RyR2 near the myocyte surface using our custom-built superresolution microscope in near-TIRF mode . The resImages of tetramer clusters near the cell surface were placed into an array with columns designating the treatment , and rows indicating whether the cells were (bottom) or were not (top) exposed to the phosphorylation cocktail. The images demonstrate dramatic, widespread, changes in the appearance and shape of the clusters. The immunophilins appeared to have fractured the clusters into smaller pieces, while all of the phosphorylated cells appeared to have larger clusters than their non-phosphorylated counterparts.As demonstrated in To quantify the dSTORM images, we used the higher resolution tomographic images to guide our analysis. We first drew alpha shapes around the tetramer arrays in the tomograms  to calcuMedian mean cluster areas and number of clusters examined. The alpha shapes and areas were generated using CGAL.Median tetramer coverage per cluster recorded from the indicated number of tomograms using the RyR_fit program . Mean anWe have provided compelling evidence that inhibitory factors, such as the immunophilins, are associated with the RyR2 tetramers moving into a largely side-by-side configuration and decreasing the cluster size. In direct contrast, excitation due to phosphorylation, even in the presence of the inhibitory immunophilins, is associated with the tetramers moving apart into a checkerboard-like configuration and increasing the cluster size. These findings show that our understanding of RyR2 function has been incomplete and a full understanding should incorporate these dynamic effects. The results confirm and extend our earlier findings, and demonstrate that normal operation of the mammalian ventricular dyad involves movement and rearrangement of the tetramers and changes in the cluster size.The distribution of nearest-neighbor centre-to-centre distances (NND) identified in control myocytes was bimodal, comparable to our previous results, with peaks at 28 and 34 nm. The median of the two modes varied little  with a rRecently published superresolution data appeared to show mostly single and isolated tetramers in a unimodal distribution with a mean NND of 40 nm . In cont2+, which also inhibits RyR2 . All chemicals were purchased from Sigma-Aldrich  unless otherwise stated.2+ sparks were recorded on a Zeiss LSM 700 confocal microscope in line scan mode (63x objective) using 30 µmol/L Fluo-4 (Invitrogen). Sparks were analyzed using SparkMaster  1.8 FKBP12.6/RyR2 (45% occupancy) and no FKBP12 ; (ii) Atd spread .2+ sparks had been recorded, and they were fixed, embedded, sectioned and ready for tomographic analysis The 2D EM images demonstrate that the ultrastructure was well preserved regardless of the experimental protocol  using our established protocols. We used Inspect 3D to align and Amira 5.3  to visualize the data sets, and employed no image processing steps other than a contrast stretch . 2D elecprotocol . AdditioAn individual tetramer’s position relative to its neighbors was classified using Amira and a custom-written C++ program, ‘RyR_fit’, as previously described . BrieflyE. coli Rosetta (DE3) cells with 0.4 mmol/L isopropyl-β-D-thiogalactoside (IPTG) at 0.6 optical density at 600 nm. After growth for 4 hr at 37°C, the cells were collected, re-suspended in the buffer A  with 100 μM PMSF, 25 μg/ml DNaseI and 25 μg/ml lysozyme and 10% glycerol, and disrupted by sonication. Cell debris was removed by centrifugation at 40,000 g for 30 min. Cell lysate was applied to a PorosMC column (Thermo Fisher Scientific), were washed with five column volumes (CV) of buffer A and 5 CV of buffer A plus 10 mmol/L imidazole, and were eluted with buffer A plus 300 mmol/L imidazole [pH 7.4]). The protein was dialyzed overnight against buffer A and was cleaved simultaneously with recombinant TEV protease. The samples were then run on another PorosMC column in buffer A, and the flow-through was collected and dialyzed against buffer C , applied to a Q Sepharose column , and eluted with a gradient from 0% to 50% buffer D . Finally, the samples were run on a Superdex 200 16/60  column in buffer A plus 5 mmol/L 2-mercaptoethanol, which separated dimeric and monomeric fractions. The monomeric fractions were pooled, aliquoted and flash-frozen for further use.FKBP12 and FKBP12.6 were cloned to obtain a fusion protein with N terminal histidine and maltose binding protein (MBP) tag. FKBPs were induced in To visualize and better understand the 3D architecture and arrangement of the t-tubules, jSR and ryanodine receptors, we manually segmented these compartments on the dual tilt tomograms using Amira (FEI). Using the segmentation module, the contours were hand drawn, creating a 3D model, by tracing the individual membranes in every slice of the tomograms using a Wacom Cintiq 27QHD drawing tablet. In each dyad, a single RyR2 tetramer was traced, then copies of it were placed in the proper position and orientation; this simplified the drawing. As displayed in the supplementary videos, t-tubules were traced in blue, jSR in red and RyR2 tetramers in green. Automatic smoothing was applied to the surface models to reduce the number of polygons and improve the models’ appearance. When drawing, the tomogram cannot be rotated, so to ensure that we identified the correct position and orientation of the tetramers we used the Slice option to superimpose the tomographic data onto the model. This simultaneous visualization ensured accurate positioning of the tetramers in the 3D data set. Movies of the complete models were created using Amira’s MovieMaker module.2 for storage at −80°C until use. Protein  was separated on 12% SDS-polyacrylamide gels and transferred to Immobilon-P transfer membrane  overnight at 4°C. Membranes were then blocked in skim milk in Tris-buffered saline with 0.1% Tween (TBS-T Sigma) at room temperature for one hour. Primary antibody incubation was performed at 4°C overnight and secondary antibody incubation at 37°C for one hour. Protein bands were visualized using the SuperSignal West Femto Chemiluminescent Substrate kit .We used a standard Western Blot protocol to confirm that exogenous FKBP12 or FKBP12.6 raised the cellular levels beyond control values . The 6X We used the following antibodies: Anti-FKBP12  to detect both FKBP 12 and FKBP12.6, anti Calsequestrin  and anti-RyR2  were used as loading controls. Secondary antibodies: HRP-Goat anti-mouse IgG  and HRP-Goat anti-rabbit IgG .2+ load was normally distributed, virtually none of the other parameters were, so, similar to Sikkel et al., we used the log transformation to analyze the data. We treated the p value generated by the R-program differently from Sikkel et al, using the Holm-Bonferroni correction .To interpret the images, we defined a cluster as consisting of pixels where no pixel was more than 50 nm distant from its nearest neighbor. The area of each cluster was determined by defining the boundary with an alpha shape . Cluster In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:2+ spark properties and CRU configurations are altered in myocytes when exposed to saturating concentrations of FKBPs and/or a phoshporylation cocktail. Ca2+ spark properties change as well as RyR orientations within clusters. The RyR orientations are determined from EM tomograms. Additional data on peripheral cluster outlines is provided by optical super-resolution microscopy.Asghari et al. describe new results concerning the variable/changeable distribution of RyRs in response to a number of stimuli and chemical modifications. The manuscript shows that both CaDecision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kenton Swartz as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Bjorn Knollmann (Reviewer #3). The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Thank you for submitting your article "Cardiac ryanodine receptor distribution is dynamic and changed by auxiliary proteins and post-translational modification" for consideration by Summary:This manuscript by Asghari et al. reports the distribution, arrangement, and cluster size of the type II ryanodine receptor (RYR2) tetramers under various conditions. This group has previously reported that phosphorylation and low concentrations of magnesium moved the tetramers into a predominantly checkerboard arrangement, whereas high concentrations of magnesium induced a dense side-by-side configuration. The present work confirms and extends their early findings and shows that FK506 binding proteins (FKBP12 and FKBP12.6) moved the tetramers into a largely side-by-side configuration and decreased the cluster size. In contrast, phosphorylation even in the presence of FKBPs moved the tetramers into a checkerboard-like configuration and increased the cluster size. By correlating the effects of FKBPs and phosphorylation with their tetramer arrangements, the authors propose that the side-by-side configuration of tetramer arrangement is associated with channel inhibition, whereas the checkerboard-like configuration is associated with channel activation.Essential revisions:1) The authors should show cluster rearrangements together with changes in Ca sparks in intact cells in response to strong phosphorylation. This would go a long way to demonstrate that the structural rearrangement modulate RyR function in a more physiologic setting. Importantly, these results show that hyperphosphorylation takes precedence over excess FKBP12 or 12.6." How do you reconcile this statement with some reports that phosphorylation itself leads to reduced binding of FKBPs to RyR2? Is it phosphorylation itself or is it consequent unbinding of FKBP's. How much FKBP is still bound to phospho-RyR2? I think this is shown in Supplementary Figure 6, but that figure is not referenced in the text. Is it a co-IP? This is a critical issue that needs to be addressed. Basically, does phosphorylation remove the immunophilins from the RyR2 tetramers or not? In addition to co-IP, this could/should be done with fluorescent-labelled FKBP.2+ spark experiments and RyR distribution experiments. Recently, Sikkel et al. [1] published an analysis of Ca2+ spark data and included a discussion of general experiments with myocytes. They concluded that it may be required to conduct a hierarchical analysis to draw valid conclusions from myocyte experiments. The authors should show that their results hold up to such a hierarchical data analysis.2) Statistical evaluation of Ca3) Determination of tetrad position and orientation in tomograms. The procedures used to determine these parameters are not clear to this reviewer. In the Materials and methods the authors say "An individual tetramer's position relative to its neighbors was classified using a custom-written C++ program, 'RyR_fit', as previously described .". I consulted Asghari et al., 2014, and could not see any reference to this or a similar software in the main text or supplementary of Asghari et al., 2014. I downloaded the C++ source of RyR_fit from the supplementary of this manuscript to learn more but had difficulty to decipher answers to my questions from the lengthy source listing. In particular, is the extraction of tetrad positions and orientations fully automatic? Alternatively, does the software require the user to choose positions and orientations? A mixture of the two? In addition, with what precision/error can these parameters be determined? For example, looking at Figure 2F: for several tetrads it is not clear how the "smudges" shown can be related to a precise orientation? A more detailed description of the procedures used, particularly the role of a human operator or the robustness of an automatic procedure, should be added – including a procedure to estimate the precision with which the orientation and centre of a tetramer can be determined.4) Actual resolution in dSTORM data. There are now a set of tools, in particular Fourier ring correlation, that can estimate the actual resolution in super-res images. This would be important to carry out to establish at what resolution level this data provides reliable information. In my experience dSTORM data often has resolution worse than 50 nm which limits conclusions that can be drawn about detailed cluster shape changes. It is important to add such estimates and then possibly re-evaluate the information that is accessible from this data.5) "Importantly, these results show that hyperphosphorylation takes precedence over excess FKBP12 or 12.6." How do you reconcile this statement with some reports that phosphorylation itself leads to reduced binding of FKBPs to RyR2? Is it phosphorylation itself or is it consequent unbinding of FKBP's. How much FKBP is still bound to phospho-RyR2? I think this is shown in Supplementary Figure 6, but that figure is not referenced in the text. Is it a co-IP? This is a critical issue that needs to be addressed. Basically, does phosphorylation remove the immunophilins from the RyR2 tetramers or not? In addition to co-IP, this could/should be done with fluorescent-labelled FKBP.6) The major novelty of the work is the discovery of the tremendous plasticity of the RyR2 cluster. The authors should quantify the degree of movement in more detail. What is the time course? Distances?7) Determining the mechanism responsible for the RyR2 movements is beyond the scope of the work but the authors should discuss possible mechanisms in more detail. For example, what is the role of Bin-1 or other molecules postulated to be important for dyad formation/assembly. Essential revisions:1) The authors should show cluster rearrangements together with changes in Ca sparks in intact cells in response to strong phosphorylation. This would go a long way to demonstrate that the structural rearrangement modulate RyR function in a more physiologic setting.We are preparing a manuscript to address the hypothesis that phosphorylation of RyR2 alone, rather than other targets, is required for the functional and structural changes that we’ve observed. We used 300 nM isoproterenol as the stimulus, rather than the phosphorylation cocktail, and the data will be in that paper. The results are identical to what we have reported in this manuscript and the Asghari et al., 2014 paper.Importantly, these results show that hyperphosphorylation takes precedence over excess FKBP12 or 12.6." How do you reconcile this statement with some reports that phosphorylation itself leads to reduced binding of FKBPs to RyR2? Is it phosphorylation itself or is it consequent unbinding of FKBP's. How much FKBP is still bound to phospho-RyR2? I think this is shown in Supplementary Figure 6, but that figure is not referenced in the text. Is it a co-IP? This is a critical issue that needs to be addressed. Basically, does phosphorylation remove the immunophilins from the RyR2 tetramers or not? In addition to co-IP, this could/should be done with fluorescent-labelled FKBP.Multiple pieces of information indicated that RyR2 phosphorylation, or hyperphosphorylation, takes precedence. Neither protein dissociated from RyR2 to a significant extent as demonstrated by our Western Blot , and by our spark analyses and superresolution results. These are discussed in the subsection “Phosphorylation”. They agree with others who have performed co-IP experiments and have examined fluorescent FKBP derivatives concluding there was no significant dissociation.2+ spark experiments and RyR distribution experiments. Recently, Sikkel et al. [1] published an analysis of Ca2+ spark data and included a discussion of general experiments with myocytes. They concluded that it may be required to conduct a hierarchical analysis to draw valid conclusions from myocyte experiments. The authors should show that their results hold up to such a hierarchical data analysis.2) Statistical evaluation of Card author on the Sikkel et al. manuscript), and he agrees with us that the modified correction has greater statistical power. The statistical analysis is discussed in the subsection “Statistical Analyses”. There were minor changes in the results and these are reflected in Figure 1B and its source data, and in various places throughout the text.Our results do hold up and the conclusions were unchanged. We reanalysed our data using the Sikkel et al. methodology as requested, but we differed in our handling of the probability with multiple comparisons and used the sequential Holm-Bonferroni rather than the simple Bonferonni correction. We have been in contact with the R-program’s author, Dr. James Howard (the 33) Determination of tetrad position and orientation in tomograms. The procedures used to determine these parameters are not clear to this reviewer. In the Materials and methods the authors say "An individual tetramer's position relative to its neighbors was classified using a custom-written C++ program, 'RyR_fit', as previously described . ". I consulted Asghari et al., 2014, and could not see any reference to this or a similar software in the main text or supplementary of Asghari et al., 2014. I downloaded the C++ source of RyR_fit from the supplementary of this manuscript to learn more but had difficulty to decipher answers to my questions from the lengthy source listing. In particular, is the extraction of tetrad positions and orientations fully automatic? Alternatively, does the software require the user to choose positions and orientations? A mixture of the two? In addition, with what precision/error can these parameters be determined? For example, looking at Figure 2F: for several tetrads it is not clear how the "smudges" shown can be related to a precise orientation? A more detailed description of the procedures used, particularly the role of a human operator or the robustness of an automatic procedure, should be added – including a procedure to estimate the precision with which the orientation and centre of a tetramer can be determined.We have made adjustments to Figure 2 and to the text in the Results to clarify the procedure.RyR_fit is used to manually place a square of the appropriate size and orientation over a tetramer once the operator has identified it using Amira. RyR_fit is essential for classifying the tetramers’ positions , for generating the NND and for determining the area occupied by the tetramers using α shapes .We estimated that the positioning error for a tetramer was ~3 nm; the basis for this estimate, and other possible errors, is discussed in detail in Asghari et al., 2014, pg 258. We have added this information to the subsection “Classifying RyR2 Positions”.2+) and those that brought them together (immunophilins and high Mg2+). These are large changes, and an error of a few nm in determining the centre of the tetramer, or of degrees in their angle, has no impact on our conclusions.New high resolution cryo-EM structures of RyR2 show that the tetramers are 27 nm on a side , rather than 29 nm, which was the best estimate at the time Asghari et al. was published. It is notable that this difference has not affected our conclusions. Comparing the results from Asghari et al., 2014 to the results presented here, Control cells still show a bimodal distribution in their NND, and we see the same changes in distribution in response to agents that moved the tetramers apart (phosphorylation and low Mg4) Actual resolution in dSTORM data. There are now a set of tools, in particular Fourier ring correlation, that can estimate the actual resolution in super-res images. This would be important to carry out to establish at what resolution level this data provides reliable information. In my experience dSTORM data often has resolution worse than 50 nm which limits conclusions that can be drawn about detailed cluster shape changes. It is important to add such estimates and then possibly re-evaluate the information that is accessible from this data.Thank you for this suggestion. Fourier ring correlation indicated that the resolution of our images was between 16 and 25 nm. This is stated in the first paragraph of the subsection “Super-resolution immunofluorescence microscopy”, and an image of the program’s output is included in Figure 6—figure supplement 1.5) "Importantly, these results show that hyperphosphorylation takes precedence over excess FKBP12 or 12.6." How do you reconcile this statement with some reports that phosphorylation itself leads to reduced binding of FKBPs to RyR2? Is it phosphorylation itself or is it consequent unbinding of FKBP's. How much FKBP is still bound to phospho-RyR2? I think this is shown in Supplementary Figure 6, but that figure is not referenced in the text. Is it a co-IP? This is a critical issue that needs to be addressed. Basically, does phosphorylation remove the immunophilins from the RyR2 tetramers or not? In addition to co-IP, this could/should be done with fluorescent-labelled FKBP.Please see the answer to question 1 and the subsection “Phosphorylation”.6) The major novelty of the work is the discovery of the tremendous plasticity of the RyR2 cluster. The authors should quantify the degree of movement in more detail. What is the time course? Distances?Comparisons of tetramer and cluster positions before and after treatment aren’t possible because we fix the myocytes for examination by both tomography and superresolution immunofluorescence microscopy. Each of the experimental treatments involves different cells, so we cannot track individual tetramers with either technique, making it impossible to quantify the distance and time course of their movements.Since the first time point we examined was 10 minutes after the application of the treatments we must assume that the changes occurred within this period. This is now stated in the last paragraph of the Discussion.7) Determining the mechanism responsible for the RyR2 movements is beyond the scope of the work but the authors should discuss possible mechanisms in more detail. For example, what is the role of Bin-1 or other molecules postulated to be important for dyad formation/assembly.A brief examination of the role of BIN1 as a possible mechanism of tetramer translocation has been added to the last paragraph of the Discussion.