Data were collected on 85 patients who were randomized to a traditional walking program (n = 40) or walking-with-poles program (n = 45) of exercise training. Patients walked for 3 times weekly for 12 weeks. Seventy-one patients completed both the baseline and the 12-week follow-up progressive treadmill tests . Using the near-infrared spectroscopy measures, StO2 was measured prior to, during, and after exercise. At baseline, calf muscle oxygenation decreased from 56 ± 17% prior to the treadmill test to 16 ± 18% at peak exercise. The time elapsed prior to reaching nadir StO2 values increased more in the traditional walking group when compared to the walking-with-poles group. Likewise, absolute walking time increased more in the traditional walking group than in the walking-with-poles group. Tissue oxygenation decline during treadmill testing was less for patients assigned to a 12-week traditional walking program when compared to those assigned to a 12-week walking-with-poles program. In conclusion, the 12-week traditional walking program was superior to walking-with-poles in improving tissue deoxygenation in patients with PAD.This randomized trial proposed to determine if there were differences in calf muscle StO Walking ability is impaired in patients with peripheral arterial disease (PAD) due to decreased blood flow to the skeletal muscle tissue used during walking. It has been demonstrated that walking distance is associated with the degree of impairment in the affected leg as measured by the ankle-brachial index (ABI) and the time to onset of claudication pain –6. Likew2) during walking exercise [2 was significantly associated with initial claudication time and absolute claudication time in patients with PAD [2 in 15 patients after a 3-month treadmill and calf exercise training program. Manfredini et al. [2 increased in those assigned to arm-cranking and there were no changes in the control group. No studies however compared leg muscle responses after a traditional walking program compared to a walking-with-poles program of rehabilitation in patients with claudication.  Recently, several investigators have used near-infrared spectroscopy (NIRS) to dynamically study the effect of walking on oxygen desaturation of the gastrocnemius muscle of the impaired leg during exercise , 10–14. with PAD –14. Figowith PAD  used a pi et al.  studied i et al.  studied 2 parameters before and after 12 weeks of a traditional walking program versus a walking-with-poles exercise program. We reasoned that by using the poles to offload the legs, patients would have less claudication pain and be able to walk longer distance thus achieving a greater training effect. We hypothesized that the calf muscle tissue deoxygenation would be less in both groups after 12 weeks of exercise training than at baseline and that the decrease would be greater in those patients assigned to the walking-with-poles group. These data represent a subanalysis of data from a larger clinical trial comparing traditional walking to walking with poles [The purpose of this study was to determine if there were differences in calf muscle StOth poles .n = 91 patients). Of those 91 patients, 3 did not have StO2 analyses on their baseline progressive treadmill test due to equipment malfunction and 3 had questionable data resulting in a final sample of 85 patients. Of those 85 patients, 45 were assigned to the walking-with-poles group and 40 patients were assigned to the traditional walking group. Patients with PAD were screened and recruited to participate in a randomized clinical trial designed to compare the effects of a traditional walking training program versus walking-with-poles exercise training program. A total of 2296 patients were screened for eligibility, 146 enrolled in the study [2 responses to exercise training. Our primary outcome focused on changes the patients realized walking on the constant work rate treadmill test whereas this analysis used data from the progressive treadmill test. At two minutes on the constant work rate test, the speed and incline increased to 85% of the patient's workload on the progressive treadmill test resulting in a sudden decline in StO2. Due to the gentle nature of the progressive treadmill protocol used, we were able to detect more subtle changes in StO2 and onset of claudication pain. The major study was a randomized clinical trial to examine the effects of a traditional walking program compared to walking with poles on walking endurance and perceived physical function in patients with PAD. This subanalysis provides an in-depth examination of StO2, moderate intensity was 45–59% peak VO2, and hard intensity was 60–84% VO2 peak. The program was designed so that patients walked on the treadmill two days/week and outdoors or in the hospital corridors one day/week. The exercise interval was adjusted based on the patient's pain tolerance. For example, some patients could only walk for one to two minutes at the higher intensities. In order for them to achieve 20% total time at the harder intensities, multiple short intervals were completed. Exercise intervals were gradually lengthened and rest times between intervals were gradually decreased. The intervention consisted of traditional walking or walking with poles three times weekly. Patients were randomized using computer-generated permuted blocks and group assignment was managed by the study statistician. The training programs for both groups were identical except that one group exercised with poles and the other group did not. Patients assigned to the walking-with-poles group received training on the use of the poles prior to beginning the exercise program. Additionally, patients were coached on proper poling mechanics during the training period if needed. The training program incorporated interval training whereby exercise consisted mostly of low-to-moderate intensity training at the start of the program and progressed to moderate-to-high intensity. Exercise intensity was determined by heart rate associated with a percentage of oxygen uptake obtained during the metabolic exercise treadmill tests. Exercise duration and intensity was adjusted every three weeks. During the first weeks, patients walked for 30 minutes . By weeks 10–12, patients walked for 55 minutes . Light intensity was defined as 25–44% peak VODemographic information, height, weight, comorbid conditions, and medication history were obtained from the patient and reviewed in the patient medical record. A physical exam was completed prior to participation in the study.The ABI was measured in the more severely affected leg at baseline and 12 weeks after training. The dorsalis pedis or posterior tibial arteries were used for measurement. The location of the signal was recorded and used consistently throughout the study for that individual subject. After the patient rested comfortably in a supine position for 15 minutes, Doppler ultrasound was used to measure the systolic pressure in the right and left arms and in the ankle of the most severely affected leg. The arm with the highest systolic pressure was used to calculate the ABI [2 of the gastrocnemius muscle were monitored continuously throughout the test. Initial claudication time was defined as the time during the treadmill test when the subject experienced pain in the affected leg. The absolute walking time was defined as the time walked by the patient on the progressive treadmill test.Patients were tested using a gentle treadmill protocol that was developed for patients with PAD , 16. IncPeak oxygen uptake was measured using the MedGraphics CPX/D System . The metabolic cart was calibrated using a 3 L syringe prior to each test and the analyzers were calibrated using references gasses and room air. Patients breathed through a mouthpiece and their nose was clipped with a standard nose clip. Oxygen uptake was averaged over 30 s and the highest value at peak exercise was recorded.2 measures were recorded every 3.5 seconds using the InSpectra software. The StO2 values used in this analysis were the StO2 value recorded after 2 minutes of standing prior to exercise (StO2 rest), time walked when the patient reached the nadir StO2 value , StO2 value at peak exercise (StO2 peak), and absolute and relative drop in StO2 .Skeletal muscle tissue oxygenation was measured prior to, during, and after exercise using the NIRS spectrometer . A 25 mm probe attached to an optical cable was used to noninvasively measure the percentage of hemoglobin oxygen saturation of the tissue beneath the near-infrared light. The InSpectra machine was calibrated prior to each test according to known high and low reference standards . The NIRRatings of perceived leg pain were obtained every minute using the Borg ratio scale . Patientt-test for independent samples and Mann Whitney U test. Data that were not normally distributed were log transformed. Regression analyses were used to compute the initial decline in slopes for StO2 values . Pearson r correlations were completed to examine relationships between the variables. Paired t-tests were used to describe changes in measures from baseline to post-12 weeks of training. Independent t-tests were used to examine differences between groups. Intent-to-treat analyses using the baseline value carried forward were completed for the comparisons between groups. The criterion for significance was P < .05. Measures of central tendency were completed to describe the sample. The two groups were compared at baseline using the −1 · min−1. The walking-with-poles group was older than the traditional walking group. No other differences in baseline demographic characteristics were identified. The sample consisted of primarily older (age = 69.4 ± 9.2 years) men. The ABI was .63 ± .19. Over one-third of the patients were current smokers, half were diabetic, and the majority of patients received treatment for hypertension and dyslipidemia . Patient2 reached its nadir value. There was no difference between the time elapsed when the StO2 reached its nadir value and pain onset (P = .87). Although all patients reported claudication pain, exercise was not limited by claudication pain for all patients. Therefore, we examined the onset of claudication pain and time to nadir StO2 for those whose exercise was limited by claudication pain only (n = 72). Patients walked 3.35 ± 2.57 minutes before experiencing claudication pain and 3.67 ± 3.49 minutes when the StO2 reached its nadir value. There was no difference between the time elapsed when the StO2 reached its nadir value and pain onset (P = .40). After exercise, the time for the StO2 to return to half of its resting value was 1.3 ± 1.3 min and 2.2 ± 2.1 minutes to return to resting values. The initial claudication time and absolute walking time were not associated with the StO2 values at rest . There was also a small to moderate relationship between onset of claudication pain and the exercise slope decline in StO2 . Relationships were similar between initial claudication time and absolute walking time and the time elapsed prior to reaching the nadir StO2 values when the analysis was restricted to those whose exercise was limited by claudication . At baseline, calf muscle oxygenation decreased from 56 ± 17% prior to the treadmill test to 16 ± 18% at peak exercise (71% relative decline from rest to peak exercise). Patients walked 4.45 ± 3.67 minutes before experiencing claudication pain and 4.39 ± 3.99 minutes when the StO at rest  but were2 values between baseline and 12 weeks was related to the change in absolute walking time between baseline and 12 weeks  and initial claudication time at 12 weeks . Analysis of covariance was completed to compare the groups with age as the covariate since there was a difference in age at baseline  and absolute walking time (P = .002) were greater in the traditional walking group when compared to the walking-with-poles group. There was, however, no difference in the change in initial claudication time between the two groups (P = .38). Data comparing groups after exercise training were analyzed using intent-to-treat analysis for all patients. The change in time elapsed prior to reaching nadir StObaseline . The chan = 71), the change in time elapsed prior to reaching nadir StO2 values between baseline and 12 weeks was related to the change in absolute walking time between baseline and 12 weeks  and initial claudication time at 12 weeks . Data from a sample patient are depicted in P = .81). After 12 weeks of exercise training, within-group changes in initial claudication time, absolute claudication time and time elapsed prior to reaching nadir StO2 values increased . Similarly, absolute walking time increased more in the traditional walking group than in the walking-with-poles group . There was no difference between the two groups in initial claudication time . Since there was a significant difference in age between the two groups, age was entered as a covariate yet it did not contribute significantly to the model. No differences in recovery time were noted between the traditional walking and walking-with-poles groups at the 12-week follow-up time period . No significant differences were identified in peak oxygen consumption or ABI within or between the two groups from baseline to 12 weeks using intent-to-treat analysis or restricting the analysis to those who completed the training only.When the analyses were restricted to those who completed the exercise training only  there was no difference in the time associated with the nadir of StO2 and the onset of claudication pain, and (3) the time to nadir StO2 of the gastrocnemius muscle was greater after 12 weeks of exercise training in the traditional walking group when compared to the walking with poles group.  There are three major findings of this study: (1) the time elapsed before reaching nadir StO2 values were associated with shorter initial claudication time and total walking duration. Second, in Gardner's study, relationships between initial claudication time (r = .339), absolute walking time (r = .68) and measured time to minimal exercise StO2 were similar to our relationships between initial claudication time (r = .42), absolute walking time (r = .58) and measured distance to nadir StO2. These findings suggest that our sample patients were similar to patients in other studies [ As Bauer notes, during higher work rates and apparent blood flow limitation, muscle oxygen desaturation becomes more rapid in PAD subjects . Thus, o studies .2 of the gastrocnemius muscle was less after 12 weeks of exercise training when compared to baseline. Wang and colleagues recently demonstrated in 17 subjects with PAD who completed a 24-week walking program that an increase in the number of capillaries in contact with type IIx and IIa calf muscle fibers was related to an improved pain-free walking time  [2 area under the curve postexercise training when compared to preexercise training. In our study, an increase in the time to nadir StO2 values was associated with delayed initial claudication time and improved absolute walking time. Our findings provide further evidence that walking exercise improves tissue muscle oxygenation in patients with PAD. Likewise, our findings support the possibility that the onset of claudication and walking duration are linked by the decline in tissue oxygenation. The onset of claudication and the time elapsed prior to reaching the nadir StO2 values were nearly identical. Thus, the patient's perception of pain is a good indicator of StO2 in the calf muscle. Exercise-associated decline in StOP < .05) . InvestiP < .05) . Figoni P < .05)  both rep2 was less in patients assigned to the traditional walking program than in patients assigned to the walking-with-poles exercise training program. We had originally hypothesized that a walking training program with poles for patients would be advantageous because the poles would “offload” the legs during periods of claudication allowing additional walking exercise. Indeed, Oakley et al. [P < .0001) and the level of claudication pain also decreased (P = .0002). Our group has also previously reported that patients had decreased calf pain when walking with poles. Oakley et al. [2. The explanation for these findings may be that leg metabolic changes in the calf muscle occurred to a greater extent in those assigned to the walking-with-poles group or we may not have had enough statistical power to detect changes that were present.Exercise-induced decline in gastrocnemius muscle StOy et al.  investigy et al.  comparedy et al.  providesAnother explanation for this finding may be that the intensity of our training program was based on oxygen uptake associated with heart rate response to exercise. With the addition of the upper body to the exercise regime for patients assigned to the walking-with-poles group, exercise intensity was augmented using the upper body. Thus, the work accomplished by the leg muscles may have been less even though overall exercise intensity was consistent between the two groups of patients. Both groups trained on the treadmill and outdoors. Patients assigned to the walking with poles group were unable to use the handrails on the treadmill for balance; added anxiety may have increased the cardiovascular response thus keeping the overall work accomplished lower in some patients. Our study had several limitations that may limit the generalizability of the findings. First, although age was not a contributing factor to the differences between the groups, the walking-with-poles group was older than the traditional walking group. We cannot exclude that the differences in exercise tolerance and willingness to push limits may differ between someone who is 67 years old  and someone who is 72 years old . This five-year age difference may have influenced our outcomes in ways that we did not measure. Next, the sample was primarily men and thus cannot be generalized to women. Additionally, in order to include only those with compressible vessels, we excluded 17 patients who were initially randomized to our study. Although this limited the variability, it also reduced power in our statistical testing. Lastly, although all of our patients experienced claudication pain, not all patients stopped exercise due to claudication pain. Including patients that did not stop exercise due to claudication pain may have affected the study results.Additional studies examining different walking intensities in patients with peripheral arterial disease should be tested. Further, it may be that use of walking poles early in the rehabilitative effort may augment traditional walking training later by assisting patients to train longer early on. Further research in this area is warranted.2 in the gastrocnemius muscle during walking exercise in patients with peripheral arterial disease reflecting muscle tissue ischemia. Contrary to our hypothesis, after 12 weeks of exercise training, absolute walking time and time to reach nadir muscle tissue oxygenation were prolonged in the patients assigned to the traditional walking program more than those assigned to a walking-with-poles exercise training program reflecting greater tissue perfusion and enhancement of arteriovenous oxygen extraction. The precise mechanism for these differences remains unknown but these findings may provide some insight to tissue adaptations to exercise stimulus and may therefore be useful to PAD rehabilitation programs.In summary, we conclude that there is a significant decline in StO


Meeting sessions examined the epidemiology of Type 1 diabetes; possible underlying causes of the continuing and rapid increase in Type 1 diabetes incidence at younger ages; and lessons learned from previous prevention trials. Consensus recommendations from the meeting were:European experts on autoimmune Type 1 diabetes met for 2 days in October 2010 in Cambridge, to review the state-of-the-art and to discuss strategies for prevention of Type 1 diabetes  and immune responses to them; and the manipulation of the environment, as early as possible in life. Combinations of interventions should be considered as they may allow targeting different components of disease, thus lowering side effects while increasing efficacy. Type 1 diabetes occurs when the body's immune system turns against itself so that, in a very specific and targeted way, it destroys the pancreatic islet β-cells, the only cells in the body that produce the vital hormone insulin. This autoimmune destruction is irreversible and the disease incurable. If new pancreas or islets are transplanted they too are destroyed, unless heavy immunosuppression is applied. Recent genetics and mechanistic studies in humans are indicating major pathways that play roles in early disease development, especially in the interleukin-2 (IL-2) and type 1 interferon systems, probably in concert with the immune response to preproinsulin [Longitudinal studies of children born with high genetic risk of Type 1 diabetes showed that circulating autoantibodies to islet antigens can be seen as early as 6 months. A large proportion of childhood cases has seroconverted by age 3 years, and almost all cases by age 7 years, even although median age-at-diagnosis is approximately 12 years . ChildreNational diabetes registries, especially those with available biosamples, provide Europe with important population-based research resources, for studying disease epidemiology, or for testing hypotheses about disease initiation and progression. Registry data where available provide evidence that the incidence of Type 1 diabetes in children under age 15 years has increased. However, there is a shortage of data on Type 1 diabetes over age 15, although at least half of new cases occur after age 15. Data from registries should be accessible to all bona fide researchers, provided that necessary confidentiality protections are in place. Common standards regarding data fields would add value and also aid data access.The appearance of autoantibodies, the first of which chronologically are usually against insulin itself, is a secondary or downstream event in the autoimmune process. Identification and validation of biomarkers that precede autoantibody appearance are urgently required. Incentives to provide access to these collections should be implemented . Useful 1c levels become abnormal. Screening for risk of diabetes can avoid suffering in children, as diabetic ketoacidosis prevalence was 4% in children enrolled in prevention trials compared with 15–20% in new-onset children in the general population [Despite the limited success of previous prevention trials, they have provided valuable lessons, combined with correctly interpreted data from the animal models. Predictions of the natural history of disease based on combined genetic (HLA) and serological markers (autoantibodies) are accurate. C-peptide is a surrogate marker for loss of β-cell function and progression to diabetes; and the oral glucose tolerance test is a sensitive screening tool for detecting individuals with diabetes before fasting blood sugar or HbApulation . Interve


Poor childhood and adult socio-economic conditions, lower childhood cognitive ability and cigarette smoking are all associated with adult mortality risk. Using data on 4458 men and women aged 60 years from a British birth cohort study, we investigated the extent to which these risk factors are part of the same pathway linking childhood experience to adult survival. Compared with women from non-manual origins, men from non-manual origins, women and men from manual origins, and those with missing data on father's social class had about double the risk of mortality between 26 and 60 years. Cox proportional hazards models showed that these differences were reduced but remained significant after adjusting for childhood cognitive ability, adult socio-economic conditions and smoking. Higher childhood ability increased survival chances by securing better adult socio-economic conditions, such as home ownership, which was strongly associated with survival. These findings were similar for cardiovascular and cancer mortality. Childhood socio-economic conditions are associated with adult mortality risk in historical and contemporary populations . In the Within a life course framework, several compatible pathways have been identified as central to the link between child disadvantage and poor adult health ; poor phSmoking is potentially an important causal link on the pathway between lifetime socio-economic circumstances, cognition and premature mortality. It is the biggest single cause of premature death in the industrialised world , and shoThe Medical Research Council's National Survey of Health and Development is a prospective national cohort of 2547 women and 2815 men, a socially stratified sample of all births that took place in England, Scotland and Wales during the week 3–9 March 1946 . Start oThe underlying cause of death between 26 and 60 years was coded using either ICD9 or ICD10 disease classifications according to standard rules, which distinguished mortality from cardiovascular diseases (ICD9 codes 401-454 and ICD10 codes I10-I89) and cancers (ICD9 codes 140-239 and ICD10 codes C00-C97).Indicators of childhood socio-economic conditions, based on information provided by the mother at home interviews undertaken by health visitors, included father's social class at age 4 years  and parental education (more than a primary education or not). A score of housing quality (range 0–3) allocated one point for each of the following: dwelling in very good repair, dwelling built since 1919, no overcrowding . Those who scored 0 and those scoring 1 were compared with those scoring 2 or 3 (the best conditions). A score of care of the home and child (range 0–5) allocated one point for each of the following ratings by the health visitor: very clean house, very clean child, at least adequate shoes, at least adequate clothes, mother coped well. Those who scored 0–2 and those scoring 3 or 4 were compared with those scoring the highest (the best care).Indicators of adult socio-economic conditions, based on information provided by the cohort member at ages 26, 36, 43 and 53 years, were two measures of household social class , net income (bottom third/top two thirds) and home ownership (yes/no).Childhood cognitive ability was measured at age 8 years, 11 and 15 years using tests designed by the National Foundation of Education Research. These tests have been described in detail elsewhere . At age Educational progress was measured by the level of qualifications obtained at age 26 years. Men and women with degrees and other higher level qualifications were grouped with those who left school  with advanced secondary qualifications as there were no deaths in women with the highest level of qualifications. Men and women who gained ordinary level qualifications  were distinguished from those who left school with no qualifications.Smoking status was defined by the most recent behaviour identified for each individual before death or censoring occurred and was categorised as never smoker, former smoker or current smoker. Never smokers were taken as the reference group.Survival curves, obtained using the Kaplan–Meier method, were first used to compare the cumulative death rate between 26 and 60 years for those in the most socio-economically disadvantaged groups in childhood and adulthood with the rate for the most advantaged group. Cox's proportional hazards models were used to investigate the relationships between socio-economic conditions and adult mortality rates in the whole sample, and in men and women separately. Those with missing data were included as a separate group but were excluded in the analyses stratified by sex because of the small number of deaths in these groups. In all analyses, the highest socio-economic group was taken as reference. The proportional hazards assumption was checked. Follow-up time (in months) was from the cohort's 26th birthday until the first of death, emigration, or the end of March 2006, the cohort's 60th birthday. If death had not occurred, follow-up was treated as censored. For adult socio-economic indicators, the most recent measure recorded for each individual before death or censoring occurred was used in analyses.Using all cause mortality as the outcome, we included father's social class and the most powerful adult socio-economic measures in the same model, identified by including various pairs of adult indicators in hazards models and seeing which had the strongest independent effects on mortality. We tested whether the patterns seen for all cause mortality were also seen for cardiovascular mortality and cancer mortality using competing risks Cox's proportional hazards models, which involved censoring deaths from other causes, at time of death. We tested for sex interactions and, where significant, report the results for men and women separately in the text.We then investigated whether childhood cognitive ability and educational qualifications were related to all cause mortality, and if these relationships were independent after mutual adjustment. We tested whether they accounted for the effects of childhood or adult conditions on all cause mortality. Finally, we investigated the effect of smoking on all cause mortality and tested whether smoking accounted for the effects of socio-economic conditions or childhood cognitive ability on mortality. These analyses were repeated using cardiovascular disease mortality and cancer mortality as outcomes.All models were conducted in STATA 8.2 and repeated allowing for the initial sampling procedure. As the results of these models were very similar, the unweighted results are presented.Between age 26 and 60 years 332 people (137 women and 195 men) died. The death rate was higher for men compared with women (hazard ratio (HR) 1.3, 95% confidence intervals 1.1, 1.7).p-value = .03 for the interaction between sex and father's social class). Men from non-manual origins, women and men from manual origins, and those with missing data on father's social class had about double the risk of adult mortality compared with women from non-manual origins  than those from non-manual origins (5.4%) (log rank test = .005). This was confirmed by sex-adjusted estimates from a Cox's proportional hazards model . There w origins . A variaThe hazard ratios (worst versus best) for the scores for housing quality and care of house and child were similar to the ratio associated with social class of origin . The effStudy members living in manual households in adult life had a 60% greater death rate of those in non-manual households  . The genIn a model containing the groups based on father's social class and gender, and adult household social class and home ownership, women from non-manual origins retained their significantly lower mortality rates over others even after accounting for both indicators of adult socio-economic conditions . In thisp-value for interaction = .09). For cancer mortality, there was an effect of father's social class for men  as well as women .Of the 332 deaths between 26 and 60 years, 81 (24.4%) were from cardiovascular diseases (CVD) and 136 (41.0%) were from cancers. The effects of socio-economic conditions were in the same direction for CVD and cancer mortality . The socHigher scores on the cognitive tests at 8, 11 and 15 years were associated with lower adult mortality . Those wIncreasing levels of educational qualifications by age 26 were also associated with lower adult mortality . In a mop < .001). When entered into a model with father's social class and gender .p = .17 for the interaction between smoking and sex). When smoking was added to the separate multivariable models for men and women  than non-smokers (5.1%) (log rank test = <.001). Smokers had a HR of 2.4  compared with never smokers. The effects were stronger in women  than in men  and cancer mortality  and its effects in the multivariable models were similar to its effects for all cause mortality (not shown).Smoking was strongly associated with cardiovascular mortality  . This diThe persistent protective effect of home ownership on mortality, even after adjustment for cognitive ability and smoking, may be because it more accurately reflected socio-economic conditions throughout life than a one off measure of social class. Home ownership reflects past, current and future wealth, and home owners in this cohort benefited from the rapidly rising British house prices since the 1970s . This maOur findings show the importance of not relying only on measures of adult social class when elucidating pathways between lifetime socio-economic conditions and survival. The highest risk of mortality is not necessarily captured by a measure of social class; those for whom these data were missing, either because there was no occupation or because the occupation was unknown, had a particularly high risk of subsequent mortality in this study.The effect of early cognitive ability on adult mortality has been shown elsewhere . Only a A study of 1347 men and women in the West of Scotland  showed tIn a study of all Swedish individuals born between 1944 and 1960 and followed until 1990, adjustment for educational attainment resulted in a more marked attenuation of the effect of childhood socio-economic conditions on later life mortality than later life social class . The autThe effects of smoking on mortality are well known, although there are substantially more data on men  than on The two-fold risk of all cause mortality from smoking in this cohort up to age 60 years is slightly lower than the three-fold difference in mortality in middle age (35–69 years) reported by Doll et al.  for smokPatterns of smoking by social class and gender vary over time within a population; variations across populations reflect the timing of the secular increase and subsequent decrease in smoking behaviours. Changing patterns in smoking behaviour are reflected in changing patterns of cause specific mortality , and wouIn an early post war cohort early socio-economic differences in all cause mortality were striking in women but not in men and were not fully explained by childhood cognitive ability, adult socio-economic conditions or smoking. The strongest adult socio-economic indicator of mortality in this cohort was home ownership. Higher childhood cognitive ability influenced mortality risk by helping to secure better adult socio-economic conditions, such as home ownership, which strongly improved survival chances. Better conditions were associated with a lower risk of smoking and this also improved survival, and partly explained socio-economic differences in mortality. These findings were similar for cardiovascular and cancer mortality.


The socio-economic impact of critical illnesses on patients and their families in Europe has yet to be determined. The aim of this exploratory study was to estimate changes in family circumstances, social and economic stability, care requirements and access to health services for patients during their first 12 months after ICU discharge.Multi-center questionnaire-based study of survivors of critical illness at 6 and 12 months after ICU discharge.Data for 293 consenting patients who spent greater than 48 hours in one of 22 UK ICUs were obtained at 6 and 12 months post-ICU discharge. There was little evidence of a change in accommodation or relationship status between pre-admission and 12 months following discharge from an ICU. A negative impact on family income was reported by 33% of all patients at 6 months and 28% at 12 months. There was nearly a 50% reduction in the number of patients who reported employment as their sole source of income at 12 months (19% to 11%) compared with pre-admission. One quarter of patients reported themselves in need of care assistance at 6 months and 22% at 12 months. The majority of care was provided by family members , for half of whom there was a negative impact on employment. Amongst all patients receiving care, 26% reported requiring greater than 50 hours a week. Following discharge, 79% of patients reported attending their primary care physician and 44% had seen a community nurse. Mobility problems nearly doubled between pre-admission and 6 months (32% to 64%). Furthermore, 73% reported moderate or severe pain at 12 months and 44% remained significantly anxious or depressed.Survivors of critical illness in the UK face a negative impact on employment and commonly have a care requirement after discharge from hospital. This has a corresponding negative impact on family income. The majority of the care required is provided by family members. This effect was apparent by 6 months and had not materially improved by 12 months. This exploratory study has identified the potential for a significant socio-economic burden following critical illness. Over 100,000 patients are admitted to intensive care units (ICUs) in the United Kingdom (UK) per year. Over the 5 years following an ICU admission there is an excess mortality for these patients compared to an age and sex matched population . In addiUp to two thirds of ICU survivors may experience significant problems with physical and psychological health as well as social functioning -10. HoweThus, the aims of this exploratory study of a representative sample of UK survivors of adult, general intensive care treatment were to: (1) explore the use of a novel questionnaire set; (2) estimate changes in family circumstances and social and economic stability; (3) assess what additional assistance  patients believed they required and whether these requirements had been delivered and (4) place the findings in the context of the health-related quality of life (HR-QoL) during the year of follow-up.This study was conducted by members of the Intensive Care After Care Network , a groupThis prospective cohort study recruited patients admitted to the ICUs of 22 UK hospitals  from 31 August 2008 until 28 February 2010. This multi-center study was approved by an appropriate multi-center research ethics committee (Ref 08/H0502/90). All patients that had received at least 48 hours of level 3 dependency care  were eligible for inclusion . Patientpro bono from QualityMetric Inc, Lincoln, RI, USA) [This was a self-completed questionnaire-based study   and a noFor those patients who had initially returned the questionnaire booklet at 6 months, and remained alive, but had failed to respond at 12 months, a protocol amendment was sought and approved by the research ethics committee to allow the patient's primary care physician to be contacted. Death was determined using the NHS Strategic Tracing Service (NSTS). Primary care physicians were contacted after two failed mailing attempts and no response to a home phone call (14-15 months post-ICU discharge). Each primary care physician was asked to clarify whether the patient had died, changed address or had been admitted into a long-term care facility.Patients were first asked to complete the EQ-5D  relating to their health state prior to ICU admission. The questionnaire booklet then included a further EQ-5D, EQ-VAS, SF-36v2 and our novel socio-economic question set to be completed with respect to status at the time of receipt -21. The The SF-36 is a comprehensive, generic 36-item questionnaire that is used extensively in clinical practice to describe HR-QoL . It has The demographic, social and economic impact question set was developed in conjunction with the Department of Public Health, University of Oxford. The question set was specifically designed to determine changes in family circumstances, social and economic stability, and working lives and to assess what additional assistance  patients received or believed they required and whether this had in fact been delivered. The demographic questions were adapted from a draft of the Office of National Statistics' questionnaire intended for use during the 2011 national census . A potenInformation about the acute illness and ICU stay were derived from the Intensive Care National Audit and Research Centre's (ICNARC) Case Mix Programme data for those units contributing to the Case Mix Programme; a similar data set was retrieved manually from non-ICNARC-contributing ICUs.Returned questionnaires were read using an electronic form reader  and the data uploaded into an SQL database , which automated the follow-up process. Data was subsequently exported to Microsoft Excel and IBM SPSS v20 for analysis.The data underwent a quality assurance process and outlying or inconsistent data points were verified with the contributing investigators. All data were assessed for normality, with subsequent analyses performed using t-tests, non-parametric equivalents or analysis of variance.Study enrolment is summarized in the CONSORT diagram Figure . The stuOf the responders, 97% reported no change in their relationship status. There was little evidence of a change in accommodation status between ICU discharge and the 6-month and 12-month follow-up time points with the proportion of patients either owning their property outright or with a mortgage (71%) or living in rented accommodation (25%) remaining stable during this period.Prior to admission 40% of patients reported themselves as being in full or part-time employment. A further 46% were retired, 6% were unemployed, 6% on long-term sick leave and the remainder opted not to disclose. For the purposes of this study, a negative impact in employment status was defined as becoming unemployed, taking early retirement, switching to part-time work or taking long-term sick leave during the study follow-up period. A negative impact on employment was reported by 33% of all patients at 6 months and 28% at 12 months. In parallel, critical illness also impacted on reported family earning source and income. Excluding those patients who were retired prior to ICU admission, there was a demonstrable reduction in reported income from wages or self-employment at 6 months  of patients reported themselves in need of help with the activities required for daily living (referred to as care assistance) at 6 months. This had fallen marginally to 22% at 12 months. For those patients receiving care at 12 months following discharge from ICU, 37% needed 0-19 hours a week, with a further 26% needing greater than 50 hours. The majority of this care was provided by family members (80% at 6 months and 78% by 12 months), with approximately 25% of responders receiving care at both 6 months and 22% at 12 months post-ICU discharge. In addition, 51% and 47%, respectively of those families providing care had to make a major adjustment to working life , a family member had been given long-term leave, had to leave a job, reduce hours or stop work to provide care. By 12 months, 7 patients no longer declared a family care requirement and four of these family members had been able to return to work after a year. Thus in 8% of all cases, a family member had experienced a significant reduction in employment activity for at least a year.Of the patients reporting a need for care, 23 patients (31%) at 6 months and 25 (38%) at 12 months had had to spend savings, borrow money, look to charity or remortage/sell their house (or a combination of these) to pay for care.Following successful discharge to home, 79% of ICU patients reported attending a clinic appointment with their primary care physician and 44% had seen a community-based nurse Figure ; the majP = 0.10). The individual domain scores are summarized in Table The median scores for overall quality of life using the EQ-5D visual analogue scale was 64 (interquartile range 46 to 80) at 6 months and 66 (interquartile range 44 to 80) at 12 months For the 74 patients who were lost to follow-up between the 6- and 12-month follow-up period, additional data was gathered from the respective general practitioners (GPs) for 65 (88%). For 46% of these patients their GP was unaware of any reason that would prevent them from responding to the study mailings. A further 23% were receiving home assistance, 15% had died without the knowledge of the investigators and 4% were in sheltered accommodation or homeless. These patients were not reported in the final cohort of 293 patients.This is the first UK (or indeed European) study to estimate changes in family circumstance, describe social and economic stability, and quantify the care requirements amongst survivors of ICUs. From this exploratory study we report that approximately one quarter of patients who had received more than 48 hours of level 3 care report being in need of care assistance during the first 12 months after ICU discharge. Family members provide the majority of this care, and half of these family members have made a major adjustment to their own working lives. These ICU patients report reduced HR-QoL compared to population norms and three-quarters experience moderate or extreme pain. Following successful discharge to home, the former ICU patients have a high utilization of a variety of health services. The majority of these patients report attending either their primary care physician or their practice/district nurse.One quarter of patients followed up by this study reported themselves in need of assistance with care at 6 months, falling marginally at the 12-month follow-up period. For those patients receiving care at 12 months, a quarter needed greater than 50 hours. Family members provided the majority of this care. Other studies have shown a similar need for care assistance. In 1994, Covinsky et al. demonstrated that a third of seriously ill hospitalized patients required considerable care-giving assistance from a family member in the 12 months following hospital discharge . SwobodaWe found that of those families providing care at 6 months and 12 months, around half had to make a major adjustment to their working lives. Van Pelt et al. also showed that a reduction in employment and disruption in lifestyle were common . At 26 mIt is only over the last few decades that an increased awareness of the potential distress experienced by families of ICU patients has kindled strong interest in family-centered care -33. HoweThis UK study has demonstrated a negative impact on employment and on reported family earning sources and income; this resonates with the North American data in spite of the different social support and economic background. The study by Covinsky et al. reported that a family member had to leave work or make another major life change in 20% of cases , and manWe demonstrated an increase in reported use of state financial support at 6 months Figure  and a doThe HR-QoL data were collected to provide a context for the social and economic data; the SF-36 data demonstrate a marked deficit in all domain scores at both 6 and 12 months and were consistent with previous reports ,35,36. CThe EQ-5D data show many need assistance with everyday activities and medical care after they are discharged back to their homes. Family caregivers take on this new and unfamiliar role, and many will experience stress and negative health outcomes themselves. Caregiver distress scores are highest if the people that they are looking after are in obvious pain or discomfort : signifiPatients who survive a prolonged stay in an ICU present many challenges to the health-care teams who assume responsibility for their subsequent care. We found that the majority of survivors in our study report visiting their primary care physicians and utilizing community nursing services. It is clear that the foundation of care following critical illness is provided in the community. Thus, primary care physicians are increasingly faced with assuming the responsibility for managing these complex patients.A third of these patients are referred to specialist medical services and a further third to psychological services. Due to limitations in questionnaire design we cannot exclude a potentially unmet need for specialist care in this patient group. Of particular interest is the access to psychological services - this study demonstrated that 44% of patients report themselves to be moderately or extremely anxious or depressed at 12 months following discharge. However, the number of patients who were able to access psychological services was appreciably lower than the apparent demand. If psychological morbidity is not addressed then return to work may be delayed or prevented .This study was a comparatively large cohort study ,29,30. IThe characteristics of the study group are similar to previously published longer-term outcome studies of ICU survivors in the UK and allow comparison with previously published work ,13,20. LThis study has several limitations. All follow-up studies are vulnerable to enrolment bias. Patients were consented during the final days of their hospital admission or in an ICU follow-up clinic. This process has clear pragmatic advantages but also includes a potential bias towards those who are more physically or mentally able at this relatively early stage in their recovery.As with any follow-up study the loss-to-follow-up rate may limit the generalizability of any results. We were unable to record the full characteristics of those patients who opted not to consent or withdrew during follow-up, but where data are available these are presented in Table Limited data were collected on the patients and their family status before ICU admission. As ICU admission is generally an unplanned event, it was necessary to rely on patients' recall of their prior functional status, which may have been subject to recall bias. However, others have used a similar approach to tackle this challenge . The lacThis was a targeted exploratory study using a novel written question set. Although reviewed by patient and family groups, the question set had not been formally piloted. Following analysis we have excluded a variety of questions that were found to be inconsistent in terms of their response or application. Whilst we have succeeded in advancing our understanding of the socio-economic and family effects of surviving critical illness, we now recognize the limitations of some questions.Such limitations include our definitions of care-giving and their relevance to the level of care being provided. A more specific assessment of activities of daily living (ADLs) using standardized instruments, such as the Caregiver Assistance Scale, Care-giving impact scale or Personal Gain Scale ,42, may per se and the impact described; effects in individuals may be due to their acute illness or progression of pre-existing disease. Similar effects may possibly be observed in a population of survivors of serious illness who did not spend time in intensive care; future studies should perhaps enroll such populations as comparator groups.As the study was conducted in the UK, data concerning access and provision of state financial/social support and health care may not be relevant elsewhere. Our findings concerning changes in employment status for this general ICU population do echo those of the Canadian ARDS cohort, although our population was around 10 years older and thus retirement was more common . ImportaThe results of this exploratory study suggest that many patients surviving an intensive care admission have significant functional disabilities, may face a change in their employment and commonly have a care requirement after discharge from hospital. A significant proportion of those who do return to work do not return to their pre-existing level of income or workload commitment. The majority of the care required is provided by family members. In addition, the reduction in patients who return to employment at 6 months and the corresponding use of state financial support remains fairly static at 12 months. This suggests that those family members who become informal caregivers make adjustments to their working lives, resulting in a reduction in financial status. More positively, we did not observe patterns of intimate relationship breakdown or loss of original home, but we cannot exclude this being a major issue in other populations or occurring at a later date. We would cautiously conclude that in families who were experiencing the greatest disruption, this effect was apparent by 6 months and had not materially improved by 12 months. This exploratory study has demonstrated a significant socio-economic burden following critical illness. As important are the methodological lessons learnt, which will hopefully guide future research and ultimately intervention studies aimed at improving these important outcomes.APACHE II: Acute Physiology and Chronic Health Evaluation II; EQ-VAS: EuroQol Visual Analogue Scale; GP: general practitioner; HR-QoL: health-related quality of life; ICNARC: Intensive Care National Audit and Research Centre; IQR: interquartile range; SF-36v2: Short Form 36 Version 2SJB, JG and BHC conceived and designed the study. SJB, JG and CJ designed the questionnaire. RAH designed the database and questionnaire analysis system. KM and JB coordinated the study and supervised questionnaire dispatch, retrieval and follow-up. SJB, RAH and JG conducted the analysis and produced the first draft of the manuscript. All authors critically revised the manuscript. All authors have seen and approved the final draft of the manuscript.Questionnaire bookletClick here for fileCritique of questionnaire instrumentClick here for file


In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae. Here we have developed a novel single-molecule fluorescence approach to directly observe interactions and stoichiometries in protein complexes from cell extracts and from in vitro synthesized components. We show that up to 50 cavins associate on a caveola. However, rather than forming a single coat complex containing the three cavin family members, single-molecule analysis reveals an exquisite specificity of interactions between cavin1, cavin2 and cavin3. Changes in membrane tension can flatten the caveolae, causing the release of the cavin coat and its disassembly into separate cavin1-cavin2 and cavin1-cavin3 subcomplexes. Each of these subcomplexes contain 9 ± 2 cavin molecules and appear to be the building blocks of the caveolar coat. High resolution immunoelectron microscopy suggests a remarkable nanoscale organization of these separate subcomplexes, forming individual striations on the surface of caveolae.DOI:http://dx.doi.org/10.7554/eLife.01434.001 If you could look closely enough at the surface of some animal cells, especially fat or muscle cells, you would see that they are covered with pocket-like indents called ‘caveolae’. These structures are thought to help the cells communicate with the outside world, but they can also be used by viruses to gain entry into living cells.Examining these caveolae even closer would reveal that these pockets contain proteins called caveolins that bind to each other—and also to cholesterol and fatty acids—to form a scaffold that help to maintain the shape of the caveolae from inside the cell. Each caveolae in a mammalian cell typically contains over 100 caveolin proteins. Caveolar coat proteins, or cavins for short, are also important building blocks for caveolae: however, we know relatively little about the interactions between caveolins and cavins.Now, Gambin et al. have used powerful new single-molecule techniques to study these interactions. These experiments looked at the three main types of cavin proteins that associate with caveolae, and by tracking individual protein molecules they showed that cavin1 can interact with either cavin2 or cavin3, but that cavin2 and cavin3 do not interact with each other. Furthermore, cavin2 and cavin3 exist in separate stripes on a caveolae. Gambin et al. also stretched the cell membrane by forcing cells to take in extra water, and showed that this caused the cavin coat to peel away from the caveolae and break down into distinct cavin1-cavin2 and cavin1-cavin3 building blocks.Faulty versions of caveolins and cavins have both been associated with several diseases in humans, including heart disease and muscle disorders. As such, an improved understanding of the formation and break down of caveolae may prove useful for developing treatments for these conditions.DOI:http://dx.doi.org/10.7554/eLife.01434.002 Caveolae are an abundant feature of the plasma membrane of many vertebrate cells. The surface of adipocytes, endothelial cells, smooth muscle, skeletal muscle and many other cell types is characterized by a dense covering of these small invaginations with a characteristic striated coat, as viewed by electron microscopy, and by the presence of membrane proteins termed caveolins . Three cRecent years have seen a dramatic increase in our understanding of caveolae with the characterization of a family of caveolar coat proteins . Cavin1/Caveolae possess a unique cytoplasmically facing striated coat enriched in CAV1 . Given tHere we have developed new methods that allow reconstitution of the cavin complex and performed a quantitative assessment of cavin complex formation. We use single-molecule fluorescence for its proven ability to directly observe multiple populations and quantify interactions in complex mixtures. These techniques are especially well suited for the study of coat proteins and have been used to study the mechanisms of clathrin assembly/disassembly . Those sFormation of higher order structures through oligomerization is a common behaviour of proteins involved in control of membrane dynamics. Here we set out to determine what role oligomerization of cavins plays in the biogenesis of caveolae. To this end we sought a technique that would allow rapid and quantitative analysis of homo- and hetero- interactions of proteins in vivo and in vitro. We chose single-molecule fluorescence spectroscopy as a way of directly assessing interactions between cavin proteins in complex mixtures.Escherichia coli. This is typically not possible with cavin proteins, which show a tendency to degrade upon expression in a bacterial host , which displays a well-defined residence time in the focal volume of 100 μs. The residence times measured for cavin1 oligomers are approximately 10 times slower than GFP. The structure of GFP is well known and the near-spherical β-barrel has a typical diameter of 5 nm. If we assume that the cavin1 oligomer forms an isotropic complex and if we simplify its shape to a sphere, we can estimate the diameter of the diffusing object to approximately 60 nm . These dAlthough cavin1 and CAV1 appear to be sufficient to form caveolae, cells generally express 3 cavin family members that all associate with caveolae . After tWe performed dual colour co-transfection of MCF-7 cells and single-molecule coincidence analysis of all three cavins. As shown in Leishmania tarentolae–based cell-free system (LTE) as it is derived from a eukaryotic organism and is capable of producing complex eukaryotic proteins in functional form  . Yet theThe hypotonic treatment mediated release of cavin protein from the membrane provides an opportunity to test the cohesion of the observed cavin1, 2 and 3 caveolar coat. Examination of the cavins released in the cytosol of hypo-osmotic cells showed that all cavins now existed as small oligomers. Remarkably we observed a complete segregation of cavin2 and cavin3. We observed rare oligomers (>15%) made of cavin1-only (red peak in histograms), but not oligomers made of cavin2-only or cavin3-only. The single-molecule coincidence histogram shows clearly that cavin3 remains bound to cavin1, and cavin2 bound to cavin1, and that these two subcomplexes detach separately from the membrane. This was confirmed by classical biochemistry and pull-downs as shown in The identification of two distinct cavin complexes raised the question of their assembly and spatial organization on the cytoplasmic face of caveolae. An immuno-EM method was developed using an ‘unroofing’ protocol modified from To determine if subcomplexes of cavin2 and cavin3 could be resolved on intact caveolae, immuno-EM was performed on the basolateral surface of the plasma membrane in cells expressing cavin2 and cavin3. 3T3-L1 fibroblasts were transfected with either GFP- or Cherry-tagged cavin constructs and were ‘unroofed’. Pre-embedding labeling was performed using with two different directly conjugated antibodies: 3 nm-α-RFP and 7 nm-α-GFP. In accordance with the single-molecule fluorescence data, cavin1-Cherry (3 nm gold–coloured red) and cavin3-GFP (7 nm gold–coloured green) demonstrated a close spatial association and co-labeled the same caveolae with no perceivable separation . Dual-laThese results, together with single-molecule fluorescence studies, demonstrate that cavin1-cavin2 and cavin1-cavin3 complexes form sub-caveolar complexes on the cytoplasmic face of caveolae. These subcomplexes form distinct striations that we propose can shape caveolae and peel away as caveolae flatten  in association with caveolae in cell extracts and on intact caveolae at the plasma membrane; (2) in the cytoplasm, in the absence of caveolin and caveolae; and (3) upon dissociation from membrane-bound caveolae in response to hypotonic medium.Leishmania cell-free system, MCF-7 cells that lack endogenous cavin proteins, and MDCK cells that natively express cavins). The comparison of results obtained in in vitro and in vivo systems shows clearly that each of the cavin proteins has an intrinsic property for self-association to form stable oligomers. After synthesis, in vitro large oligomers are formed at very low concentration  Our data suggest that the cavin complex associated with caveolae in the cell extract is made of typically 50 cavin proteins. We believe that the contribution of endogenous proteins must be minor because of the striking consistency in the size of the complexes observed both for cavins and caveolins in our different systems  Even in the absence of the scaffolding element CAV1, cavins can assemble in the cytoplasm forming a complex that has a similar size to a caveola assembly. But this does not mean that the cavin complex will be fully formed before interacting with caveolae. Expression in cells lacking caveolins may reflect an artificial situation as cavins and caveolins are generally expressed together in vivo. We believe the large complexes formed under these conditions may be rare in vivo, possibly explaining the slow association of expressed cavin with newly-arrived caveolin at the plasma membrane (25 min) when fluorescently-tagged cavin is expressed in mammalian cells . The siz3) The use of hypotonic treatment to swell cells allowed us to gain insights into the complexes that dissociate from membrane-bound caveolae in a reversible fashion . We showThe mode of assembly of cytoplasmic caveolar coat proteins shown here is distinct from that of any other known coat complex. Cavins form characteristic homo- and hetero-oligomeric complexes, which associate to form striated nanodomains that can wrap around caveolae. In contrast to other coat proteins, such as clathrin, which form a very dense basket-like network in a cooperative and stepwise process through protein–protein interactions and binding to membrane components and then is disassembled into individual triskelia in an ATP-dependent process, we hypothesize that caveolar assembly relies on association of pre-oligomerized cavin subcomplexes that give rise to the unique caveolar striations. Further functional characterization of the cavin subcomplexes should prove illuminating in understanding the diverse roles of caveolae.MCF-7 and MDCK cells were maintained as previously described . MDCK ceLeishmania tarentolae is as described in Cell-free expression of proteins for interaction mapping used the eukaryotic Leishmania cell-free system. Manufacture and supplementation of lysate from Single-molecule spectroscopy was performed based on , 2011. 2The single-molecule multicolour detection method is based on a simple principle: the two excitation lasers are focussed within the same point, creating a very small observation volume (∼1 fl). The proteins are tagged with genetically-encoded GFP and Cherry fluorophores. Proteins diffuse freely by Brownian motion, and they enter and exit the confocal volume constantly. To reach single-molecule detection, the samples were diluted to approximately 100 pM concentration, so that only single proteins or protein complexes are present in the confocal volume.For single-molecule coincidence, the time-trace of GFP and Cherry intensity was binned in 100 microseconds time ‘bins’. That is, we calculate the number of photons collected in 100 ms. When the fluorescence is greater than the defined threshold (20 photons), we consider that fluorophores are present in the focal volume. When a burst lasts for more than one time bin, we ‘stitch’ the bursts together. That is, we calculate the total fluorescence emitted by the diffusing objects as long as they are in the focal volume. For single-molecule coincidence, the coincidence ratio is calculated as The brightness analysis is calibrated using GFP expressed in the same MCF-7 cells or cell-free extracts. Due to random diffusion, most of the events are the result of incomplete transfer through the focal volume, and few bursts represent the maximal number of photons that the GFP-tagged proteins can emit.The perfect trajectory, maximizing the time spent in the middle of the focal volume, is extremely rare, and the protein complex is more likely to exit the detection volume quickly. The escape from the detection volume can be modeled as a first order decay: the probability of a long residence in the focal volume is the combined effect of a succession of random events, each with a successful outcome. In this simple model, following the laws of probability, the longer bursts will be exponentially less frequent.The probability of detecting a burst of a given brightness  at short timescales is analysed for correlation as <G(T))>=<I(t).I(t + T)>/<I(t)>.<I(t + T)> using the FCS (Fluorescence Correlation Spectroscopy) software (Zeiss710 Confocor) . This siThis dwell time measured at the single-molecule level for GFP monomers corresponds perfectly to the one measured at a 10 nM concentration using FCS. We did not attempt single-molecule FCS using Cherry fluorophores, as the FCS curves typically display an important contribution from triplet states with a characteristic time of 30 μs. This phenomenon interferes with the accurate determination of residence times.Leishmania tarentolae-based cell-free system  The mixture was incubated for 3.5 hr at 27°C. Four serial dilutions of the proteins of ¼ were made in buffer A . The AlphaScreen Assay was performed in 384-well plates. Per well, 0.4 μg of the Anti-Myc coated Acceptor Beads  was added in 12.5 μl reaction buffer B . 2 μl of the diluted proteins and 2 μl of biotin labeled GFP-nanotrap (diluted in reaction buffer A to a concentration of 45 nM) were added to the acceptor beads, followed by incubation for 45 min at room temperature. Then 0.4 μg of Streptavidin coated donor beads diluted in 2 μl buffer A were added, followed by an incubation in the dark for 45 min at room temperature. ALPHAScreen signal was recorded on a PE Envision Multilabel Platereader, using the manufacturer’s recommended settings . The signals were then averaged and normalized to background.ALPHAScreen cMyc detection and Proxiplate-384 Plus 384-wells plates were purchased from Perkin Elmer . Proteins  were co-expressed in the cell-free system by adding mixed DNA vectors in 10 μl of the For hypo-osmotic treatments, cells were treated with 100% DMEM (iso-osmotic) or with a dilution of DMEM (1:10) in sterile water. The duration of the hypo-osmotic treatment was 20 min at 37°C.MCF-7 or MDCK cells were transfected with equal amounts of cavin constructs as specified in the pertinent figure legends. The soluble cytoplasmic fractions were prepared by extensive washing of cells in PBS. Cells were then scraped into PBS containing protease  and phosphatase  inhibitors. Cells were mechanically disrupted by syringe lysis using a 1 mL syringe and a 25-gauge needle . Cells were then pelleted at 2000 rpm, 10 min at 4°C. 1/120th of the supernatant was retained as the starting material. The remaining supernatant was mixed with 20 μl of prewashed sepharose conjugated antiGFP beads  for 30 m2 at pH 6.9) and sonicated with a probe tip sonicator (Vir Sonic Ultrasonic Cell Disruptor 100–setting 3) at an angle of 45°. Baso-lateral membranes were selected using fluorescence microscopy . Areas with fluorescent membranes but lacking nuclei were selected and washed repeatedly with PHEM buffer, blocked in 0.2% bovine serum albumin and 0.2% fish skin gelatin then incubated with α-GFP and α-RFP antibodies. Dual labeling was performed as follows: unroofed cells were labeled first with the large 7 nm α-GFP antibody for 30 min, washed with block and then re-labeled with the 3 nm α-RFP antibody for 30 min. Basal membranes were then fixed in 2.5% glutaraldehyde  for 1 hr at room temperature and then post-fixed in 1% OsO4 for 1 hr, also at room temperature. Dishes were serially dehydrated in increasing percentages of ethanol in a BioWave microwave and LX-112 resin was infiltrated at ratios of 1:2, 1:1, 2:1 (resin to ethanol) and 100% resin twice in a BioWave microwave. Resin was polymerized at 60°C overnight. Ultrathin sections (55–65 nm) were then cut, such that only the very first sections were collected  and imaged in an JEOL1011 electron microscope at 80 kV equipped with a Morada Soft Imaging System 4K × 4K camera at twofold binning under the control of iTEM .Cells were grown as described above, plated onto 6 cm tissue culture dishes (TRP), transfected overnight with the plasmid of interest, trypsinized (TryplExpress–Gibco) and replated onto 3 cm tissue culture dishes (TRP) that had been pre-coated with Poly-L-Lysine . Cells were incubated for 4 hr to ensure attachment and flattening of cells to the coated dish, washed with PHEM buffer . Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  In general there was very little explanation about how the data presented related to the conclusions that were drawn.1) The data in 2) Problems arise with the quantitative analysis. a) b) The residence times shown in c) These diffusion times are then converted to a size of a particle by a method that is not explained. Is the particle modeled as a linear polymer, as a sphere, or as some type of ellipsoid? What does the single size mean? I would guess that an effective radius of gyration is meant, but it would be good to know.d) The analogy with true FCS measurements does not seem correct – again at least as explained. Usually, FCS measures the fluctuations in the number of particles in a measuring volume as a function of time. This study seems designed to measure intensity correlations as a single particle diffuses into and out of the measuring volume. There is a rigorous theory for interpreting FCS data in terms of diffusion coefficients. I do not know what is the basis for interpretation of the data in this study.3) In the Results section, assertions are made about the number of CAV1-GFP molecules in a fluorescent particle and the association with cavin1-cherry with CAV1-GFP. What is the evidence supporting these statements, and how were the data interpreted?4) The size for a CAV1 particle (caveola) is the same as for the cavin1 complexes, which seems surprising. This needs to be explained since the caveola also includes the cavin1.5) 6) Other comments that also need to be addressed:7) The authors conclude they have determined the composition of caveolae yet their analyses may be entirely focused on cytosolic subassemblies rather than membrane associated caveolae. The authors are encouraged to revise the text very carefully to ensure that they are accurately describing what they are monitoring.8) In August a paper appeared in PLOS Biology from Ben Nichols using crosslinking followed by detergent extraction to characterize the same complexes. The authors should include a discussion of how their findings corroborate or differ from those reported in that study.9) Please clarify the basis for the statement that most cavin1 assembled into oligomers within 15 minutes.10) The manuscript slightly hypes the novelty of the methods used . In this light, the last paragraph of the Discussion should be modified. Many methods already achieve what the authors write there.11) Please include discussion of the consequences of the presence of endogenous untagged versions of the studied proteins in cells. 1) The data inclearly show that all three cavins can self-associate and that cavin1 can associate with cavins 2 and 3, but cavins 2 and 3 do not co-polymerize. The data inalso show qualitatively that polymerized cavins diffuse more slowly than monomeric GFP.2) Problems arise with the quantitative analysis. a)shows an exponential fall-off in the fraction of occurrences of different numbers of photons per burst. It is asserted that this is consistent with GFP monomer diffusion. The basis for this assertion is not explained.We now have a better description of the burst profile in the main text and in the Methods section.Cell (25 May 2012). Instead, our analysis conservatively states that the highest brightness reached by the oligomers gives an indication of the maximal number of proteins included in the oligomers. Because the profile of bursts remains exponential  The residence times shown inshow a sharp boundary with a minimum residence time. This seems unlikely since by Brownian motion some particles should barely enter the measurement volume and then exit rapidly. Thus, it seems likely that this lower limit for residence time is due to some filtering of the data that is not explained well. The impact of this filtering on the calculated diffusion coefficients is not known.As described in the Methods section, we indeed have to use a threshold to detect a burst, and the events considered correspond to trajectories passing through the middle of the focal volume. This approach proved to be valid for the GFP-only construct, as the residence times measured at single-molecule resolution matches perfectly the average GFP diffusion times measured by FCS at higher concentrations. When performed on A488-only molecules, the diffusion times were about 50 microseconds, also matching the ones observed at higher concentration using regular FCS. In the case of the cavin oligomer, the threshold does not create the lower limit for the residence times, as shown by the dissociation experiments in This remark is actually extremely relevant and we now included it in the manuscript.c) These diffusion times are then converted to a size of a particle by a method that is not explained. Is the particle modeled as a linear polymer, as a sphere, or as some type of ellipsoid? What does the single size mean? I would guess that an effective radius of gyration is meant, but it would be good to know.As understood by the reviewer, the size described in the paper referred to the radius of gyration of a sphere-like object. This seems to make sense for the caveola-sized oligomer formed by cavin1; it is probably more approximate in the case of the sub-complexes. As we do not know at this stage a possible diameter of rod-like sub-complexes, we cannot determine the length of these possible oligomers. The average size indicated in the manuscript correspond to the size of a sphere, and we clarified this point in the text.d) The analogy with true FCS measurements does not seem correct – again at least as explained. Usually, FCS measures the fluctuations in the number of particles in a measuring volume as a function of time. This study seems designed to measure intensity correlations as a single particle diffuses into and out of the measuring volume. There is a rigorous theory for interpreting FCS data in terms of diffusion coefficients. I do not know what is the basis for interpretation of the data in this study.In fact, there is no requirement for a high number of proteins to be present in the focal volume in the mathematical theory of FCS; it is only performed as such due to signal-to-noise issues. A single protein will diffuse in the same manner, whether proteins are also present in the focal volume, and the fluctuations in its burst profile are still due to the local excitation of the laser . The analysis of the stacks of burst profiles (events before and after detected burst) was also performed using the Zeiss710 confocal FCS software. We found a perfect match between FCS numbers and curves obtained at various GFP dilutions.3) In the Results section, assertions are made about the number of CAV1-GFP molecules in a fluorescent particle and the association with cavin1-cherry with CAV1-GFP. What is the evidence supporting these statements, and how were the data interpreted?The number of CAV1-GFP molecules on caveolae was determined using the single-molecule brightness analysis, as shown in 4) The size for a CAV1 particle (caveola) is the same as for the cavin1 complexes, which seems surprising. This needs to be explained since the caveola also includes the cavin1.The text contains a clarified statement in the Results. We also discuss this observation in more detail in the manuscript.5)is a cartoon, but it seems to be cited in the text as providing information about a mixed coat of cavins on a caveola.The panel 3E is only a schematic explaining the data observed in panel 3D: the coincidence between cavin2 and cavin3 when both cavin1 and CAV1 are co-expressed suggest that all proteins are now gathered on the same caveolae. We clarified this point in the Results.6)shows an EM of caveolae stained for various cavins. It is not clear to me how these were interpreted to produce the predicted orientations on the right. “These are simply predicted orientations based on a model of caveolar striations. We have now clarified this in the text and figure legend.Other comments that also need to be addressed:7) The authors conclude they have determined the composition of caveolae yet their analyses may be entirely focused on cytosolic subassemblies rather than membrane associated caveolae. The authors are encouraged to revise the text very carefully to ensure that they are accurately describing what they are monitoring.We revised the text to specify where our measurements describe cytoplasmic components and where the results apply to membrane-bound proteins.8) In August a paper appeared in PLOS Biology from Ben Nichols using crosslinking followed by detergent extraction to characterize the same complexes. The authors should include a discussion of how their findings corroborate or differ from those reported in that study.We now discuss our results in the light of this new study; see also comment #11 below.9) Please clarify the basis for the statement that most cavin1 assembled into oligomers within 15 minutes.We now refer to 10) The manuscript slightly hypes the novelty of the methods used . In this light, the last paragraph of the Discussion should be modified. Many methods already achieve what the authors write there.We deleted the whole statement and concluded the study instead with discussion of the specificity of the cavin coat compared to clathrin and other known protein assemblies.11) Please include discussion of the consequences of the presence of endogenous untagged versions of the studied proteins in cells.We now discuss the possible impact of endogenous proteins on our results.


Drug addiction results from the interplay between social and biological factors. Among these, genetic variables play a major role. The use of genetically related inbred rat strains that differ in their preference for drugs of abuse is one approach of great importance to explore genetic determinants. Lewis and Fischer 344 rats have been extensively studied and it has been shown that the Lewis strain is especially vulnerable to the addictive properties of several drugs when compared with the Fischer 344 strain. Here, we have used microarrays to analyze gene expression profiles in the frontal cortex and nucleus accumbens of Lewis and Fischer 344 rats. Our results show that only a very limited group of genes were differentially expressed in Lewis rats when compared with the Fischer 344 strain. The genes that were induced in the Lewis strain were related to oxygen transport, neurotransmitter processing and fatty acid metabolism. On the contrary genes that were repressed in Lewis rats were involved in physiological functions such as drug and proton transport, oligodendrocyte survival and lipid catabolism.These data might be useful for the identification of genes which could be potential markers of the vulnerability to the addictive properties of drugs of abuse. Drug addiction arises from the interplay between social and biological factors. Among the later, genetic variables are thought to play a major role , 2. ThisOn the other hand, LEW and F344 rats differ in several neurochemical parameters which are related to reward processes. For instance, LEW animals have higher levels of tyrosine hydroxylase in the ventral tegmental area (VTA) but lower levels in the nucleus accumbens (NAcc) when compared with F344 rats -14. TherAnother important difference between both strains is the reactivity of the hypothalamic-pituitary-adenal (HPA) axis which displays a higher activity in the F344 strain when compared with the LEW strain , 17.Notwithstanding all the data referenced above, the differential gene expression pattern in areas of the reward circuit of these two strains was unexplored. Therefore we designed the following experiment where we used Affymetrix cDNA microarrays to study gene expression in two key components of the reward system, the NAcc and frontal cortex (FC)  of the aMale F344 (n=9) and LEW (n=9) rats weighing 300-320 g at the beginning of the experiments were used.All animals were maintained at a constant temperature (20±2º C) in a 12 hours light-dark cycle (lights on at 08:00 hours), with free access to food and water . All animals were maintained and handled according to European Union Laboratory Animal Care Rules (86/609/EEC Directive).Two weeks after arrival in the vivarium, the rats were lightly anesthetized with isoflourane and decapitated. Brains were quickly removed and the FC and NAcc dissected out on ice. After weighed, the tissue was preserved in RNAlater solution  at 4ºC overnight and then at -20ºC until RNA extraction. All the procedures were performed under the maximum sterility and dissection instruments and work surfaces were thoroughly cleaned with RNAase Zap  to prevent RNAase activity. Total RNA extraction was performed with RNAeasy extraction kit with in-column DNAase digestion , according to manufacturer’s instructions. RNA quantity was determined by 260 nm absorbance and integrity was determined with an Agilent Bioanalyzer 2100 .For microarray studies we made 3 pools of RNA per structure and strain, resulting on 12 different pools of RNA . For each strain, RNA from 9 rats was used.Probe preparation, hybridization and analysis were carried out in the genomics core facility of the “Universidad Complutense de Madrid”. Probes for the Affymetrix Gene 1.0 ST were prepared and hybridized to the array using the “GeneChip Whole Transcript Sense Target Labeling Assay” (Affymetrix) according to the manufacturer’s suggestions. Briefly, for each sample of 200 ng of total RNA, cDNA was synthesized with random hexamers tagged with a T7 promoter sequence. The double-stranded cDNA was used as a template for amplification with T7 RNA polymerase to create antisense cRNA. Next, random hexamers were used to reverse transcribe the cRNA to produce single-stranded sense strand DNA. The DNA was fragmented and labeled with terminal deoxynucleotidyl transferase. The probes from each pool were hybridized to the Affymetrix Gene 1.0 ST array for 16 h at 45ºC. Subsequently, arrays were scanned and gene expression indexes were calculated with the RMA software .Expression ratios higher than 2 were considered to reflect induction of gene expression whereas ratios lower than 0.5 were taken as indexes of inhibition of expression. Significance was set to p<0.05.1-6).For the sake of clarity, we have organized the data according to induction/inhibition and anatomical localization criteria  (See Tables 1). Only a limited number of the genes studied were significantly different between strains. LEW rats showed higher expression in both the NAcc and FC in only 5 of the genes included in the array. These genes were related to different functions such as protein transport, lipid metabolism, nitrogen metabolism, hydrolase activity or organismal development (Table 2 for further details).The number of genes that were significantly induced in the FC and not in the NAcc was higher in the LEW strain compared with the F344 strain. Two of these genes were related to oxygen transport (LOC689064 and MGC72973) and another pair was related to ion transport . The rest of the genes induced in the FC were involved in different biological processes such as ADP-ribosylation (Adprhl1), regulation of cell cycle (RGD1310778) and signal transduction (Gpr103) to mention just a few examples (see Table 3).As for the NAcc, only a few genes were induced in this structure in the LEW strain, without alteration in FC expression patterns. These genes were involved in sodium transport (Slc10a4), sphingolipid metabolism (Fa2h_predicted//Wdr59), cytokinesis (Anln), organismal development (Hydin) and apoptosis (Perpr) (see Table 4).With regard to inhibited expression, the genes that were repressed in both the NAcc and FC in LEW rats compared with F344 were more numerous than those which were induced. Interestingly, two of these genes (Nqo2 and Akr1b10) were involved in oxidation-reduction processes while the rest of the inhibited genes had diverse functions, such as calcium binding , sphingolipid metabolism (Sgms2) or drug transport (Abcg2) among others (see Table 5).Specific gene expression inhibition in the FC comprised more genes, with a broader range of functions. More specifically, three of the genes that were inhibited in the FC but not in the NAcc were related to neurotransmitter regulation  and two of them were involved in organismal development (Dlx5 and Cml2). The others were related to biological processes such as membrane organization (Ap1s2), regulation of DNA damage (Chd1l), regulation of oligodendrocyte prolongations (Ermn) and apoptosis (Alox15) among others , cell growth (Igfbp2 and Igfbp6), DNA replication (Rad1) or proton transport (ATP8).In this work we have used cDNA microarrays analysis to study the differential gene expression profile in the NAcc and FC of the addiction-prone LEW strain and its histocompatible control the F344 strain. Although further real-time PCR studies must now validate these preliminary results, it seems that there are four set of genes differentially expressed in both inbred rat strains.1.1.1.To the best of our knowledge, neither of the genes that were induced in LEW rats as compared with F344 rats in both the NAcc and FC had any known relationship to drug addiction and were mainly involved in general homeostatic processes.1.2.Within the genes that were induced in the FC but not in the NAcc of LEW rats compared with F344 animals, Atp5g2 could be related to drug addiction phenomena since it has been shown to be up-regulated in the pancreatic cells of alcohol-consuming rats, a fact which parallels with mitochondrial damage . In nervi protein inactivation by pertussis-toxin-induced ADP-ribosylation is able to reverse some of the behavioral responses elicited by dopaminergic agents during cocaine withdrawal [ADP-ribosylation is a major mechanism for G-protein inactivation. In fact, G proteins have been shown to be altered in opiate-dependent patients  and Gi pthdrawal . As regaThe rest of the genes induced in the FC but not in the NAcc have either unknown functions or no clear relationship with drug addiction processes.1.3.The next set of genes comprises those induced in the NAcc but not in the FC. Among these, one gene that merits mention here is the apoptosis-related gene Perp which is induced in degenerating dopamine neurons . Given t2.2.1.Among the genes that were inhibited in both the FC and NAcc of LEW rats compared with F344 animals, Pvalb is of special interest given that it is a marker for GABAergic neurons and there are several reports in the literature showing that drugs of abuse alter the number of parvalbumin-containing GABAergic neurons in several areas of the brain , 29. Ano2.2.5). Therefore, three neurotransmitter release-related genes seem to be affected in the LEW strain. The relevance of this fact to normal synaptic function and addiction phenomena remains to be determined. The expression of Mme gene, which codes for the membrane metallo endopeptidase  enzyme, was repressed in the FC but not the NAcc of LEW rats. This enzyme is responsible for the degradation of several endogenous peptides including the enkephalins [We then examined the genes that were inhibited in the FC but not NAcc of LEW rats as compared with F344 rats. In this set of genes, Sv2c, the gene coding for synaptic vesicle glycoprotein 2c was repressed. This protein binds to synaptotagmin and regulates exocytosis , a proceephalins . A lowerephalins  or morphephalins  injectioephalins . Chd1l gephalins . Nur77 iephalins . Interesephalins , highlig2.3.Lastly, we found a reduced set of genes that were inhibited in the NAcc but not FC of LEW rats as compared with F344 rats. ATP8 (coding subunit 8 of the ATP synthase enzyme) inhibition could have some importance in explaining the vulnerability to the addictive properties of drugs of abuse. In this sense, it has been already found that the alpha-subunit of ATP synthase is differentially modulated in two subsets of rats which differed in the extinction of cocaine-induced conditioned place preference . AnotherIn conclusion, we have found four set of genes (each one only including a limited number of examples) that may be useful markers of vulnerability to addiction. Further studies should validate these results and test the implication of each of the genes reported here in addiction-related phenomena.