Beta‐cell autoantibodies are established markers of autoimmunity, which we compared between Ghanaian adults with or without diabetes, living in rural and urban Ghana and in three European cities.In the multicenter cross‐sectional Research on Obesity and Diabetes among African Migrants (RODAM) study (N = 5898), we quantified autoantibodies against glutamic acid decarboxylase (GAD65Ab) by radioligand binding assay (RBA) and established cut‐offs for positivity by displacement analysis. In a subsample, we performed RBA for zinc transporter‐8 autoantibodies (ZnT8Ab). Associations of environmental, sociodemographic, and clinical factors with GAD65Ab were calculated.P = .25). This was also true for ZnT8Ab positivity.In this study population , 9.2% had diabetes with adult‐onset. GAD65Ab concentrations were the highest in Ghana‐rural , followed by Ghana‐urban  and Europe  with no differences between European cities. These distributions were similar for ZnT8Ab. Current fever, history of fever, and higher concentrations of liver enzymes marginally explained site‐specific GAD65Ab concentrations. GAD65Ab positivity was as frequent in diabetes as in nondiabetes (5.4% vs 6.1%; Geographic location determines the occurrence of GAD65Ab and ZnT8Ab more than the diabetes status. Beta‐cell autoimmunity may not be feasible to differentiate diabetes subgroups in this population. Among 5898 Ghanaian adults living in rural Ghana, urban Ghana, and three European cities, GAD65Ab was strongly determined by the environment. Thus, GAD65Ab may not differentiate autoimmune subgroups of adult‐onset diabetes. According to the World Health Organization (WHO), the number of adults with diabetes has doubled to 422 million between 1980 and 2014.The occurrence of GAD65Ab constitutes an essential decision criterion for diabetes management,Therefore, we aimed at determining the population‐specific GAD65Ab prevalence as a marker for autoimmune diabetes among a representative sample of Ghanaian adults, and identifying the relationships with geographic location, sociodemographic and clinical factors.22.1The study protocol and procedures of the Research on Obesity and Diabetes among African Migrants (RODAM) study have been published elsewhere.All blood samples were collected, handled, processed, and stored according to standardized procedures and were analyzed in the same laboratory in Berlin (Charité). In this cross‐sectional study, diabetes was defined as fasting plasma glucose (FPG) ≥ 7.0 mmol/L or documented glucose‐lowering medication or self‐reported diabetes. Medical history, lifestyle, and socioeconomic factors were recorded either by an ethnically matched staff in questionnaire‐based interviews or by self‐report.2.2The RODAM study was conducted according to the guidelines laid down in the 1964 Declaration of Helsinki and its later amendments. All procedures involving human subjects were reviewed and approved by the respective ethics committees in Ghana, the Netherlands, the United Kingdom, and Germany. Written informed consent was obtained from all participants.2.3Sociodemographic data were collected as previously described2.42).Anthropometric measures were taken in light clothing without shoes and to the nearest decimal. Weight was assessed with a person scale  and standing height with a portable stadiometer . Waist circumference was measured with a measuring tape. Body mass index (BMI) was calculated as weight (kg) divided by height (m2.52) was calculated using the abbreviated Modification of Diet in Renal Disease (MDRD) formula: 186 × (creatinine in mg/dL) − 1.154 × (age in years) − 0.203 ×  × . Insulin concentrations were assessed using the Mercodia ELISA kit  with a lower detection limit of 76 pmol/L. HbA1c (mmol/mol) was determined using the TOSOH G8 HPLC Analyzer .Fasting venous blood samples were collected and aliquoted immediately after collection according to standard operating procedures and temporarily stored at −20°C. First early morning urine was collected. The separated blood samples and urine samples were stored at −80°C. FPG, C‐reactive protein (CRP), liver enzymes , and biomarkers of kidney function  were measured using an ABX Pentra 400 Chemistry Analyzer . For diabetes definition, we applied FPG because of the questionable utility of HbA1c in populations with frequent hemoglobinopathies and conditions that reduce erythrocyte lifespan. The estimated glomerular filtration rate  as previously described.35S]‐GAD65 in the absence or presence of rhGAD65 (200 ng/mL). Samples whose binding to [35S]‐GAD65 were reduced by 40% in the presence of rhGAD65 were considered as having specific GAD65Ab (true positive) . This binding strength was achieved at higher GAD65Ab concentrations in samples from Ghana than in samples from Europe, and was set as the gold standard in subsequent receiver operating characteristic analysis . At 78% sensitivity and 70% specificity, the GAD65Ab cut‐offs were 121 and 97 U/mL for the Ghanaian and European study sites, respectively.To establish specific cut‐off values for GAD65Ab positivity in this population, we refrained from using arbitrary cut‐offs that are highly laboratory specific. Rather, a competition assay was performed in 59 participants in Ghana without diabetes and in 58 participants in Europe without diabetes, employing recombinant human (rh) GAD65 .In subsamples, we assessed the robustness of GAD65Ab detection using alternative laboratory methods (Table S1). These methods used for GAD65Ab detection include (a) enzyme‐linked immunosorbent assay (ELISA) using a kit by Kronus (Boise) (n = 124), (b) ELISA using a kit by DRG International Inc (n = 84), and (c) luciferase immunoprecipitation system (LIPS) (n = 140) performed in an independent laboratory at Helmholtz Centre Munich, Germany.Lastly, isotype‐specific identification of immunoglobulins (Ig) was performed using an ELISA (Bio‐Rad) in 316 samples  to account for isotype shifts during different phases of the humoral immune response. Specifically, we measured total IgG 1, 2, 3, 4, IgE, IgA, and IgM. This analysis was performed because the RBA for GAD65Ab measurement does not discriminate GAD65Ab of different Ig isotypes.2.7t test, the Wilcoxon rank‐sum test, and χ2 test, respectively. Spearman correlations were used to investigate the relationships of GAD65Ab concentrations between different assays and with different isotypes. Next, we examined associations of place of residence, sociodemographic and clinical factors with GAD65Ab concentrations. Linear regression models were used to calculate adjusted means of GAD65Ab concentrations with their 95% confidence intervals. Study site and length of stay in Europe were included as important sociodemographic factors. Clinically relevant factors comprised duration of diabetes and insulin treatment. Particularly, we hypothesized that infectious agents may serve as potential environmental triggers for islet autoimmunityFigure S3 shows the flow chart of imputed data, resulting in a final analytical sample size of 5898 participants. Multiple imputations were applied to estimate the missing values . The characteristics of the study population are presented as mean ± standard deviation for normally distributed continuous variables, as median (interquartile range [IQR]) for non‐normally distributed continuous variables, and as the number of individuals (percentage) for categorical data. Comparisons across study sites and by diabetes status were made by Student's P < .05 was considered statistically significant.All analyses were performed using SAS 6.1  and a 2‐sided 33.1Table S2 displays the characteristics of the study population according to sex and place of residence. The mean age of the study population was 46.1 ± 11.9 years and the majority was female (62%). Individuals in Europe were mainly first‐generation migrants (98%) and their mean length of stay in Europe was 17.3 ± 8.8 years. Most participants had a lower or intermediate education (71%) and worked in manual jobs (68%). With regard to diabetes, the crude prevalence was 9.2% in the total study population, and the vast majority had an adult disease onset . Diabetes was more common in men than in women (11% vs 8%), and more frequent in migrants than in nonmigrants . More than two‐thirds (69%) were reported to have diabetes, and 58% had an FPG ≥ 7 mmol/L. The median duration of diabetes was 5.0 years (IQR: 1.0‐11.0 years) and this was similar between study sites. Fifty‐four percent of the diabetes patients received medication: oral glucose‐lowering drugs (80%), lifestyle therapy (55%), and insulin treatment (15%).3.2P = .002) and Europe . No differences in GAD65Ab concentrations were seen between Amsterdam , London  or Berlin . Therefore, we combined the European study sites in the following analyses. Correspondingly, the proportions of individuals with GAD65Ab positivity was the highest in rural Ghana, followed by urban Ghana and Europe , respectively.The results obtained by the Kronus ELISA in 124 samples were similar to the RBA‐based measurements: the median GAD65Ab concentrations for Ghana and for Europe were 64.5 U/mL (IQR: 54.9‐101.8 U/mL) and 58.5 U/mL . Yet, as shown in Figure P < .0001). ZnT8Ab correlated moderately with GAD65Ab concentrations .The Pearson correlation between the Kronus ELISA and the RBA was 3.3P < .0001 and Figure S5). This was also true for IgM  and IgE concentrations . The correlations for GAD65Ab concentrations with Ig isotypes and IgG subclasses are presented in Figure S6. IgM, IgG, and IgE levels correlated with GAD65Ab concentrations in the 316 samples analyzed for all four parameters. However, when stratified by study site, only IgM levels correlated with GAD65Ab concentrations and only in Ghana .The additional isotype analyses revealed that the most prevalent serum autoantibody isotype in all study sites was IgG, with IgG1 being the major subclass . IgG1 concentrations in samples from Ghana  were significantly higher compared with those from Europe  gave rise to the hypothesis that GAD65Ab might be related to an infectious environment. In Table  gave ris3.5P = .48). In addition, there were no differences per study site in GAD65Ab positivity between diabetes and nondiabetes groups. These findings were confirmed when excluding participants with self‐reported diabetes (Table P = .62). This translated into 11% (8 out of 70) GAD65Ab‐positive individuals in the diabetes group and 10% (7 out of 70) GAD65Ab‐positive participants in the nondiabetes group (P = .79). For Ig isotypes and IgG subclasses, there were no differences between individuals with or without diabetes. Lastly, none of the individuals with diabetes‐onset before the age of 18 years (n = 11) were GAD65Ab‐positive.Table 44.1The present study aimed at determining the potential of beta‐cell autoantibodies to classify diabetes and to guide its therapy among 5898 Ghanaian adults. Using a highly specific detection method for GAD65Ab, surprisingly, we observed a similar prevalence of GAD65Ab positivity between individuals with and without adult‐onset diabetes. There was a geographic trend in GAD65Ab concentration with the highest concentrations in rural Ghana, followed by urban Ghana and Europe. However, this was only partly explained by markers of infection.4.2The present study provides first‐time findings from a large sub‐Saharan African sample regarding biomarkers of adult‐onset autoimmune diabetes that have been established in mainly European populations. Nevertheless, our results need to be interpreted with caution. Participation in the study was limited to adults aged 25 to 70 years. Formerly, individuals with T1D in sub‐Saharan Africa had a poor prognosis.4.3P = .08), whereas the figures for non‐Hispanic whites were clearly distinct: 6.3% vs 2.0%; P = .001.Previous reports indicate that the occurrence of GAD65Ab and their association with diabetes is ethnicity specific. The prevalence of GAD65Ab positivity in individuals without diabetes spans from 1% in Greenland Inuit through 2% to 3% in individuals with European descent up to 1% to 7% in non‐Hispanic blacks.4.4Plasmodium falciparum is dominated by high IgG and IgM serum levels.Plasmodium sp., are known to initiate autoimmune responses in previously nonautoimmune individuals.The different distributions of GAD65Ab across study sites were striking, and we hypothesized that exposure to infectious environments could contribute to this observation. IgM concentrations were the highest in rural Ghana, and only there, IgM moderately correlated positively with GAD65Ab. High IgM concentrations characterize acute and recurrent infections.5Pending verification in independent sub‐Saharan African populations, our findings may have important implications for clinical management and health care planning for these population groups. Our results highlight the need to validate established markers for autoimmune diabetes in different ethnic populations and to develop new ones. Until then, for the growing group of migrants from sub‐Saharan Africa to Europe, other factors than autoimmune status may be more relevant for efficient and effective disease management and for the identification of novel diabetes subgroups.The authors declare that there are no conflict of interests.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.


Left ventricular ejection fraction (LVEF) and end diastolic volume (EDV) are measured using Simpson's biplane (SB), 3-dimensional method (3DE), and speckle tracking (STE). Comparisons between methods in routine practice are limited. Our purpose was to compare and to determine the correlations between these three methods in clinical setting.  LVEF and EDV were measured by three methods in 474 consecutive patients and compared using multiple Bland–Altman (BA) plots. The correlations (R) between methods were calculated.  Median (IQR) LVEF_SB, LVEF_STE, and LVEF_3DE were 63.0% (60–69)%, 61% (57–65)%, and 62% (57–68)%. Median (IQR) EDV_SB, EDV_STE, and EDV_3DE were 85 ml (71–106) ml, 82 ml (69–100) ml, and 73 ml (59–89) ml. R between LVEF_SB and LVEF_3DE was 0.65 when echogenicity was good and 0.43 when poor. R for EDV_SB and EDV_3DE was 0.75 when echogenicity was good and 0.45 when poor. On BA analysis, biases were acceptable (<3.5% for LVEF) but limits of agreement (LOA) were large: 95% of the differences were between −15.4% and +18.8% for LVEF as evaluated by SB in comparison with 3DE, with a bias of 1.7%. In the comparison EDV_SB and EDV_3DE, the bias was 14 ml and the LOA were between −24 ml and +53 ml. On linear regressions, LVEF_3DE = 17.92 + 0.69 LVEF_SB and EDV_3DE = 18.94 + 0.63 EDV_SB.  The three methods were feasible and led to acceptable bias but large LOA. Although these methods are not interchangeable, our results allow 3DE value prediction from SB, the most commonly used method. Transthoracic echocardiography (TTE) is the most commonly used diagnostic tool for left ventricular (LV) systolic dysfunction. The new guidelines state that LV systolic function should be routinely assessed using two-dimensional echocardiography 2DE) or three-dimensional echocardiography 3DE) . In laboDE or thrDE . In lFew data exist regarding the agreement between LVEF and EDV as determined by these different methods, in clinical practice.We aimed to assess the impact of the method used on LVEF and LVED values as part of a routine practice and to establish correlations between these three methods.The study population comprised consecutive patients undergoing TTE at the A. de Rothschild Foundation Hospital, Paris, France, between March 2015 and August 2016. TTEs were performed by an expert cardiologist. Hospitalized patients comprised approximately 90% of our population.TTEs were performed using a commercially available ultrasound system  with a 1.3–4.2 MHz phased array transducer.LVEF and EDV were calculated by the three methods almost simultaneously, in a predefined sequence, in all patients: Simpson's biplane method, 3DE, and STE, were performed according to current guidelines .Simpson's biplane method: LVEF was calculated using the manual tracing on apical four and apical two-chamber views.Speckle tracking method: the three apical views  were recorded with a frame rate between 70 Hz and 80 Hz. Careful manual tracking of the endocardial contour was performed. Myocardial deformation and LVEF were assessed in a semiautomatic manner, based on greyscale images.Three-dimensional echocardiography: LVEF and EDV measurements were performed after a 3D image acquisition including the entire LV within the pyramidal data set (4 beats volume acquisition). The 3D-guided biplane analysis was the method used. The different steps were described elsewhere .Patient's echogenicity was reported as good, moderate, or poor, to allow further analysis by “acoustic window” subgroups. The quality of the acoustic window had to be sufficient to allow the calculation of LVEF and EDV by at least two of the three methods. Otherwise, the TTE was excluded.As the aim of this study was to compare three echocardiographic methods for LVEF and LVED measurement as part of an everyday practice, no rereading by a second observer was done.Pearson's correlation coefficient was used to analyse correlations between LVEFs and EDVs when calculated by two different methods.As SB is the most available method, simple linear regression was used to assess the relationship between LVEF_SB and LVEF_3DE or LVEF_STE, and between EDV_SB and EDV_3DE or EDV_STE.Analysis of variance was used to compare the three LVEF means  and the three EDV means.Bland–Altman analysis, in which the mean of two measurements was plotted against the difference, was used to measure the variability between two techniques. Differences between methods are in absolute units.Among the consecutive TTEs performed during the study period, 39 were excluded because of atrial fibrillation and an additional 65 because more than one LVEF and/or EDV methods of measurement were not performed.Consequently, the study involved 474 TTEs. The main patients' clinical and echocardiographic characteristics are shown in n = 275), occlusion of the central retinal artery (n = 12), chest pain (n = 12), cancer therapy monitoring (n = 12), dyspnoea (n = 11), hypertension (n = 19), diabetes mellitus (n = 11), heart murmur exploration and control of valvular disease, endocarditis and suspicion of endocarditis (n = 28), preoperative assessment (n = 10), LVEF evaluation and/or heart failure (n = 5), pulmonary embolism (n = 3), subarachnoid haemorrhage (n = 3), ischaemic heart disease (n = 5), history of AF (n = 4), syncope (n = 5), and other indications (n = 59).The indications for TTE were as follows: ischaemic stroke or transient ischaemic attack and haemorrhagic stroke  revealed significant differences between the average LVEF_SB and LVEF_3DE and between the average LVEF_SB and LVEF_STE, with The modified Simpson's rule produced higher LVEFs than STE and 3DE, as shown in Median EDV_SB, EDV_STE, and EDV_3DE are shown in p values of, respectively, <0.0001 and p=0.04.ANOVA revealed significant differences between the average EDV_SB and EDV_3DE and between the average EDV_SB and EDV_STE, with The modified Simpson's rule produced higher EDVs than STE and 3DE, as shown in The highest average EDV was provided by the modified Simpson's rule and the lowest by three-dimensional echocardiography .r = 0.62, n = 450, p < 0.0001). Pearson's coefficients for the correlations LVEF_SB vs LVEF_STE and LVEF_3DE vs LVEF_STE were, respectively . Among the three echocardiographic LVEF estimates, SB and 3DE assessments had the best correlation , r = 0.88 (p < 0.00001), and r = 0.71 (p < 0.00001) when echogenicity was good, and, respectively, r = 0.80 (p < 0.00001), r = 0.90 (p < 0.00001), and r = 0.76 (p < 0.00001) when echogenicity was moderate.The correlations were quite similar when echogenicity was good or moderate and altered when echogenicity was poor. Hence, Pearson's correlation coefficients for EDV_SB vs EDV_3DE, EDV_SB vs EDV_STE, and EDV_3D vs EDV_STE were, respectively, r = 0.45 (p=0.0674), r = 0.65 (p=0.0009), and r = 0.35 (p=0.1736).When echogenicity was poor, correlation coefficients decreased and reached, respectively, Linear regressions were used to describe the relationships between the LVEF and EDV estimates from the different methods. For one unit increase in LVEF_SB, a 0.69 unit increase in LVEF_3DE and a 0.42 increase in LVEF_STE are expected .For one unit increase in EDV_SB, a 0.63 unit increase in EDV_3DE and a 0.84 increase in EDV_STE are expected .After excluding the 23 TTEs with poor echogenicity, the regression parameters were quite similar in the two cases.Bland–Altman analysis was used to evaluate the variability between LVEF_3DE and LVEF_SB, LVEF_STE and LVEF_SB , EDV_3DEPatients with normal LVEF using SB (≥50%) and misclassified by one of the two other methods were rare. Only 26 of these patients (5.8%) had LVEF <50% using 3DE or STE. Twenty patients were misclassified by 3DE (4.5%), 8 patients by STE (1.8%), and 2 patients by the two methods (0.5%).When the limit was set at 45% using SB, 16 patients (3.6%) were misclassified by one of the two other methods.This is a prospective study to compare three approaches of LVEF and EDV estimation as part of a routine echocardiographic practice. Well-designed trials are indeed essential. Although real life practice may sometimes be different, it also deserves to be explored , 8.Today, the modified Simpson's biplane rule is the most commonly used echocardiographic technique for LVEF assessment. However, recent evolutions in echocardiography have led to new techniques, which include STE and 3DE. STE-based LVEF has been shown to be feasible, accurate, and reproducible, with lower interobserver and intraobserver variability than other echocardiographic and magnetic resonance imaging methods .Three-dimensional echocardiography has become the recommended technique for repeated LVEF measurements during, and after cancer therapy, when available .However, for now, only top of the line commercial ultrasound systems are equipped with 3DE and STE software. Hence, this study which looks at an intraindividual comparison of these three echocardiographic methods provides useful data.Our results support the feasibility of these three methods during routine practice, since only 12% of the patients in sinus rhythm explored during TTE did not have at least two of the three methods done.In this study, the mean LVEFs and EDVs were significantly different. These results could be challenged, since at least one study shows no differences between the means (ANOVA) of LVEFs as measured by four different techniques  . This stIn our study, Simpson's biplane method provided the highest mean LVEF and the highest mean EDV, while 3DE provided the lowest mean LVEF. Three-dimensional echocardiography significantly underestimated EDV, which is in line with previous studies on patients with rather preserved LVEF , on patiIn our study, Bland–Altman analysis provided low biases between methods for LVEF measurements, all being less than 5% in absolute units: 1.7% between SB and 3DE, 1.8% between 3DE and STE, and 3.5% between SB and STE. This might be considered clinically acceptable.A recent study in patients with LV dysfunction reported such low variability in biases between echocardiographic modalities for LVEF measurement. The mean absolute differences between LVEF as determined by quantitative vs visual echocardiographic methods were all less than 5% .The biases were also low for EDV measurements in our study: 14 ml between SB and 3DE, 3.5 ml between EDV_SB and EDV_STE, and −11 ml between EDV_3DE and EDV_STE.Despite the documented acceptable biases, the huge LOA precludes routine interchangeability between methods in such clinical setting, unless adjustments are made.This systematic intermodality variability for LVEF and EDV measurements in our study is concordant with previous reports .r = 0.62, p < 0.00001; r = 0.56, p < 0.00001; and r = 0.45, p < 0.00001. A recent study reported similar moderate correlations between different methods for LVEF measurements [The correlations between methods were moderate see , even deurements . The corurements , depend Of note, echogenicity is often worse in hospitalized patients than in outpatients. Indeed, only 57% of the scans in our study had good echogenicity.This seems usual in such cohorts. In a previous echocardiographic study, good echogenicity was reported in only 48.1% of scans .Finally, we could obtain, from 474 scans, interesting linear regression analyses, allowing the prediction of LVEF_3D and EDV_3D, from LVEF_SB and EDV_SB: LVEF_3DE = 17.93 + 0.69 LVEF_SB and EDV_3DE = 18.05 + 0.64 EDV_SB.It is of major importance to emphasize that we do absolutely not suggest the replacement of 3DE by 2DE measurements. We only provide a tool to approach 3D LVEF and EDV, when 3D software is not available on the echo machine.All the measurements were performed online, without rereading by a second observer. However, this was part of the underlying philosophy of this study, which aimed to assess, in consecutive patients in our echo lab real-life practice, the feasibility of the three echocardiographic methods for LVEF and EDV assessment and the correlations between methods.Our results might only be true with the equipment used in this study  and might be slightly different on more recent machines.This study was exclusively driven by a practical goal of improving the assessment of LVEF and EDV in routine.The use of three echocardiographic methods  is confirmed to be possible in everyday practice.The variability in LVEF measurements did not exceed 5% and was hence clinically acceptable.When close echocardiographic monitoring is needed, longitudinal assessments of a given patient should ideally be accomplished using a single echocardiographic modality, on a single machine. This is actually very difficult and sometimes impossible in real life. Hence, there may be some room for the kind of formulas we report. The prediction of 3-dimensional LVEF and EDV from biplane ones is possible, even if in no way one modality can replace another.This may be valuable when 3DE machines are not available.


Various GPCRs have been described as being modulated in a voltage‐dependent manner. Opioid analgesics act via activation of μ receptors in various neurons. As neurons are exposed to large changes in membrane potential, we were interested in studying the effects of depolarization on μ receptor signalling.We investigated potential voltage sensitivity of μ receptors in heterologous expression systems (HEK293T cells) using electrophysiology in combination with Förster resonance energy transfer‐based assays. Depolarization‐induced changes in signalling were also tested in physiological rat tissue containing locus coeruleus neurons. We applied depolarization steps across the physiological range of membrane potentials.M). This became apparent at the level of G‐protein activation, G‐protein coupled inwardly rectifying potassium channel (Kir3.X) currents and binding of GPCR kinases and arrestin3 to μ receptors by a robust increase in signalling upon membrane depolarization. The pronounced voltage sensitivity of morphine‐induced μ receptor activation was also observed at the level of Kir3.X currents in rat locus coeruleus neurons. The efficacy of peptide ligands to activate μ receptors was not  or only moderately  enhanced upon depolarization. In contrast, depolarization reduced the ability of the analgesic fentanyl to activate μ receptors.Studying μ receptor function and signalling in cells, we discovered that morphine‐induced signalling was strongly dependent on the membrane potential (VOur results indicate a strong ligand‐dependent modulation of μ receptor activity by the membrane potential, suggesting preferential activity of morphine in neurons with high neuronal activity. Measurements from G‐protein activation, arrestin3 and GRK2 recruitment which are prone to extensive photobleaching were corrected by subtracting a mono‐exponential decay function  to maximum responses evoked by a saturating concentration of opioid . Due to a functional receptor reserve  in HEK293T cells, only those cells were taken into evaluation which gave an opioid response at holding potential  below 0.3‐fold of maximum DAMGO response. In case of high K+ measurements, this led to exclusion of eight cells under morphine application, and for DAMGO, five cells had to be excluded due to high initial responses that might run into saturation under further depolarization‐induced activation. In the morphine measurements under physiological K+ concentration, no cell had to be excluded for that reason. With morphine, we used higher concentrations of morphine (22.5 nM instead of 4.5 nM) when we measured outward K+ currents. We assumed differences in receptor expression during that time. The difference in ligand concentrations used was of minor importance to our conclusions, as we were focused on the proportional increase of responses, which were normalized to a maximum response. To prevent saturation and to enable observation of depolarization‐induced decrease in Kir3.X currents, for fentanyl , we chose to include cells which gave up to 0.9‐fold of maximum response at −90 mV. This led to exclusion of two cells, which gave higher responses in non‐depolarized condition. For evaluation of the proportional opioid response, we always calculated the mean current response during the last second before agonist withdrawal or repolarization to holding potential, respectively. Furthermore, depolarization to 0 mV caused a short current spike in the outward direction, which returned to baseline within 10 s and was therefore omitted from the calculation of current response amplitudes.We compared Kir3.X currents in HEK293T cells, we sought to use the lowest concentration possible to generate a maximum response . This concentration was determined by a concentration–response curve as shown in Figure ir3.X current response also differed from other studies investigating Kir3.X currents  in HEK293T cells by plotting background (evoked in absence of agonist)‐substracted agonist‐induced currents (evoked by 30 nM DAMGO) against respective V2.111‐mTur2 has been generated by exchange of the Gß1‐Cer , morphine sulfate (MacFarlan Smith), naloxone and tetrodotoxin (HelloBio).For HEK293T cell experiments, morphine hydrochloride was purchased from Merck, DAMGO acetate salt, [Met2.13http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY  of DAMGO , morphine‐evoked responses were increased upon depolarization from −50 to 0 mV from 16% ± 2% to 40% ± 2% of the respective maximal response, reflecting a 2.5‐fold increase in relative current response .The absence of detectable voltage‐dependence of responses to ME ‐YFP, mTurq‐Gß1 and Gγ2, a reporter system that indicates Gi‐protein activation by an increase in FRET , Gαd Figure . To avoid Figure . Depolard Figure  led to nir3.4 currents and G‐protein activation in HEK293T cells, we conducted the same protocol in both assays  of the peptide ligand DAMGO led to a weak Gi activation at −90 mV which was increased 1.4‐fold by depolarization to 0 mV  was reduced ~0.45‐fold at 0 mV compared with at −90 mV  did not change Go activation  whereas the normalized morphine‐evoked GRK2 recruitment was increased significantly upon depolarization  faster upon repolarization, compared with agonist washout  in activity at the μ receptor Figures , DAMGO s Figures  and 6F.  Figures  and 5e.  Figures  and effi Figures  of agoni Figures  and 6a,cd Figure . Third, l Figure .Based on our results that show a decrease of fentanyl‐induced GRK2 recruitment Figure  to μ rec4.2ir3.X currents were measured by means of whole‐cell patch‐clamp recording both in HEK293T cells and in LC neurons from brain slices and GRK2 or arrestin3 recruitment, as well as G‐protein activation was measured by means of single cell FRET assays under conditions of voltage‐clamp. Detectable effects of voltage on μ receptor activity were only seen in the presence of agonist  which was within the physiological range of membrane potential. The calculated net charge movement (z‐factor: 0.66) obtained from the slope of the Boltzmann function of arrestin3 recruitment was similar to previously published values from muscarinic receptors (Ben‐Chaim et al., 1‐ or α2A‐adrenoceptors; Birk et al., Even in native tissue, which required no overexpression of interacting proteins or fluorescent labelling, we could observe a significant effect of depolarization on morphine‐induced μ receptor signalling, despite experimental limitations of the LC slices (North & Williams, 4.3i/o activation is the opening of Kir3.X channels, which leads to hyperpolarization, decreased neuronal excitation and reduced propagation of action potentials (Lüscher & Slesinger, ir3.X currents relative to the response evoked by ME or DAMGO. In HEK293T cells, we observed a robust 2.5‐fold increase in the proportional morphine‐induced Kir3.X activation upon depolarization from −50 to 0 mV (Figure ir3.X currents in LC neurons upon depolarization from −80 to −40 mV, compared to those evoked by the voltage‐insensitive agonist ME (Figures One of the physiological effects of μ receptor‐induced GV Figure , which cV Figure  Also, giV Figure , we coul Figures  and S3D. Figures . The fin Figures . Further Figures  but redu Figures  at equalTaken together, the pronounced voltage sensitivity of the morphine‐activated μ receptor that we have observed in transfected cells, as well as in native neurons, demonstrates the importance of voltage sensitivity of GPCRs in living systems and suggests ligand‐specific voltage sensitivity as a novel opportunity for drug development.The authors declare no conflicts of interest.J.G.R. and S.B.K. designed, performed and analysed the FRET and electrophysiological measurements in HEK293T cells, S.K. performed the experiments in brain slices and J.G.R and S.K. analysed data from brain slices. M.B. designed the study and experiments. J.G.R. wrote the manuscript, and M.B., S.B.K. and C.B. reviewed and edited the manuscript.BJP guidelines for Design & Analysis, and Animal Experimentation, and as recommended by funding agencies, publishers and other organizations engaged with supporting research.This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the Figure S1: Membrane potential regulates MOP‐induced outward GIRK currents+ concentrationsFigure S2: The observed voltage sensitivity occurs also in physiological KFigure S3: Comparison of voltage induced current responses in LC neuronsFigure S4: Bleach correction of FRET recordings, depolarization in the absence of agonist, Reproduction of the protocol from GIRK recordings in FRET recordings from G‐protein activation, comparison of voltage induced changes in Go‐ and Gi‐protein activation assayFigure S5: Depolarization in the absence of agonist, single emission traces and Bystander FRET experiments in a GRK2‐recruitment assayFigure S6: Single emission traces and depolarization in the absence of agonists, kinetics of voltage‐induced changes, characterization of voltage induced effects on DAMGO‐ or fentanyl‐mediated Arrestin3 recruitment at equieffective concentrations to morphineClick here for additional data file.


The ongoing coronavirus disease 19 (COVID‐19) is posing a threat to the public health globally. Serological test for SARS‐CoV‐2 antibody can improve early diagnosis of COVID‐19 and serves as a valuable supplement to RNA detection.A SARS‐CoV‐2 IgG/IgM combined antibody test strip based on colloidal gold immunochromatography assay was developed, with both spike protein and nucleocapsid protein of SARS‐CoV‐2 antigen used for antibody detection. From 3 medical institutions across China, serum or plasma of 170 patients with confirmed COVID‐19 diagnosis and 300 normal controls were collected and tested with the strip. Sensitivity, specificity, kappa coefficient, receiver operating characteristic (ROC) curve, and area under the curve (AUC) were analyzed. Positive rates in different medical centers, age group, gender, and different disease course were compared.158 out 170 samples from confirmed COVID‐19 patients had positive results from the test, and 296 out of 300 samples from normal controls had negative results. The kit was 92.9% sensitive and 98.7% specific. The positive rate was 77.3% during the first week after disease onset, but reached 100% since day 9. AUC and kappa coefficient were 0.958 and 0.926, respectively, which showed the consistency of the test results with the standard diagnosis. Age or gender caused little variations in the kit sensitivity.The rapid, easy‐to‐use SARS‐CoV‐2 IgG/IgM combined antibody test kit has a superior performance, which can help with accurate diagnosis and thus timely treatment and isolation of COVID‐19 patients, that contributes to the better control of the global pandemic. A SARS‐CoV‐2 IgG/IgM combined antibody test strip based on immunochromatography assay was developed with both spike protein and nucleocapsid protein used for detection. With clinical assessment from 3 medical centers from across China, the kit was 92.9% sensitive and 98.7% specific. The positive rate was 77.3% during the first week after disease onset and reached 100% since day 9. As a highly contagious disease, it caused over 26,000,000 confirmed cases of infection and over 870,000 deaths as of early September 2020, which are still on a constant rise.The diagnosis of COVID‐19 has been largely dependent on nucleic acid RT‐PCR with patient throat swab. However, the sensitivity of this test is insufficient, due to the low viral loads in upper respiratory tract of patients, failure to correctly collect high‐quality swab specimen, and varied viral loads in different stage of infection. Liang C, et al reported that out of 22 suspected patients, 8 patients had positive results only after 2 consecutively negative results, and 3 patients turned positive after 3 consecutively negative tests.There has been quite a few detection kits for serological test of SARS‐CoV‐2 antibody.22.1https://www.ncbi.nlm.nih.gov/protein/YP_009724390 for S protein, and https://www.ncbi.nlm.nih.gov/protein/1798174255 for N protein. Mouse anti‐human IgG monoclonal antibody was purchased from GenScript Inc (Cat#: V90401); mouse anti‐human IgM monoclonal antibody was purchased from Clongene Biotech Co. (Cat#: MS00704). Chicken IgY  and goat anti‐chicken IgY polyclonal antibody  were used in the control setting. Other materials including fiberglass sample pad (Shanghai Kinbio Tech. Co), nitrocellulose membrane , conjugate pad (Ahlstrom), PVC panel (Shanghai JN Bio) were all purchased from commercial companies.The recombinant antigens of SARS‐CoV‐2 were commercially purchased—S protein (S1‐RBD) from GenScript Inc (Cat#: T80302) and N protein from Fapon Biotech Inc (Cat#: ncov‐PS‐Ag6). Both S protein and N protein were used as the antigen in the kit. Detail information including protein sequence is available on NCBI website: 2.2Purified water was brought to boil in a round‐bottom flask, when reductive agent trisodium citrate dihydrate solution was added and kept boiling for 1 minute. Tetrachloroauric acid solution was then added and kept boiling for 10 minutes. The solution was cooled down to room temperature, and purified water was added to make 800 milliliter of total volume. The final colloidal gold solution was checked, and those with good permeability under the light were deemed qualified. Collect the solution in a clean flask until use.Potassium carbonate was added to colloidal gold solution and stirred for 20 minutes. While stirring, N protein of SARS‐CoV‐2 antigen was added to make final concentration of 10μg/mL and kept stirring for 45 minutes. Centrifuge at 12 000 rpm for 50 minutes and remove the supernatant. Collect the conjugation solution, centrifuge again, and remove the supernatant, and the final colloidal gold conjugated with SARS‐CoV‐2 N protein was obtained. Apply the same protocol to make colloidal gold conjugated with SARS‐CoV‐2 S protein and with goat anti‐chicken IgY polyclonal antibody. Mix all the conjugates and spray to the conjugate pad.2.3The SARS‐CoV‐2 antibody test strip was based on colloidal gold immunochromatography assay. Colloidal gold conjugated with recombinant SARS‐CoV‐2 antigen and goat anti‐chicken IgY was coated in reagent conjugate pad. Mouse anti‐human IgG monoclonal antibody and mouse anti‐human IgM monoclonal antibody were coated in the detection area of nitrocellulose membrane. Chicken IgY was coated in the control area. During the test, specific SARS‐CoV‐2 antibody in the sample combines to the colloidal gold conjugated with recombinant SARS‐CoV‐2 antigen and forms an immune complex, which flows across the membrane due to capillary action. If the sample contains specific SARS‐CoV‐2 antibody (IgG or IgM), it will be captured by the mouse anti‐human monoclonal antibody in the test area and form a visible line (test line). Excess colloidal gold conjugated with goat anti‐chicken IgY polyclonal antibody keeps moving and specifically binds with the chicken IgY in the control area and from a visible line (control line). If the sample does not contain SARS‐CoV‐2 antibody, only control line will appear.2.4Serum or plasma samples from 470 participants including 170 confirmed COVID‐19 patients and 300 excluded cases were collected from 3 centers across China, which were 1) The Center for Disease Control and Prevention of Jiangsu Province, 2) Chest Hospital of Shandong Province, and 3) Infectious Diseases Hospital of Xuzhou City. The sample collection was performed by following the “SARS‐CoV‐2 infected pneumonia laboratory test technical guidance” protection requirement. Confirmed cases were defined based on the diagnostic criteria including nucleic acid RT‐PCR and clinical characteristics, according to the Guideline of Diagnosis and Treatment of COVID‐19 published by National Health Committee of China.2.5A dropper was inserted the into the blood collection tube and absorbed the sample automatically until it reached to 10μL. The sample was added to the sampling area on the strip, and 2 drops of sample diluent were added Figure A. 15‐20 2.6From patients with detailed clinical information, a timeline graph was illustrated by Office PowerPoint to show disease process of each patient, as well as the timepoint and result of antibody test.For all participants from three medical centers, detection kit sensitivity, specificity, kappa evaluation, receiver operation characteristic curve (ROC curve), and area under the curve (AUC) were generated with IBM SPSS Software (Ver. 23) to assess the accordance of the test result and the clinical diagnosis.Sensitivity, specificity, Youden index, positive predictive value, and negative predictive value were calculated according to formula below:Sensitivity = true positive/ .Specificity = true negative/ .Youden index = sensitivity +specificity −1.Positive predictive value = true positive/ .Negative predictive value = true negative/ .P < .05 was considered statistically significant.With clinical information for each patient, dynamic analysis of positive rate was performed. Positive rates for different clinical centers, gender, age group, and different disease course were further depicted and analyzed by GraphPad Prism 8.0 with unpaired t test, where 33.1The product of test strip for specific SARS‐CoV‐2 antibody based on colloidal gold and immunochromatography assay was completed by Changsha Sinocare Inc, China. To test the strip, serum or plasma from 170 confirmed COVID‐19 patients and 300 normal controls were collected. The appearance of both control line and test line on the strip was considered as a positive result, suggesting that IgM or IgG or both were present in the sample. The appearance of only control line was considered as negative, indicating that none of IgM or IgG was present in the sample. An example of both positive and negative result is shown in Figure 3.2The clinical information of these participants is summarized in Table 3.3Of the patients with detailed information, a timeline graph was depicted to illustrate the illness onset, hospital admission, serological test for antibody, and discharge of each patient, along with their clinical classification Figure . In all Among the total 170 samples with confirmed cases from all medical centers, 158 had positive result from the test, generating a sensitivity of 92.9%. Among the 300 samples from normal controls, 296 had a negative result, with a specificity of 98.7%  and 30% of presymptomatic cases (SARS‐CoV‐2 detected before symptom onset) in travelers and returning residents to Brunei.There has been other report on clinical evaluation of serological test kit for SARS‐CoV‐2. Lijia et al assessed a quantitative test kit for IgG and IgM, but only with a small group of patients .Admittedly, there are some limitations of the antibody test kit. First, our IgG/IgM combined kit does not distinguish IgG and IgM apart, which failed to inform the stage of infection, but on the other hand, it tends to avoid false‐negative result and have higher sensitivity. Second, same as other kits, the result cannot be used alone for decision making in the treatment or other management, but should be combined with nucleic acid test, clinical presentations, or epidemiological contact history.In conclusion, we developed a rapid and easy‐to‐use SARS‐CoV‐2 antibody test kit which is highly sensitive and specific in the diagnosis for COVID‐19. With the widespread use of the kit, earlier treatment and timely isolation can be achieved, that improves the outcome and controls the extensive transmission of the global pandemic.The authors declare no conflicts of interest.WL designed the study. ML, JY, XW, and HQ collected and processed the patient samples and tested the samples. JL and ZL performed the optimization of test kit. ML, CW, BY, BW and SL analyzed and interpreted the data. ML and CW drafted the manuscript. SL, WL, DX, QZ, MZ, and QG revised the manuscript. All the authors approved the final manuscript.Figure S1Click here for additional data file.Figure S2Click here for additional data file.


Exposure of lambs to herbage-based diets prior to weaning may facilitate the development of the rumen which may subsequently increase animal performance after early weaning. The aim of this study was to estimate the effects of varying durations of exposure of lambs to a herb–clover mix containing chicory, plantain, red clover, and white clover prior to early weaning (at ~45 days of age) on their subsequent growth and rumen development at conventional weaning age. Prolonged exposure of lambs to the herb–clover mix prior to early weaning had no impact on lamb growth or rumen development, suggesting that using this management option will not improve performance of lambs after early weaning.CW); (2) lambs born on ryegrass–clover-based pasture and early weaned onto a herb–clover mix at ~45 days of age (Grass–HerbEW); (3) lambs born on ryegrass–clover-based pasture, transferred with their dam onto a herb–clover mix at ~45 days of age until conventional weaning (Grass–HerbCW); (4) lambs born on ryegrass–clover-based pasture, transferred with their dam onto a herb–clover mix at ~15 days of age and early weaned onto a herb–clover mix at ~45 days of age (Grass–HerbD15EW); (5) lambs born and managed on herb–clover mix until conventional weaning (Herb–HerbCW); (6) lambs born on herb–clover mix and weaned early onto a herb–clover mix at ~45 days of age (Herb–HerbEW). In both years, Herb–HerbCW lambs had greater (p < 0.05) growth rates than lambs in other treatments. The liveweight gains and rumen papillae development of Herb–HerbEW, Grass–HerbD15EW and Grass–HerbEW lambs did not differ (p > 0.05). The weight of the empty digestive tract components at either early weaning or conventional weaning did not differ (p > 0.05) between treatments. Exposing early-weaned lambs to the herb mix for a prolonged period, prior to early weaning, does not improve their subsequent growth.Twin sets of lambs were randomly allocated to one of six treatments: (1) lambs born and managed on ryegrass–clover-based pasture until conventional weaning approximately at 99 days of age (Grass–Grass Plantago lanceolata), chicory (Cichorium intybus), red clover (Trifolium pretanse) and white clover (Trifolium repens) has been shown to have a greater nutritional quality compared to a ryegrass–clover-based pasture [Weaning lambs early can be a useful management option that farmers can use when herbage production is inadequate or of poor quality, to reduce the overall ewe feed demand and to support the growth of early-weaned lambs to achieve liveweight targets ,2. Lambs pasture ,6,7. Thi pasture ,9,10,11. pasture  of the hEarly development of the reticulo-rumen and its microbial population is important for efficient utilization of herbage-based diets after weaning ,14. EarlLolium perenne L.) and white clover (Trifolium repens) were used over two spring periods in 2017 and 2018. Herb–clover mix paddocks were sown during autumn in 2012 and 2013 with a seed mixture of chicory , plantain , white clover (4 kg/ha) and red clover . During the experimental period (August to December in both years), a rotational grazing system was used whereby the post-grazing sward surface heights were maintained at a minimum of 5 to 7 cm for both ryegrass–clover-based pasture and herb–clover mix, respectively, in order to provide ad-libitum dry matter intakes for lambs and ewes [Eight paddocks  of herb–clover mix and seven paddocks  of ryegrass–clover-based pasture containing perennial ryegrass . All animal manipulations were approved by the Massey University Animal Ethics Committee (MUAEC 17-40). Each year, Romney ewes, from a large commercial flock, were naturally bred during a 17-day breeding period. A random subset of ewes diagnosed as twin-bearing using transabdominal ultrasound at approximately 90 days of pregnancy, were included in each year. Throughout the gestation period, within each year, ewes were managed as a single flock (group) under commercial pastoral farming conditions on a ryegrass–clover-based pasture.CW, Grass–HerbEW, Grass–HerbCW, Grass–HerbD15EW, Herb–HerbCW and Herb–HerbEW, CW was omitted resulting in five treatments . The Grass–GrassCW treatment had previously been examined over three years [n = 117 in 2017 and n = 161 in 2018) were allocated to Grass–HerbEW, Grass–HerbCW, and Grass–HerbD15EW and Grass–GrassCW (only in 2017) treatments and grazed on a ryegrass–clover-based pasture. Similarly, on the same date, ewes (n = 80 in 2017 and n = 82 in 2018) were allocated to Herb–HerbCW and Herb–HerbEW treatments begun grazing the herb–clover mix.Each year, twin-bearing ewes were allocated to treatments three days prior to lambing using a stratified random sampling procedure, in order to balance the groups for ewe live weight. A summary of the experimental design can be found in ee years ,11 and wn = 136 and n = 138 lambs were born on herb–clover mix in 2017 and 2018, respectively. On the ryegrass–clover-based pasture n = 224 and n = 302 lambs were born in 2017 and 2018, respectively. In accordance with veterinary advice, at L27 (average 27 days of age) in 2017 and L26 in 2018, lambs were drenched with an oral triple combination drench  and thereafter at 28-day intervals throughout the study at a rate of 1 mL per 5 kg live weight to control internal parasites. This method has been successfully used in previous studies [Lambing began on 28 and 27 August in 2017 and 2018 (L0), respectively. Lambs were born over 26- and 28-day period in 2017 and 2018 . All lambs were weighed, ear tagged and identified to their dam within 24 h of birth. In total,  studies ,10,11, tD15EW treatment were transferred from ryegrass–clover-based pasture to a herb–clover mix where they remained until early weaning  that were both a minimum of 14 kg live weight  were included in the study. Any ewes and lambs that did not fulfil these criteria were excluded from the remainder of the study. Lambs in each treatment  remained on these treatments until conventional weaning at L99 in 2017 and L87 in 2018.At L15 (average 15 dayss of age) in both years, ewes and lambs allocated to the Grass–HerbEW, Grass–HerbD15EW and Herb–HerbEW treatments (weaned ewes) were managed with Grass–GrassCW treatment in 2017 until L99 or with a commercial flock under pastoral farming conditions on ryegrass–clover-based pasture until L87 in 2018, receiving similar allowances in terms of pasture offered. Early-weaned lambs  were managed with the Herb–HerbCW ewes and lambs until L99 and L87 in 2017 and 2018, respectively. Within 1 to 2 h of birth, ewes develop an exclusive bond with their lambs and reject any alien lamb that attempts to suck for the remainder of the lactation [After early-weaning, ewes in the Grass–Herbactation . TherefoLambs and ewes were weighed within 1 h of removal from herbage at L15, L51, L65 and L99 in 2017 and L15, L44, L61 and L87 in 2018. Ewe body condition score  was assn = 12) and L87 (n = 24) in 2018 to determine the effect of feed type prior to early weaning, and the combination of feed type and early weaning on the rumen development, respectively. Lambs were selected using stratified random sampling procedure, based on live weights, in order to obtain a representative sample of lambs from each treatment. At L44, lambs in the Grass–HerbEW  and Herb–HerbEW  treatments were euthanised. At L87, 24 male lambs were selected from the Grass–HerbEW , Grass–HerbCW , Herb–HerbCW  and Herb–HerbEW  treatments and euthanised. Lambs were weighed within 1 h of removal from herbage and were euthanised using captive bolt stunning and exsanguination.A subset of male lambs from the complete twin sets were selected for anatomical and histological examination of the digestive system at L44 (2 each) were collected from the right side of the caudal dorsal sac as [Immediately after euthanasia, their skins removed, they were eviscerated, and the head was separated from the carcass. Hot carcass weight was then recorded. The digestive system was removed by severing the esophagus which was tied at the cardia of the stomach. All the organs attached to the digestive system were removed and then weighed. The digestive system was then separated into its components  which were cleaned using tap water. Empty component weights were recorded to the nearest gram. The cleaned reticulo-rumen was dissected according to the procedure of  and the l sac as  reported®, Waltham, MA, USA). Dehydration was carried out by ascending graded series of ethyl alcohol  for 12 h, followed by clearing in Xylene at 60 °C. The samples were then embedded and impregnated in melted Paraffin wax at 55–58 °C using a HistoStar Embedder . Embedded samples were cut using Rotary Microtome  into sections (4 µm). Two sections were produced, 150 µm apart from each tissue sample resulting in 4 sections per animal (replicates). The sections were floated in a Tissue Bath  at 43 °C and were mounted onto adhesive pre-cleaned slides . The sections were stained using Haemytoxelin and Eosin on an Autostainer XL . The stained slides, 2 slides per lamb, each contained two sections were covered using a Leica® CV5030 cover slip. All complete papillae in each section were examined microscopically to determine their length and width at the base  were immediately fixed in a 10% formalin saline solution for 24 h. Samples were then processed using a Excelsior ES Tissue Processor (ThermoFisherthe base .2 each) were taken to ground level from each herbage type at each sampling date using an electric shearing handpiece [Herbage masses were measured at L15, L51 and L99 in 2017 and L15, L44 and L87 in 2018. Four random quadrat cuts (0.1 mandpiece . Samplesandpiece . To deteandpiece ), digestandpiece ), percenandpiece . Metabolandpiece ). These andpiece ,11,12 toThe individual animal was considered the experimental unit for these analyses. Live weight of lambs and ewes were subjected to analysis of variance for repeated measures using the MIXED procedure in SAS . Analyses were performed separately for each year due to the differences in the days on which measurements were collected, number of treatments in 2017 and 2018, differences in climate and herbage quality, and different animals used between years. The model for lamb live weight at L51 and L99 and at L44 and L87 in 2017 and 2018, respectively, included the fixed effects of weaning treatment, sex of lamb , measurement date and the two-way interactions of treatment and measurement date. The live weight of lambs at the start of the treatment period was included in the model as a covariate. The model for lamb liveweight gain from early weaning to conventional weaning included the fixed effect of weaning treatment and sex of lamb.The models for ewe live weight included the fixed effects of weaning treatment, measurement date and two-way interactions of treatment and measurement date. The model for ewe liveweight gain from early weaning to conventional weaning included the fixed effect of weaning treatment. The live weight of the ewe at the start of the treatment period was included in the model as a covariate. Ewe body condition score was analysed using a Poisson distribution and logit transformation using the GENMOD procedure in SAS. The Poisson distribution was chosen as it is a nonlinear regression model for discrete outcomes. The model included the fixed effects of weaning treatment, measurement date and the two-way interaction of treatment and measurement date.Papillae length and width were subjected to analysis of variance using the MIXED procedure in SAS. The models for papillae length and width included the fixed effect of weaning treatment and the random effect of lamb, sample and replicate (sections) for each sample. The live weight of the lamb at slaughter was included in the model as a covariate. The models for the anatomical measurements of the rumen included the fixed effect of weaning treatment and lamb live weight at slaughter as a covariate.The botanical composition of herbages was subjected to an analysis of variance for repeated measures using the MIXED procedure in SAS. The model included fixed effects of plant species and measurement date. Herbage masses were analysed using a model that included herbage type and measurement date as fixed effects. The nutritional quality data were analysed using the MIXED procedure in a model that included the fixed effects of herbage type and measurement date.p < 0.05) and plantain was 23% lower (p < 0.05) in the herb–clover mix at L44 in 2018 than at L47 in 2017 (p < 0.05) and dead matter content was 35% lower (p < 0.05) in the herb–clover mix at L87 in 2018 than at L99 in 2017. Other plant components in the herb–clover mix did not differ (p > 0.05) between years and time points. At L15, the percentage of ryegrass in the ryegrass–clover-based pasture was 22% greater (p < 0.05) in 2018 than 2017. Other plant components in the ryegrass–clover-based pasture did not differ (p > 0.05) between years and time points. In both years, herbage mass of both herbage types was above 1600 kg DM/ha at all sampling dates throughout the study (The percentage of chicory and clover was 16% greater ( in 2017 . The perhe study .p < 0.05) than herb–clover mix whereas the reverse was seen at L51. At L99, CP content did not differ (p > 0.05) between herbages. At L15 and L99, the ADF content of herb–clover mix was greater  than that of ryegrass–clover-based pasture, however, at L51 ADF did not differ (p > 0.05). The DMD, neutral detergent fibre (NDF) and ME of herb–clover mix were lower  than ryegrass–clover-based pasture at all sampling times.At L15 in 2017, the CP content of ryegrass–clover-based pasture was ~5% greater (p < 0.05) than ryegrass–clover-based pasture, whereas the CP content of herb–clover mix was ~3% greater (p < 0.05) at L44 (p > 0.05) from that of ryegrass–clover-based pasture at L15 and L87 but was ~5% lower (p < 0.05) at L44. The NDF of herb–clover mix was 10 to 20% lower (p < 0.05) than that of ryegrass–clover-based pasture at L15, L44 and L87. The DMD and ME of herb–clover mix were greater  than that of ryegrass–clover-based pasture at L15, L44 and L87.In 2018, at L15 and L87, the CP content of herb–clover mix was lower  at L44 . The ADFp > 0.05) among treatments  but were ~1 kg lighter (p < 0.05) than Herb–HerbCW, Grass–HerbCW and Grass–GrassCW lambs. At L65, Herb–HerbCW lambs were 0.8 kg heavier (p < 0.05) than Grass–GrassCW lambs but neither differed (p > 0.05) from Grass–HerbCW lambs. At L99 , Herb–HerbCW and Grass–HerbCW lambs did not differ (p > 0.05) in live weight but were 4 to 5 kg heavier (p < 0.05) than lambs in all other treatments, which did not differ (p > 0.05) from one another. Lamb liveweight gains between L51 and L99 in the Herb–HerbCW and Grass–HerbCW treatments were greater (p < 0.05) than lambs in the Herb–HerbEW, Grass–HerbD15EW, Grass–HerbEW and Grass–GrassCW treatments that did not differ (p > 0.05).In 2017, at L51 (date of early weaning), the live weights of lambs did not differ  among the treatments (CW and Grass–HerbCW lambs were ~2 kg heavier (p < 0.05) than Herb–HerbEW, Grass–HerbD15EW and Grass–HerbEW lambs that did not differ (p > 0.05). Liveweight gain of Grass–HerbCW lambs between L44 and L87 was greater (p < 0.05) than Herb–HerbCW lambs, which in turn was greater (p < 0.05) than Herb–HerbEW, Grass–HerbD15EW and Grass–HerbEW lambs, which did not differ (p > 0.05).In 2018, at L44 (date of early weaning), the live weights of lambs did not differ (eatments . At L61 p > 0.05) between treatments . Herb–HerbEW ewes were 3 kg heavier (p < 0.05) than Grass–HerbEW and Grass–GrassCW ewes whose live weight did not differ (p > 0.05). At L99 , Herb–HerbCW, Herb–HerbEW, Grass–HerbD15EW, Grass–HerbCW ewes did not differ (p > 0.05) in live weight but were 2 to 5 kg heavier (p < 0.05) than Grass–HerbEW and Grass–GrassCW ewes. Between L51 and L99, the liveweight gain of Herb–HerbCW, Herb–HerbEW, Grass–HerbD15EW, Grass–HerbCW and Grass–HerbEW ewes did not differ (p > 0.05) but were at least 30 g/day greater (p < 0.05) than Grass–GrassCW ewes.In 2017, at L51 (date of early weaning), the live weights of ewes did not differ  (p > 0.05).In 2018, at L44, L61 and L87, the live weights of ewes in all five treatments did not differ ( > 0.05) . Similarp > 0.05) between treatments . The BCS of Grass–HerbCW and Grass–GrassCW ewes did not differ (p > 0.05) but were lower (p < 0.05) than Herb–HerbEW, Grass–HerbD15EW and Grass–HerbEW ewes. At L99 , the BCS of Herb–HerbCW, Herb–HerbEW, Grass–HerbD15EW and Grass–HerbEW and Grass–HerbCW were greater (p < 0.05) than Grass–GrassCW ewes.In 2017, at L51 (date of early weaning), the BCS of ewes did not differ  than the Grass–HerbCW and Grass–HerbEW ewes that did not differ (p > 0.05). At L61, the BCS of Grass–HerbCW ewes was lower (p < 0.05) than Herb–HerbEW and Grass–HerbD15EW ewes but was similar (p > 0.05) to Herb–HerbCW and Grass–HerbEW ewes. At L87 , the BCS of Herb–HerbCW, Herb–HerbEW, Grass–HerbD15EW and Grass–HerbEW ewes did not differ (p > 0.05). The BCS of Grass–HerbCW ewes was lower (p < 0.05) than Grass–HerbD15EW and Grass–HerbEW ewes but was similar (p > 0.05) to that of Herb–HerbCW and Herb–HerbEW ewes.In 2018, at L44 (date of early weaning), the BCS of Herb–Herbp > 0.05; EW and Herb–HerbEW lambs did not differ  papillae than Grass–HerbCW and Herb–HerbCW lambs, however, the width of ruminal papillae did not differ (p > 0.05) between any of the treatments.The weight of the empty digestive tract components of lambs between treatments at either L44 (at early weaning) or L87  did not differ  to early weaning, had no effect on lamb growth rates post-early weaning or their live weights at conventional weaning age, in either year of the current study. This suggests that this management option is not needed to improve the growth of lambs after early weaning onto herb–clover mix. This finding is supported by the limited differences in rumen measurements observed in 2018. In early life, lambs rely primarily on milk to fulfil their nutritional requirements and herbage consumption is low . In the Lambs weaned early onto a herb–clover mix grew at a similar rate, and were as heavy at conventional weaning, as unweaned lambs on a ryegrass–clover-based pasture in 2017. This was the only year that this later treatment was utilised. The growth rates achieved in the current study for early-weaned lambs on a herb–clover mix, were similar to those previously reported for commercially-reared-twin lambs on ryegrass white clover in New Zealand . This suIn both years of the current study, lambs weaned early onto a herb–clover mix had lower liveweight gains to a conventional weaning age, than unweaned lambs on a herb–clover mix. This was likely due to unweaned lambs having greater total nutrient intake from both milk and herbage, compared to early-weaned lambs that had access only to herbage post-early weaning. Interestingly, the papillae length of early-weaned lambs was 200 μm greater than unweaned lambs on the herb–clover mix at conventional weaning age in 2018. The longer papillae of early-weaned lambs were likely driven by the need for greater dry matter intake to fulfil their nutritional requirements. Dry matter supplementation early in life is known to improve dry matter intake, rumination, the establishment of rumen microflora and alter feeding behaviors of neonatal ruminates . PreviouLambs left unweaned on the herb–clover mix were 4 kg heavier at conventional weaning than those on ryegrass–clover-based pastures in 2017, supporting previous findings ,10,11. IThis study also allowed for the examination of the impact of moving ewes and lambs from a ryegrass pasture onto herb–clover mix at approximately 45 days of age without early weaning. The results in 2017 indicated that lambs were heavier and grew faster to conventional weaning than unweaned lambs that remained on ryegrass–clover-based pastures. Farmers, therefore, could consider moving ewes and lambs onto the herb–clover mix as an alternative to lambing ewes on a herb–clover mix. Further, unweaned lambs moved to a herb–clover mix at approximately 45 days of age grew at similar rates in 2017 and faster in 2018 than lambs reared with their dams on herb–clover mix from birth to conventional weaning. This suggests that farmers could use ryegrass–clover-based pasture for the lambing period and then move ewes and lambs to a herb–clover mix at 45 days of age to improve lamb weaning weights.Ewes whose lambs were weaned early and offered ryegrass–clover-based pasture until conventional weaning had greater liveweight gains and BCS than unweaned ewes on ryegrass–clover-based pasture. This was likely due to the cessation of lactation, allowing ewes to partition nutrients to gaining live weight and was consistent with previous studies ,11. In bEarly-weaned lambs on a herb–clover mix had similar liveweight gains to conventional weaning as lambs left unweaned on a ryegrass–clover-based pasture. Prolonged exposure of lambs to the herb–clover mix prior to early weaning, however, had no impact on their rumen development or subsequent growth, therefore, this management option cannot be used to improve performance post-early weaning. Ewes whose lambs were weaned early gained greater live weights and BCS compared to ewes which remained with their lambs on a ryegrass–clover-based pasture. Early weaning of lambs with a herb–clover mix is a management tool that farmers can use to achieve adequate growth in their lambs to a conventional weaning age whilst allowing their ewes to gain additional live weight. Greater lamb performance to conventional weaning, however, was achieved when lambs were left unweaned on the herb–clover mix.


OPA1‐Dominant Optic Atrophy (OPA1‐DOA) led to some degrees of visual improvement in seven patients. We here present the results of a cohort study, which investigated the effect of off‐label idebenone administration in a larger OPA1‐DOA group compared with untreated patients. Inclusion criteria were: OPA1‐DOA clinical and molecular diagnosis, baseline visual acuity (VA) greater than/equal to counting fingers and treatment duration greater than 7 months. We found a significant difference between the last visit and baseline VA in favor of stabilization/recovery in idebenone‐treated as compared to untreated patients. This effect was retained after controlling for confounders.We previously documented that idebenone treatment in  OPA1 haploinsufficiency heterozygous mutations, who were treated for at least 1 year with idebenone.OPA1‐linked DOA is characterized by defective mitochondrial fusion and cristae derangement, which in turn lead to defective oxidative phosphorylation with reduced ATP synthesis driven by complex I substrates.OPA1‐mutant DOA (OPA1‐DOA) patients pointed to a possible beneficial effect of idebenone therapy documenting the improvement of VA in these patients.The results of a double‐blind, placebo‐controlled, randomized clinical trial,OPA1 mutations leading to haploinsufficiency or missense point mutations have been treated with idebenone. Thus, through this observational cohort study, we aimed at investigating the effect of off‐label idebenone administration on visual outcome in OPA1‐DOA patients compared to untreated OPA1‐DOA individuals, considered as controls.After this pilot report, a larger group of DOA patients carrying either This study has a historical cohort design and follows the STROBE guidelines.OPA1‐DOA diagnosis were enrolled and divided in those who were treated with off‐label idebenone for at least 7 months between April 2007 and April 2017, compared with those untreated (controls). Similarly to our previous retrospective analysis of idebenone use in LHONhttps://www.ema.europa.eu/en/medicines/human/EPAR/raxone#authorisation-details-section). The dosage in individual cases was modulated to avoid the occurrence of specific clinical side effects or blood exam abnormalities , thus adjusting to maximal dosage without side effects, as required for off‐label drug administration. Patients with evidence or previous history of glaucoma or with any optic neuropathy other than DOA, and with a baseline best‐corrected‐visual acuity (VA) less than counting fingers (CF) were excluded.Patients satisfying inclusion criteria for a molecular and clinical defined OPA1‐DOA patients and stratified them into 37 untreated subjects and 50 idebenone‐treated.We identified 87 OPA1‐DOA is characterized by a relentless visual loss progression without spontaneous recovery.‐stabilization/recovery;‐worsening.The current knowledge of the natural history of For treated patients, baseline was considered as the last VA before the start of idebenone therapy, whereas in untreated baseline was the first available VA.logMAR VA: the subject’s eye with lower logMAR VA was the better‐seeing eye. If both eyes had an equal logMAR acuity, the following criterion was used;Mean Deviation (MD) for VF ;if the eyes were equal based on criteria 1 and 2, the best‐seeing eye was randomly selected.For statistical analysis, eyes were classified into the best‐ and worst‐seeing based on baseline visual examination. The pre‐defined algorithm for determining the best‐seeing eyes was the following:We followed a “one‐eye” approach by evaluating the best‐seeing eye as the one assumed to have more benefit from the idebenone therapy. We also assessed the worst‐seeing eye, to check for the degree of concordance of the two eyes as non‐independent variables.OPA1 mutation type, baseline VA, baseline age, observation time). Two‐sided P‐values and 95% CIs are presented. For statistical analyses, SPSS  and Stata SE  softwares were used.Chi‐square, Wilcoxon, Mann‐Whitney U‐ tests were used to compare variables among groups. We performed univariate and multivariate logistic regression analyses to study the association between idebenone administration (exposure), the outcome of interest (VA stabilization/recovery) and confounding factors . Most patients  took 540 mg/day of idebenone, ten 270 mg/day, ten 405 mg/day, five 675 mg/day, and only one 10‐year‐old patient received 135 mg/day. According to the established criteria we classified 74 eyes as stable/recovery and 13 as worsening. Considering the 74 stable/recovery best‐seeing eyes, 94.5% of the corresponding worst‐seeing eyes were stable/recovery as well (data not shown).In this study we included 87 patients from 69 unrelated pedigrees Table . Demograuntreated: Mdn, Q1–Q3 = 0.52, 0.2–0.9; VAidebenone: Mdn, Q1–Q3 = 0.52, 0.3–1.0) and last visit  time‐points was significantly different only in the treated group, which showed stability of visual acuity in most cases  which further confirms the tendency towards VA‐stabilization in idebenone‐treated group   Fig..OPA1‐DOA (Table We also used a univariate logistic regression model Table  to evaluOA Table .OPA1 pathogenic mutations was significantly associated with stabilization/recovery of visual acuity. In fact, DOA patients taking idebenone benefit four times more than untreated ones in terms of visual stabilization/recovery, even after controlling for confounders. Despite the major limitations due to the study’s retrospective nature, such as possible sample bias and not‐homogeneous between‐groups observational time and not‐homogeneous idebenone dosage, these findings point to a probable benefit of idebenone therapy for DOA patients.The key finding of our study is that off‐label idebenone administration in DOA patients carrying This study deserves a few comments. Data were collected from both eyes, but we did not combine them using a binary correlation, due to small sample size.OPA1‐DOA may be ameliorated by idebenone, as for LHON.OPA1‐related DOA would represent a very relevant option to fight blindness in the largest categories of inherited optic neuropathies. This is an important opportunity for this rare and currently untreatable disease while waiting for other therapeutic options such as gene therapyIn conclusion, this study strengthens the initial positive trend we have reported in 2013The authors declare that they have no conflict of interest related to the content of this article.MR, VC, wrote the first draft of the manuscript. MR, CLM, MC, PB, VC, contributed to the conception and design of the study. MR, CLM, MC, LDV, GA, MLC, PB, VC, contributed to acquisition of the data. MR, CZ, contributed to the statistical analysis of data. MR, CLM, MC, LDV, GA, MLC, PB, VC, contributed to the interpretation of the data. MR, CLM, MC, CZ, PB, VC, contributed to drafting a significant portion of the manuscript or figures or tables. MR, CLM, MC, LDV, GA, CZ, MLC, PB, VC, reviewed the manuscript and provided revisions for intellectual content.Figure S1. Visual acuity outcome for the worst‐seeing eyes in untreated and idebenone‐treated OPA1‐mutant DOA patients.Click here for additional data file.Table S1. Wilcoxon signed‐rank test to compare paired samples, before and after idebenone off‐label administration, in OPA1‐mutant DOA patients.Click here for additional data file.


To evaluate the impact of parental myopia and outdoor time on myopia among students in Wenzhou.We examined 1388 primary students from first grade to third grade in Wenzhou from September 2012 to March 2015. We performed noncycloplegic refractometry on each student every six months and axial length (AL) measurements every year. At the commencement of our study, children were asked to complete a questionnaire regarding near work activity and outdoor activity, whereas parents were asked to complete a self-administered questionnaire regarding their background circumstances and their history of myopia.p = 0.022), and elongation of AL in children with a high level (> 2.5 h/day) of outdoor time (0.22 ± 0.13 mm/Y) was less than those with a low level (≤ 1.5 h/day) of outdoor time . The proportion of rapid myopia progression (≤-0.5D/Y) was 16.7%, 20.2% and 31.5% among the children with no myopic parent, one myopic parent and two myopic parents, respectively , and the elongation of AL in children among different numbers of myopic parents was significantly different (p < 0.001). A high level of outdoor time was a protective factor for children with one myopic parent .A total of 1294 students (93.2%) returned for follow-up examinations. Children with initial and final no myopia spent more time on outdoor activities than those with new onset myopia (1.92 vs. 1.81 h/d, In this sample, parental myopia and outdoor time were associated with myopia in children. A high level of outdoor time was a protective factor for children with one myopic parent.The online version contains supplementary material available at 10.1186/s12886-020-01763-9. Myopia is a severe public health problem, especially among Asian students . In ChinSeveral myopia-related environmental factors, such as prolonged near work, lack of outdoor exposure and inappropriate reading posture, have been reported . A numbeSince parents and their children have similar family environments and behavioural habits, parental myopia should not be simply classified as environmental or genetic factors . Saw’s sThe purpose of this study was to evaluate the interactive efficacy of parental myopia, near work and outdoor activity time on myopia onset and progression in Chinese children.This study was a school-based longitudinal cohort study. Three primary schools were selected in Wenzhou, China with cluster sampling based on similar socioeconomic status and campus culture. Grade 1–3 children were investigated at baseline and were followed for 2.5 years.A total of 1388 grade 1–3 students were included in the study, excluding objects with ocular inflammation, trauma, dysgnosia and ill-matching. A total of 1294 students and their parents completed the eye examinations and questionnaire survey at the beginning of every semester from September 2012 to March 2015.Students and their parents completed the self-administered questionnaire at the beginning of the study. The questionnaire for children included basic questions about gender, grade and near work activity  and outdoor activity. The questionnaire was distributed by taking class as a unit before the eye examinations. The children needed to complete the questionnaires after we gave them the questionnaire and told them how to complete it. Then, the questionnaires were returned it at once.Parents completed the questionnaire that included basic questions about age, family income, education level, occupation and refractive status. Parents need to answer the following questions: “Did child’s father (or mother) have the following vision problems?” (Myopia/Hyperopia/Astigmatism/NO), “What’s the most myopic or hyperopic refractive of the two eyes?” (<− 6.0D/− 6.0 to − 3.0D/> − 3.0D). At the first time of investigation, children took the questionnaires for their parents and returned it the day after tomorrow. The investigators checked and audited all the questionnaires to increase their validity.Before the start of the study, every member of the research team was trained, including 2 senior ophthalmologists, 4 experienced ophthalmic nurses and 3 postgraduates. All children included in the study were examined for visual acuity, noncycloplegic refraction and optical biometers. All instruments were checked and adjusted before the examination.Visual acuity and noncycloplegic refraction were examined by experienced senior ophthalmologists from each term. The examination process began with testing visual acuity at 5 m using a standard logMAR chart. Visual acuity was tested with and without refractive correction for those wearing spectacles. An autorefractor (TOPCON-RM8900) was used to measure noncycloplegic refraction. Each eye of each student was measured without cycloplegia at least thrice, and three repeated measurements were averaged for analysis.AL, corneal radius of curvature and anterior chamber depth were performed with an optical biometer . We used the average of five valid AL measurements for analysis.p-values were 2-sided and considered statistically significant when less than 0.05.Statistical analysis was performed using SPSS 20.0. SER = sphere + 1/2 cylinder. Myopia was defined by an SE of − 1.0 diopters (D) or more myopic. Parental myopia collected from the questionnaire was classified by the number of myopic parents . The average daily time spent near work and outdoor activities was calculated using the formula: [(hours spent on a weekday) *5+ (hours spent on a weekend day) *2]/7. Outdoor time was divided into three groups as low: ≤ 1.5 h/day (25th percentile), moderate: 1.5–2.5 h/d (50th percentile), and high > 2.5 h/day (75th percentile) for analysis. Chi-square and T-tests were used to compare near work time, outdoor time and proportion of children with different number of myopic parents between children with initial and final no myopic and new set myopia. The comparison of myopia progression and elongation of AL between different numbers of myopic parents by Chi-square test and ANOVA. Kaplan-Meier failure curves to display event rates. Hazard ratios (HRs) with 95% CIs were estimated using the Cox proportional hazards model between children with different levels of time spent on outdoor activities and the number of myopic parents. We used the Cox proportional hazards model for odds of new onset myopia in children to assess the interaction of the number of myopic parents and outdoor activity. All p > 0.05). The proportion of myopic parents was significant different between children who was myopia and those was no-myopia(p < 0.001).A total of 1388 children were enrolled in this study, and 1294 (93.2%) completed eye examinations and self-administered questionnaires six times over a 2.5-year period. Socio-demographic characteristics were compared between those children who was myopia and those was no-myopia (Table p = 0.708). The time spent on outdoor activities was higher in children with initial and final non-myopia than in children with new onset myopia . The proportion of rapid myopia progression (≤-0.5 D/Y) decreased with time outdoors with no significant difference .As shown in Table p < 0.01) (Table n = 44), one myopic parent (n = 99) and two myopic parents (n = 171), respectively . A significant elongation of the AL was noted during the follow-up that was associated with an increasing number of myopic parents during this period   Table . However1) Table . As note Table . p = 0.027, supplemental Table p < 0.001, supplemental Table p < 0.05). A high level of outdoor time was a protective factor for children with one myopic parent . A high level of outdoor time exhibited a 31%  reduced risk in new myopic children compared with a low level of outdoor time. However, myopia progression was not related to different levels of time spent on outdoor activities (p = 0.521) in our study. This finding was consistent with a meta-analysis [p < 0.05, [p = 0.051), but significantly quicker elongation was noted compared with children with lower levels of outdoor time (p = 0.045). This finding indicates that a high level of outdoor time inhibited AL changes in children.Our study reported that myopia in children was significantly related to outdoor activity time. Epidemiological studies , 26 foun < 0.05, . Given tp = 0.708).Near work was also a risk factor for myopia in school children , 29, 30.p < 0.001) compared with no myopic parent. This trend was similar to previous studies. For example, one large-sample study among Chinese children [Our study showed that parental myopia was significantly associated with child myopia. Children with new-onset myopia had a higher proportion of two myopic parents than those with initial and final non-myopia (51.5% vs 34.3%). Moreover, our study showed that two myopic parents represented an increased risk in new myopic children . The finding that myopia developed faster in children with myopic parents was consistent with some previous studies [p < 0.001), especially in children with two myopic parents.In addition, as the number of myopic parents increased, the proportion of myopia progression (≤ − 0.5 D/Y) increased remarkably  reduction in the risk of new myopia for children with one myopic parent.Furthermore, the interaction between parental myopia and outdoor time among children’s myopia was significant. Outdoor activity time was significantly associated with myopia in children within myopic parents. Our study results suggested that a high level of outdoor time resulted in a 58%  study Questionnaire


Wide QRS‐T angles and inflammatory activity are markers of future cardiovascular events including sudden cardiac death (SCD). The association between wide QRS‐T angles and inflammatory activation is however not fully understood.1,094 study participants of both sexes, 50–64 years old, were included from a randomly selected population‐based cohort as a part of the Swedish CArdioPulmonary bioImage Study (SCAPIS) pilot study. Serum samples were analyzed for markers of inflammation, cardiac wall stress/injury, and the metabolic syndrome. Wide QRS‐T angles were defined using Frank vectorcardiography. Variables were analyzed through unsupervised principal component analysis (PCA) as well as Orthogonal Projections to Latent Structures (OPLS) modeling. In addition, a subset of study participants was analyzed in a post hoc matched group design.Wide QRS‐T angles correlated positively with markers of inflammation, cardiac wall stress/injury, the metabolic syndrome, and male sex in both PCA and OPLS models. In the matched post hoc analysis, participants with wide QRS‐T angles had significantly higher counts of white blood cells (WBC) and neutrophils in comparison with matched controls. WBC as well as the number of neutrophils, monocytes, basophils, eosinophils and levels of C‐reactive protein, IL‐1, IL‐4, IL‐6, TNF‐α, and NT‐pro‐BNP were also significantly higher in comparison with healthy controls.Markers of inflammatory activation and cardiac injury/wall stress were significantly higher in the presence of wide QRS‐T angles. These results corroborate an association between abnormal electrophysiological function and inflammatory activation and may have implications for the prediction of SCD. Participants of both sexes, aged 50–64 years, underwent extensive standardized characterization according to a 2‐day program as described previously . Two age‐ and sex‐matched groups with the same number of participants were then established. One of the two groups was further matched with regard to the presence of hypertension, diabetes, both, or neither ("HT/DM"). The second group was not matched for disease and therefore constituted a healthy control group . For a detailed comparison of the three study groups, see online Table Among all participants eligible for matching, 311 had hypertension, 33 had diabetes and 53 had both diagnoses. Among the remaining participants, 320 "healthy" participants without acute or chronic disease, chronic medication, or abnormal laboratory tests requiring medical attention were identified. 52 participants eligible for matching had wide QRS‐T angles and constituted the study group with wide QRS‐T angles  recordings according to Frank was used, which is regarded as the “gold standard” for assessing the spatial QRS‐T angles Frank, . The VCG2.4We computed two measures of the relation between the dominant depolarization (QRS) and repolarization (T) forces in space (3‐D): (a) the spatial peak QRS‐T angle between the maximum QRS‐ and T‐vectors inscribed in the QRS‐ and T‐vector loops ; and (b) the spatial mean QRS‐T angle between the QRS area and the T area vectors . HemoglobinA1c (HbA1c) were analyzed by the approved reference method using high pressure liquid chromatography system with UV detection , neutrophils, lymphocytes, monocytes, and eosinophils were measured with the auto‐hematology analyser ADVIASerum levels of alanine aminotransferase (ALT), cholesterol, triglycerides, high‐density lipoprotein cholesterol (HDL), low‐density lipoprotein cholesterol (LDL), apolipoprotein A (apoA), apolipoprotein B (apoB), glucose, creatinine, cystatin C, C‐reactive protein (CRP), cardiac troponin t (cTnT), and N‐terminal pro b‐type natriuretic peptide (NT‐pro‐BNP) were analyzed using standard laboratory techniques and automated Cobas instruments (Roche Molecular Diagnostics). All analyses were performed at the Department of Clinical Chemistry, Sahlgrenska University Hospital. Estimates of glomerular filtration rate were calculated based on creatinine and cystatin C as previously described . The human V‐PLEX Pro‐inflammatory Panel 1 Human Kit (#K15049) for the precise quantitative determination of interferon gamma (IFN‐γ), interleukin (IL)‐1β, IL‐2, IL‐4, IL‐6, IL‐8, IL‐10, IL‐12p70, IL‐13, and tumor necrosis factor‐alpha (TNF‐α) were applied. Intensity of the emitted light was measured according to the manufacturer's instructions on an MSD QuickPlex SQ120 plate reader .2.6Imaging for coronary artery calcification score (CACS) was performed using a dedicated dual‐source CT scanner equipped with a Stellar Detector  and a validation set (n = 548). Principal component analysis (PCA) was performed to determine the correlation structure of 31 variables reflecting impaired glucose tolerance and diabetes, hypertension, cardiovascular disease, age, inflammation, heart disease, and wide QRS‐T angles. Orthogonal Projections to Latent Structures (OPLS) models for the prediction of QRS‐T angles were also determined to calculate the regression coefficients of the respective X‐variables. Outliers were determined as individuals with scores >Hotellings T2 99% level and/or a DmodX level >2 times 95% Dcrit level. A few outliers were also excluded based on t1/u1 plots for the OPLS models, as these individuals clearly deviated from the majority of observations. The PCA and OPLS models were firstly calculated based on the training data set, secondly based on the validation data set to determine the reproducibility of the models. PCA and OPLS models were calculated with as well as without outliers.Nondetectable levels of analytes and coronary calcium scores were assigned numerical values corresponding to half of the lowest level detected for the respective variables. All data were log transformed prior to further statistical analysis. The study participants were randomly divided into a training set  or Simca v. 15 . 33.1Table 3.2Principal component analysis was calculated based on 539 randomly selected study participants . The second principal component of the PCA model was not fully reproducible, which corresponded to a lower explanatory value of the component in both calculated models. Diabetes, impaired fasting glucose, HbA1c, hypertension, male sex, BMI, cTnT, CACS, WBC count, the number of all leukocyte types analyzed, and the levels of CRP, IL‐6, and TNF‐α were reproducibly, positively correlated with the QRS‐T angles. In contrast, female sex, estimated glomerular filtration rate (eGFR), and the absence of diabetes, hypertension, and cardiovascular disease correlated negatively with the QRS‐T angles. PCA models calculated without the omission of outliers resulted in similar models Figure .To determine regressions coefficients for the analyzed variables, OPLS models predicting QRS‐T angle width were calculated for the training and validation sets Figure .3.3A post hoc study was performed to determine whether the observed positive correlations between markers of inflammation and QRS‐T angle width were independent of hypertension, diabetes, age, and sex. Three subgroups were formed—one with wide QRS‐T angles, one without wide QRS‐T angles matched for age, sex, diabetes, and hypertension, and one healthy control group matched for age and sex Table .Study participants with wide QRS‐T angles had significantly higher levels of WBC and number of neutrophils in comparison with both control groups, as well as significantly higher levels of CRP, monocytes, basophils, and eosinophils in comparison with healthy controls Figure .Participants with wide QRS‐T angles also had significantly higher concentrations of the inflammatory cytokines IL‐1, IL‐4, IL‐6, and TNF‐α in comparison with healthy controls Figure . HDL and4The main finding of this study was that wide QRS‐T angles were associated with markers of inflammation, cardiac wall stress/injury, the metabolic syndrome, and male sex. Inflammatory markers were positively correlated with increased QRS‐T angle width, independent of age, sex, hypertension, and diabetes. These results corroborate an association between inflammatory activation and abnormal electrophysiology, and may have implications for SCD prediction.Inflammatory mechanisms, for example, through activation of T cells and macrophages, have been implicated in the development and progress of ischemic heart disease as well as heart failure  were shown to correlate with risk factors of heart disease and SCD. OPLS models developed to predict QRS‐T angle width demonstrated similar results, as shown by the regression coefficients for the predictor variables. To verify our findings, all study participants had randomly been assigned to either a "training set" or a "validation set" before development of the PCA/OPLS models. The similar results obtained for both data sets suggest that the observed correlations are not due to random variation. Notably, the WBC and the numbers of neutrophils were associated with increased QRS‐T angle width in all models and data sets. WBC, and the numbers of neutrophils were also significantly increased in the group with wide QRS‐T angles as compared to matched controls. These observations indicate that inflammatory activation is not only an effect of hypertension, diabetes, the metabolic syndrome, male sex, or old age, but is specifically associated with wide QRS‐T angles. The results from the PCA/OPLS models based on the entire cohort were thus corroborated.Several in vitro studies point toward a possible causal relationship between cardiac inflammation, ischemic heart disease, and arrhythmogenesis—as discussed previously. As the current study is based on cross‐sectional observational data, it is not possible to prove any causal relationships between inflammatory activation, wide QRS‐T angles, and SCD. The role of cardiac inflammation in arrhythmogenesis and SCD therefore requires further study in humans.Previous studies on the relationship between abnormal QRS‐T angles and subsequent SCD have analyzed the QRS‐T angle as a categorical variable. As statistical power increases when variables are treated as continuous, rather than categorical, OPLS models were calculated to predict the continuous QRS‐T angle width. All other statistical models, however, included the categorical, binary, wide QRS‐T angle according to specified cutoff values. The prognostic impact of wide QRS‐T angles has not yet been tested within the SCAPIS study. The reviews by Oehler et al. and Voulgari et al., however, present compelling evidence of the predictive value of abnormal QRS‐T angles in various cohorts despite different electrocardiographic methods. Our cutoff values were similar . The study was carried out in accordance with the Helsinki Declaration, as revised 2013.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Table S1Click here for additional data file.