Nanoparticle tracking analysis (NTA) can be used to quantitate extracellular vesicles (EVs) in biological samples and is widely considered a useful diagnostic tool to detect disease. However, accurately profiling EVs can be challenging due to their small size and heterogeneity. Here, we aimed to provide a protocol to facilitate high-precision particle quantitation by NTA in plasma, the supernatant of activated purified platelets [the platelet releasate (PR)] and in serum, to increase confidence in NTA particle enumeration. The overall variance and the precision of NTA measurements were quantified by root mean square error and relative standard error. Using a bootstrapping approach, we found that increasing video replicates from 5 s × 60 s to 25 s × 60 s captures led to a reduction in overall variance and a reproducible increase in the precision of NTA particle-concentration quantitation for all three biofluids. We then validated our approach in an extended cohort of 32 healthy donors. Our results indicate that for vesicles sized between 50 and 120 nm, the precision of routine NTA measurements in serum, plasma, and PR can be significantly improved by increasing the number of video replicates captured. Our protocol provides a common platform to statistical compare particle size distribution profiles in the exosomal-vesicle size range across a variety of biofluids and in both healthy donor and patient groups. Extracellular vesicles (EVs) are released by nearly all eukaryotic cells and are found in a diverse range of human biofluids. They regulate a diverse range of biologic and inflammatory pathologic processes and have been implicated in myriad of diseases –3. As suNanoparticle tracking analysis (NTA) is widely used to determine the particle size distribution of a sample –9. PartiThe validity of particle size distributions for a sample depends on accurate sizing of particles as well as precise concentration measurements. Accuracy is generally within 5% of the expected particle size once correct hardware and software setting have been applied , 14, 15.Here, we sought to determine the effect of increasing video replicates on the precision of particle concentration quantitation using NTA in the biofluids of plasma, serum, and the supernatant of activated purified platelets [the platelet releasate (PR)]. Although plasma and serum are widely used in clinical diagnoses , 16, 17,Human plasma, serum, and platelets were obtained from healthy adult volunteers in accordance with approved guidelines from the UCD research, and with ethical approval from St. Vincent’s University Hospital and the Rotunda Hospital Research Ethics committees. All subjects gave their informed written consent according to the declaration of Helsinki.g for 10 min at room temperature and platelet-rich plasma was aspirated. Platelets were pelleted from platelet rich plasma by centrifugation at 720 × g for 10 min at room temperature and platelet poor plasma was aspirated. Platelets were suspended in a modified Tyrode’s buffer . Platelet count was adjusted to 1 × 109/ml and platelets were stimulated with 1 U thrombin/ml at 37°C in a PAP-4 aggregometer under constant stirring. The supernatant from the thrombin activated platelets was centrifuged three times at 10,000 × g for 10 min at 4°C to remove the aggregated platelets and cell debris, leaving the activated platelet supernatant, or PR.Isolation of platelet-free plasma and PR was as described . Brieflyg for 10 min and serum was aspirated. All plasma, serum, and PR samples were stored at −80°C prior to NTA analysis.Serum was prepared by drawing 4 ml whole blood into serum blood collection tubes  and processed according to manufacturer’s instructions. In brief, samples were rested upright for 60 min to allow RBCs to clot. The RBC clot was subsequently pelleted by centrifugation at 1,300 × Particle size distribution in PR, plasma, and serum samples was determined by NTA using a NanoSight NS300 system  configured with a 488 nm laser and a high sensitivity scientific CMOS camera. Samples were diluted  in particle-free PBS  to an acceptable concentration, according to the manufacturers recommendations. Samples were analyzed under constant flow conditions (flow rate = 50) at 25°C according to Ref. , 26. Forn = 5, 10, 15, 20, and 25 NTA recordings, which were sampled from the available 30 concentration measurements. The RMSE is the error sum of squares scaled to the data from which it was derived and was calculated as follows:Nbins is the number of bins considered, Nboot is the number of bootstrap samples taken for each bin, xij is the thj bootstrap mean for bin i and i.Particle size distributions were binned into 10 nm bin widths using NTA 3.1.54 software for all video replicates to determine concentration measurements. To understand the variability in the estimates across the complete range of bin widths, we quantified the root mean square error (RMSE) of 100 bootstrap samples of Next, to determine the precision of the concentration estimates, we quantified the relative standard error (RSE) as follows:s is the SD of measurements, n is the number of samples. Precision of our clinical cohort was quantified by RSE where n = 5 or 15 video replicates. Initially, 100 bootstrap samples were sampled from the available 30 concentration measurements and the RSE was calculated for each bin width for n = 5, 10, 15, 20, and 25 video replicates. In our validation cohort, RSE was calculated from n = 5 and n = 15 video replicates for each bin width.where All bootstrapping, RMSE, and RSE calculations were performed using the open source programming language for statistical computing R 3.3.1  and the n = 5, 10, 15, 20, and 25 video replicates. RMSE was normalized to n = 5 video replicates with n = 5 representing 100% variance. For plasma, PR, and serum increasing video replicates led to a decrease in total variance across all samples . 15 s × 60s NTA video replicates were captured for PR and plasma from 32 healthy donors. The first five videos were analyzed to determine the RSE of n = 5 video replicates for every 10 nm bin width for each donor. These five, plus an additional 10 videos were then analyzed to determine the RSE of n = 15 video replicates. By increasing the video replicates from 5 to 15, the precision of NTA concentration quantitation was improved (Figure n = 5 replicates with maximum RSE for plasma of 41.0%, and 22.3% for PR (Table n video replicates had a reproducible significant effect on maximum RSE. When video replicates were to n = 15, maximum RSE was 18.0 and 12.6% for plasma and PR, respectively, indicating a greatly improved precision of NTA concentration measurements (Table Next, we wished to determine if the increase in precision of concentration quantitation observed in our bootstrapped samples could be replicated in a larger cohort n = 3. 15 s × Here, we sought to determine the effect of increasing video replicates on the precision of EV concentration quantitation using NTA. Initially, we used a bootstrapping approach to investigate the precision of particle concentration measurements in plasma, the PR, and in serum. Our results indicated that the precision of routine NTA measurements can be significantly improved in the particle size range of 50–120 nm for all biofluids analyzed and this held true even as we extended our analysis to plasma and PR in a larger cohort of 32 donors.As the NTA method is the current gold standard to measure the size and concentration of small particles in biological samples and has the potential to be a useful diagnostic tool to detect disease , 29, ourHuman plasma, serum and platelets were obtained from healthy adult volunteers in accordance with approved guidelines from the UCD research ethics committee, and with ethical approval from St Vincent’s University Hospital and the Rotunda Hospital Research Ethics committees. All subjects gave their informed written consent according to the declaration of Helsinki.MP, DM, PS, MG, AM, and PM designed research; MP, DM, PS, MG, KO, HO, CM, FÁ, AM, and PM performed experiments; MP, DM, PS, MG, AM, and PM analyzed data; MP, DM, PS, MG, KO, HO, CM, FÁ, AM, and PM wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Acquired hemophilia A might be caused by Ginkgo‐dipyridamolum especially by Ginkgo, and it was successfully treated with hemostasis and immune‐suppression therapy including methylprednisolone and cyclophosphamide. We reported a case of 84‐year‐old women treated with Ginkgo‐dipyridamolum because of thrombus of peroneal vein. She was diagnosed with acquired hemophilia A for high‐titer factor VIII (FVIII) inhibitor and lower level of FVIII activity. aPTT returned to normal range after treated with corticosteroids and cyclophosphamide.Acquired hemophilia A (AHA) is a bleeding disorder caused by an autoantibody to factor VIII (FVIII), presenting with serious and fatal bleeding such as subcutaneous and gastrointestinal bleeding A patient  visited Peking University Shougang Hospital with swelling and pain in the right leg for 2 days on 6 May 2015. Her medical history was coronary artery disease confirmed by coronary angiography , occlusion of middle part of left circumflex artery), hypertension and hyperlipidemia, without history of stroke, diabetes mellitus, peripheral artery disease, autoimmune diseases, and tumor diseases. Her usual treatment included aspirin 100 mg/day, clopidogrel 75 mg/day, metoprolol succinate sustained release 71.25 mg/day, atorvastatin 10 mg/day, and amlodipine besylate 5 mg/day. All of these drugs had been used for 1 year. In addition, she was treated with low‐molecular weight heparin in July 2013 because of unstable angina without abnormality of coagulation function. There were no family histories of bleeding disorders and other hemophilia‐related diseases. Another medical history was that she had pain and swelling in her right knee for some days after leg pressing.9/L (reference range 3.5–9.5 × 109/L) with normal differentials, hemoglobin level of 119 g/L (reference range 115–150 g/L), and a platelet count of 151 × 109/L (reference range 125–350 × 109/L). The result of coagulation examination showed an aPTT of 20 sec (reference range 24–35 sec) with normal bleeding time, prothrombin time, and thrombin time. The results of electrolytes, urea, creatine, and liver function tests were all normal. Ultrasound showed thrombus of peroneal vein in the right leg. Low‐molecular weight heparin 3200 iu/day Whole‐blood count analysis revealed a white blood cell count of 7.5 × 10The orthopedic doctor did the joint cavity puncture on the right knee and extracted dark red liquid (about 100 mL) on 7 May 2015. This manifestation suggested there was some bleeding in the cavity, and low‐molecular weight heparin and Ginkgo‐dipyridamolum were held immediately. However, aPTT was prolonged to 126.6 sec without abnormality in bleeding time, prothrombin time, and thrombin time on the next morning. The value of aPTT was higher in the next examination on afternoon 8 May 2015 (more than 180 sec) with lower hemoglobin (70 g/L). However, there was no abnormality on the results of electrolytes, urea, creatine, liver function, and fecal occult blood tests. Physical examination showed that there were large hematomas on the right upper arm, axillary, and breast. We gave blood plasma 200 mL Qd, red blood cell suspension 2 u Qd (for 4 days), protamine sulfate injection 50 mg/day (for 2 days), and human prothrombin complex 300 iu bid (for 2 days) for symptom treatment. But the value of aPTT was still high (more than 120 sec) with normal bleeding time, prothrombin time, thrombin time, and stable hemoglobin (about 90 g/L).Further examination showed that tumor markers, lung CT, and abdominal ultrasound were normal. Thromboelastogram showed that there was no heparin effect. Coagulation tests showed markedly prolonged aPTT (129.5 sec), which was partially corrected on an immediate 1:1 mixing study with normal plasma (25.2 sec). However, the mixing test results by prolonged incubation for 2 h at 37°C showed prolongation (120.2 sec), not corrected on a 2‐h 1:1 mixing study with normal plasma. Further laboratory tests showed that FVIII activity was 0.1% (reference range 50–150%), with normal level of FXII activity , normal level of FIX activity , decreased level of FXI activity . FVIII inhibitor was 20.8 Bethesda units (BU) (reference range <0.6). The result of lupus anticoagulant (LA) test was 1.23 (reference range ≤1.2). Other autoantibodies including anticardiolipin antibody, antinuclear antibody, double‐stranded DNA antibody, rheumatoid factor, and von Willebrand factor were negative. She was diagnosed with AHA.According to the diagnosis and recommendation of AHA, she was treated with corticosteroids (methylprednisolone 60 mg intravenously/day), cyclophosphamide (400 mg twice a week). In addition, calcium and vitamin D were used to prevent osteoporosis. The level of aPTT decreased to 49.5 sec after 3 weeks. The treatment was changed to corticosteroids (methylprednisolone 40 mg intravenously/day), cyclophosphamide . aPTT returned to the normal range with normal level of electrolytes, urea, creatine, and liver function, and there were no further hemorrhagic manifestations after 4 weeks. Factor assays showed that the level of FVIII activity was 24.6%, and Bethesda assays showed a low‐titer FVIII inhibitor at 0.5 BU, both obviously improved than before. After 6 weeks, the level of FVIII activity was 138.5%, and the level of FVIII inhibitor was 0.0 BU, completely restored to normal.This patient was treated by aspirin 100 mg/day, clopidogrel 75 mg/day, metoprolol succinate sustained release 71.25 mg/day, atorvastatin 10 mg/day, and amlodipine besylate 5 mg/day because of coronary artery disease, hypertension, and hyperlipidemia after hospital discharge on 5 August 2015. And aPTT was 33.9 sec (reference range 24–35 sec) with normal bleeding time, prothrombin time, and thrombin time on 27 November 2015 for re‐examination.Acquired hemophilia A is a bleeding disorder caused by an autoantibody to factor VIII (FVIII), presenting with serious and life‐threatening bleeding and associated with significant morbidity and mortality. There is low incidence of AHA in population (0.2–1 million/year) but high mortality (7.9–22%) according to a recent review This patient did not have causes of AHA such as autoimmune diseases, malignancy, dermatological disorders Ginkgo‐dipyridamolum was compound preparation of Ginkgo and dipyridamolum. Ginkgo biloba extract is one of the few Chinese herbal preparations that is recognized by the international medical community Bleeding control and inhibitor‐removing therapy should be started as soon as possible after AHA is confirmed As far as we know, this is the first idiosyncratic acquired hemophilia that might be related with Ginkgo‐dipyridamolum especially Ginkgo and it was successfully treated with hemostasis and immune‐suppression therapy.No conflict of interests, financial or otherwise, are declared by the authors.


Therapeutic response to metformin, a first‐line drug for type 2 diabetes (T2D), is highly variable, in part likely due to genetic factors. To date, metformin pharmacogenetic studies have mainly focused on the impact of variants in metformin transporter genes, with inconsistent results. To clarify the significance of these variants in glycemic response to metformin in T2D, we performed a large‐scale meta‐analysis across the cohorts of the Metformin Genetics Consortium (MetGen). Nine candidate polymorphisms in five transporter genes  were analyzed in up to 7,968 individuals. None of the variants showed a significant effect on metformin response in the primary analysis, or in the exploratory secondary analyses, when patients were stratified according to possible confounding genotypes or prescribed a daily dose of metformin. Our results suggest that candidate transporter gene variants have little contribution to variability in glycemic response to metformin in T2D. WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?☑ Published studies on the impact of the polymorphisms in organic cation transporter genes on metformin response have been inconsistent and mostly hindered by small sample size.WHAT QUESTION DID THIS STUDY ADDRESS?☑ This study explored whether variations in metformin transporter genes affect glycemic response to metformin through a large‐scale meta‐analysis.WHAT THIS STUDY ADDS TO OUR KNOWLEDGE1c reduction, in patients with T2D.☑ Nine candidate variants in membrane transporter genes showed no significant effect on metformin response, assessed as HbAHOW THIS MIGHT CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE☑ Candidate variants in transporter genes, despite their role in metformin pharmacokinetics, showed no relevant contribution to variability in metformin efficacy and might not be useful as predictors for individualized treatment with metformin.1c <7% (53 mmol/mol).1c reduction explained by common genetic variants, each conferring a small to moderate impact.Metformin is the first‐line pharmacological therapy for type 2 diabetes (T2D) and the most widely prescribed antidiabetic drug. The glycemic response to metformin is, however, highly variable. In patients receiving metformin as an initial treatment for T2D, less than two‐thirds achieve acceptable glycemic control or a target HbA1c <7% 5 mmol/molSLC22A1) is expressed on the sinusoidal membrane of hepatocytes and is the main transporter of metformin into the liver.SLC22A2) is expressed primarily at the basolateral membrane in the kidney tubular cells and facilitates the uptake of metformin from the blood into the kidneys.SLC47A1) and MATE2‐K (encoded by SLC47A2), are H+/drug antiporters located in the apical membrane of the renal tubular cells, and facilitate metformin excretion from tubular cells into urine.SLC22A4).Previously published studies of metformin pharmacogenetics have mostly focused on the candidate genes involved in drug pharmacokinetics, with the expectation that these might have a large clinical effect. For example, polymorphisms in the transporters, organic anion‐transporting polypeptide 1B1 and breast cancer resistance protein, have been associated with large effect sizes with the pharmacokinetics and pharmacodynamics of several drugs, including statins.To clarify the role of genetic variants in these transporters on metformin clinical response, we performed a large‐scale meta‐analysis of the impact of known candidate variants on metformin efficacy in T2D, across the cohorts of recently established Metformin Genetics Consortium (MetGen). This resource now has in excess of 10,000 individuals in whom metformin response can be defined, and offers a unique opportunity for a highly powered pharmacogenetic meta‐analysis of glycemic response to metformin.TableTableFigureFigure1c reduction (Supplementary Table S1 online). The results from the HOME and SDDS studies, two MetGen cohorts in which metformin was added to insulin therapy, did not show significant impact on metformin response assessed as HbA1c reduction (Supplementary Table S2 online).We studied the effect of nine candidate variants in transporter genes OCT1,TableSupplementary Table S3a online). Likewise, no significant associations were found in the separate meta‐analyses of the effects of variants on metformin response in individuals treated with low  or high daily doses of metformin .As the transport of metformin could depend on its concentration, we next explored possible gene by dose interactions, using a dose as a proxy of metformin concentration. In the meta‐analysis of SNP × dose interaction effects, none of the interactions showed significant effect in the monotherapy group Table or the tTablesandSupplementary Tables S4a and S4b online show the meta‐analyses results for all participants, including both monotherapy and dual therapy. These exploratory subgroup analyses revealed no significant interactions between the SNPs that affect metformin glycemic response.We next explored the potential interactions between these transporters that might affect metformin response. The interactions were tested by examining the impact of one SNP in two separate subgroups of participants, homozygotes for the reference, and variant allele of a potential confounding SNP. P > 1.6 × 10−4; Supplementary Figure S1).In the supplemental locus‐wise meta‐analysis of the association of all common SNPs in transporter gene regions with metformin glycemic response, none of the variants showed significant signal after correction for multiple testing (1c reduction >0.14% (1.5 mmol/mol) for any of the candidate SNPs at the nominal significance level of α = 0.005. Thus, none of the SNPs reported as being associated with metformin response in previous literature is likely to have an allelic impact on HbA1c reduction of >0.14%. Furthermore, it is unlikely that other SNPs, such as cis‐regulatory variants, in these genes could have a significant impact on metformin glycemic response, as shown by a locus‐wise meta‐analysis of all common SNPs within genes' proximity in 6,964 patients.Despite the established role of cation‐selective transporters in metformin pharmacokinetics, polymorphisms in these transporters showed no significant impact on glycemic response to metformin in this large study of patients with T2D. This meta‐analysis had 80% power to detect an allelic effect of HbA1c reduction rather than other surrogates of metformin response. SomeOur findings contrast to most of those previously reported in healthy subjects,Transporters exhibit asymmetry in their kinetic properties; thus, for facilitated transporters that are bidirectional, the direction of the transport will depend on the substrate concentration.Studies of small cohorts have previously reported gene‐gene interactions affecting glycemic response to metformin.in vivo.This is the largest metformin pharmacogenetic study reported to date, despite a few limitations due to the need to align cohorts. Our finding that there is no significant role for metformin transporter variants in mediating glycemic response to metformin challenges our understanding of metformin action in patients with T2D chronically treated with metformin. For example, there is increasing recognition that metformin works presystemically in the gut, via a number of mechanisms, to improve glycemia.1c reduction smaller than 0.06–0.14% , however, these small effect sizes may be unlikely to have any clinical importance. Second, the meta‐analysis included population‐based observational studies, which could be confounded by a number of factors, such as patient adherence or frequency of HbA1c measurements in the cohort. However, in four cohorts, including two largest cohorts, GoDARTS and PMT2‐EU, adherence could be calculated from the drug dispensing records, and was added as a covariate in the model. Likewise, although we could not completely harmonize the definition of on‐treatment HbA1c due to different frequency of HbA1c measurements available in the cohorts, four studies, including two of the largest studies, used minimal HbA1c achieved within 18 months as outcome. Third, concomitant medications, which affect metformin transport, are well established to cause changes in its pharmacokinetics,Although the current study demonstrates that genetic variants in transporters that play a role in metformin pharmacokinetics have no significant effect on metformin glycemic response in large cohorts of patients with diabetes, there are some limitations to our study. First, despite the large sample size used in the current study, we did not have the statistical power to detect an allelic effect size for HbAIn conclusion, in our large meta‐analysis, including almost 8,000 individuals across 10 international cohorts of the MetGen Consortium, variants in metformin transporter genes have shown no relevant contribution to variability in metformin response in patients with T2D, although we cannot rule out gene‐concentration or more complex multiple gene‐gene interactions that may be required to account for transporter redundancy. As has been recognized now for a number of years in disease and complex trait genetics, this study shows the importance of large sample sizes, usually only available to international consortia, for robust pharmacogenetic studies. Future even larger consortia efforts are required to corroborate these findings and to unravel genetic variations that could be used as better predictors for personalized metformin therapy.Supplementary Table S5 online. The studies were approved by relevant institutional review boards, and all participants gave written informed consent.The MetGen Consortium consists of research groups from Europe and the United States with available data for studies of metformin pharmacogenetics from population observational studies and clinical trials. The 10 MetGen cohorts with available data for the study of metformin transporter gene variants are presented in TableSupplementary Table S6 online. All SNPs were in line with Hardy‐Weinberg equilibrium (P > 0.01). The variants were first analyzed individually for association with metformin response. The reduced‐function OCT1 polymorphisms, R61C and M420del, were analyzed individually, and also together as a combined genotype, according to the number of haplotypes carrying reduced‐function alleles: 0, 1, or 2, in line with previous studies in patients of European ancestry.Nine SNPs in five transporter genes, reported to be associated with metformin response or pharmacokinetics in previous studies, were included in the meta‐analysis Table. The num1c during treatment with metformin: pretreatment minus on‐treatment HbA1c. As such, a positive β in the regression models indicates an association of the effect allele with greater glycemic response to metformin. Inclusion criteria for all participants included in the meta‐analysis were continuous treatment with metformin for at least 3 months, pretreatment HbA1c measured within 6 months prior to metformin therapy and <14%, measurement of on‐treatment HbA1c within 18 months of metformin commencement, and no treatment with other glucose‐lowering agents except stable sulfonylurea treatment before and during metformin therapy. Hence, two cohorts from randomized controlled trials, HOME and SDDS studies, in which metformin was added to insulin therapy, were not included in the meta‐analysis, and the results are shown separately.Metformin response was defined as a reduction in HbA1c was defined as minimal HbA1c measured within 18 months of metformin commencement in the GoDARTS, PMT2‐EU, Riga, and Rotterdam studies, as HbA1c measured after 6 months (Košice and Sarajevo studies) or 12 months (DCS study) of metformin treatment, and as HbA1c measured within the first 3–9 months of metformin therapy in the PMT1‐EU study.On‐treatment HbA1c as outcome . The pretreatment HbA1c, metformin daily dose, adherence, creatinine clearance, baseline gap (time between pretreatment HbA1c measurement and start of metformin therapy), and treatment group , were added as covariates, when available/applicable (Supplementary Table S7 online). In the HOME study, analyses were adjusted for pretreatment HbA1c, metformin daily dose and creatinine clearance, and in the SDDS study, pretreatment HbA1c and randomization groupIn each cohort, the effects of individual SNPs on metformin response were assessed in additive genetic model using linear regression with reduction of HbA1c reduction ∼ pre‐treatment HbA1c + adherence + creatinine clearance + baseline gap + treatment group + dose + SNP + SNP × dose.As the effects of transporter SNPs could depend on metformin level, or they could be confounded by the effect of other variants, secondary analyses were performed to explore possible gene‐dose and gene‐gene interactions. For gene‐dose interactions, we first examined interaction models that included SNP × dose interaction term with dose as a continuous variable and then dose coded as a dichotomized variable (low or high dose) in the following model: HbA1c reduction ∼ pre‐treatment HbA1c + adherence + creatinine clearance + baseline gap + treatment group + dose + SNP.Next, we assessed the association of SNPs with metformin response separately for patients taking low  or high doses  of metformin. The cutoff value of 1,000 mg was chosen based on the median dose in the largest cohort. The analyses were performed using the same basic regression model: HbATo examine potential interactions between the variants, additional exploratory analyses of the effects of individual SNPs were carried out in the subgroups of individuals who were homozygous for wild‐type allele and of the individuals who were homozygous for variant allele of possibly confounding SNPs, assuming that impact of the variants could be more pronounced in more extreme genotype groups. Possible 1 × 1 interactions between SNPs in metformin liver transporters (OCT1 and MATE1), and SNPs in metformin kidney transporters  were explored.http://pngu.mgh.harvard.edu/purcell/plink/).2 heterogeneity index. In case the substantial heterogeneity was detected , the results of the random‐effects model were presented. Power analyses were performed using Quanto (http://biostats.usc.edu/Quanto.html). Forest plots were created using RevMan 5.3 software .All analyses were carried out for the total group of patients treated with metformin, including monotherapy and dual therapy group (metformin added to stable sulfonylurea therapy), and also separately for the metformin monotherapy group. The results from individual studies were combined in the meta‐analysis using PLINK software under fixed‐effects model (−3 (P = 0.05/10), and a study‐wise statistical significance threshold to 2 × 10−4 to correct for the overall number of tests performed (P = 0.05/236).As the primary analysis included nine SNPs and one combined OCT1 genotype, a nominal statistical significance threshold was set to 5 × 10P = 1.6 × 10−4.In addition to our main hypothesis, we performed a supplemental, locus‐wise meta‐analysis of all common SNPs within the proximity of the transporter genes to examine the possibility that original associations reported in previous studies might be driven by other SNPs in these gene regions. A total of 3,471 variants with minor allele frequency ≥1% were tested for association with metformin glycemic response in 6,964 participants from two MetGen cohorts with available genome‐wide association study imputed data, the GoDARTS and PMT2‐EU. The details of genotyping, quality control, imputation, association analysis, and meta‐analysis were described in our previous study.T.D., K.Z., K.M.G., and E.R.P. wrote the manuscript. T.D., K.M.G., and E.R.P. designed the research. T.D., K.Z., S.W.Y., N.v.L., C.E.d.K., M.J., S.G., L.Z., M.M.H.C., M.O., R.T., M.K., M.M.H., A.A.v.d.H., L.K., V.P., A.K., K.B., J.K., S.S., I.T., B.H.S., C.N.A.P., L.M.t.H., K.M.G., and E.R.P. performed the research. T.D., K.Z., S.W.Y., N.v.L., C.E.d.K., M.J., S.G., L.Z., M.M.H.C., and M.O. analyzed the data.The authors declared no conflict of interest.Supporting InformationClick here for additional data file.


Danio rerio) under moderate but not higher levels of stress improved their performance on instinctive activities driven by visual, hydrodynamic and thermal inputs. Our findings reveal, for the first time, the existence of an inverted-U shaped performance function according to stress level during early development in a series of innate behaviours.What is the relationship between the level of acute stress and performance on innate behaviour? The diversity of innate behaviours and lack of sufficient data gathered under the same experimental conditions leave this question unresolved. While evidence points to an inverted-U shaped relationship between the level of acute stress and various measures of learning and memory function, it is unknown the extent to which such a non-linear function applies to performance on innate behaviour, which develops without example or practice under natural circumstances. The fundamental prediction of this view is that moderate stress levels will improve performance, while higher levels will not. Testing this proposition has been difficult because it entails an overall effect that must be invariant to the nature of the stressor, the behaviour under scrutiny and the stimulus that drives it. Here, we report new experimental results showing that developing zebrafish ( In general, the relationship between the level of acute stress  and cognition is believed to follow an inverted U-shaped function3. Although both components  of this non-linear relationship have been difficult to pin down together, an inverted U-shape memory function according to stressor intensity has been confirmed under the same experimental conditions4. It remains unclear the extent to which the Yerkes-Dodson law applies to the relationship between acute stress and innate behaviour, which has high heritability and occurs inexorably without example or practice. Likewise, how widespread would such a relationship be and how early could it be detected? We address these issues in larval zebrafish using novel assays and the acute glucocorticoid response as a proxy for stress level. Zebrafish larvae are chosen because their hypothalamic-pituitary-interrenal (HPI) axis is homologous to the hypothalamic-pituitary-adrenal (HPA) axis5 and their small size allows for the continuous measurement of behaviour with full environmental control, including stressor onset. Also, they offer an excellent handle for the analysis of stress reactions and for non-invasive brain imaging and behavioural genetics8. The results below provide direct evidence for the existence of an inverted-U shaped relationship between stress level and performance on innate behaviour.Behaviour arises from a myriad of processes that guide the acquisition, processing and integration of sensory inputs, the connection between these inputs, past experiences and present physiological status, and the coordination of muscular activity by the prospective neural centres. By virtue of its complexity alone, behaviour is susceptible to disturbance by stress, whose consequences are assumed to combine and make up the state that is relevant to the effectiveness of a particular activity. There is little doubt that dysfunctional responses to stress can have deleterious consequences for healthp < 0.0001, hyperosmotic medium: F = 68.8, p < 0.0001, strong hydrodynamic flows: F = 81.6, p < 0.0001, squared pulse of light: F = 100.1, p < 0.0001, followed by post hoc comparisons). These measurements were used to establish baseline, moderate and higher levels of stress caused by single stressors, as well as similar levels caused by different stressors. In selecting the stimulations producing these levels, we avoided those causing maximum levels of stressor-mediated cortisol increase (not shown), linked to severe locomotion abnormalities and possibly other impairments. Baseline levels were those of control (unexposed) larvae from each of the four stimulation groups, which were equally handled, but the stressor was not present. Levels across these groups were similar  = 0.02, p = 0.99) and gave cortisol means (±S.E.M.), in picograms (pg) per larva, of 5.01 (±0.34), 4.96 (±0.34), 4.90 (±0.35) and 5.00 (±0.34) for pH drop, hyperosmotic medium, strong flows and light pulse, respectively. Equally moderate levels  = 1.9, p = 0.15) were reached via pH 4, NaCl 25 mM, 3 V and 1 mW*cm−2, with means (±S.E.M.) of 8.82 ± (0.60), 9.42 (±0.64), 9.24 (±0.62) and 10.83 (±0.66) pg/larva for each group, respectively, whereas equally higher levels  = 0.2, p = 0.20) were reached via pH 3.3, NaCl 100 mM, 6 V and 5.8 mW*cm−2, with means (±S.E.M.) of 17.83 (±0.90), 17.07 (±1.05), 17.15 (±0.96) and 17.87 (±0.92) pg/larva for each group, respectively .We deployed two main approaches. Firstly, we exposed larvae to four different stressors of increasing intensity: pH drop, hyperosmotic medium, strong flows or light pulse . To compare the level of stress produced by these stimulations, we measured circulating levels of cortisol, a major stress hormone, directly after stressor exposure. The results confirmed that larvae can adjust whole-body cortisol to compensate for stressor intensity  water temperature. For this we monitored the percentage of time that single larvae spent in each -virtual- quadrant of an elongated chamber offering a stable temperature gradient  = 0.02, p = 0.99, level × type: F = 0.04, p = 0.99, E, level: F = 68.5, p < 0.0001, type: F = 0.37, p = 0.77, level × type: F = 0.40, p = 0.88, F, level: F = 59.2, p < 0.0001, type: F = 0.26, p = 0.85, level × type: F = 0.15, p = 0.99, followed by post hoc comparisons). To complement these assessments, we plotted relative performance values (in %), as functions of the minimum and maximum values from each of the three assays, against the level of whole-body cortisol  is homologous to the mammalian hypothalamic paraventricular nucleus, innervation of the pituitary by NPO cells is established by 3 days post fertilization, and ablation and activation of cortisol-producing interrenal cells reduces and increases cortisol upon stress, respectively20. Two-photon calcium imaging on intact larvae shows that the activity of NPO-cells producing corticotropin-releasing hormone, the primary stimulator of the HPA axis, is highly synchronized and co-varies with stressor intensity21. Interactions of GRs and serotonin signalling are present too22, and larvae show elaborate stress reactions25, including a reversible suppression of the feeding drive13, a common phenomenon among vertebrates also found in adult teleosts27. Furthermore, evidence also shows that serotonergic neurons in the dorsal raphe can modulate sensory responsiveness28 as well as light/dark preference29, depending on their activity level. The present data set shows an inverted-U shaped relationship between the level of acute stress and a larva’s performance on innate behaviour, complementing recent findings of enhanced stimulus responsiveness after stressor exposure6 and a recovery of the feeding drive that is faster under intermediate but not higher levels of salt stress13.In zebrafish, basal levels of whole-body cortisol and expression levels of genes responsible for corticosteroid synthesis and signalling increase drastically around the time of hatching, revealing an HPI axis that matures early in development30. There is, however, a complete lack of evidence linking early activation patterns of the HPA axis to direct measures of performance, resilience and fitness31. In larval zebrafish, all three elements of the HPI axis can be genetically visualized and manipulated6 and inbred populations of larvae can be used to link early environments to HPA axis activation patterns and measures of performance, survival and reproductive outcome. Our findings suggest the relationship between acute stress and behavioural performance following an inverted-U curve as a widespread phenomenon among vertebrates. Both environmental and resource uncertainty under great competition may have set the circumstances that selected for it. Elucidation of the mechanisms underlying such relationship is central to the analysis of phenotypic adaptation.The results are exceptional in identifying such a non-linear relationship so early in life. A key question now emerging is how it relates to survival in a species facing greater mortality during early development. For developing zebrafish, increasing performance under moderate stress levels may have advantages. Such ability may help larvae to better cope with environmental variability and predation risk, lessening a strong pressure for survival. Another emergent question relates to the study of behaviour, resilience and fitness as a function of maturation. Ontogeny is concerned with the roles of genetics and maturation in shaping an animal’s life history. All behaviours are influenced by an animal’s genetic background and the environments that exist during development. The extent to which the two influences determine the outcome varies from species to species, and from activity to activity within a species. It is generally assumed that the acute response of the HPA axis can be beneficial, and that its positive effects may be linked to epigenetic programming caused by coping with stress32. Wild-type embryos  were collected in the morning and raised on a 12:12 light/dark cycle in E2 medium at 28 °C. All experiments were carried out with larvae at 6 days post fertilization (d.p.f.). Tests were performed between 09:00 hours and 18:00 hours, with different experimental groups intermixed throughout the day. Zebrafish experimental procedures were performed according to the guidelines of the German animal welfare law and approved by the local government .Zebrafish breeding and maintenance were performed under standard conditions21, each based on one of four different stimulations: HCl (pH drop), NaCl (hyperosmotic medium), strong hydrodynamic flows or a squared pulse of light. They were then used for cortisol measurement or transferred to custom-made swimming chambers for behavioural testing. pH drop: larvae were incubated for 3 min in steady-state E2 medium (unexposed) or E2+ varying concentrations of hydrochloric acid  at 28 °C under white-light illumination. They were then washed three times with E2 medium and kept in a small container for cortisol detection (6 min later), or transferred to a custom made swimming chamber for behavioural testing (12 min later). The wash and transfer period lasted 3 min (±10 s) and was performed at room temperature. Hyperosmotic medium: larvae were incubated for 10 min in steady state E2 medium (unexposed) or E2+ varying concentrations of NaCl  at 28 °C under white light illumination. They were also washed three times with E2 medium and kept in a small container for immediate cortisol detection, or transferred to a swimming chamber for behavioural testing (5 min later). As before , the wash and transfer period took 3 min (±10 s) and was performed at room temperature. Strong hydrodynamic flows: larvae were presented with water motions caused by rapid lateral displacements of a rigid silica capillary tube  fixed to a multilayer piezo bender actuator  GmbH and Co. KG; Karlsruhe, Germany). The actuator had an operating voltage of 0–60 V, a maximum displacement of ±1000 µm, and an unloaded resonant frequency of 160 Hz. The bender was connected to a dual-piezo-amplifier (maximum voltage: 10 V), a pulse generator and a TTL control box  allowing for computer control. The tip of the silica capillary tube was submerged (2 mm) at the centre of a 30 mm Petri dish, half filled (1.8 ml) with E2 medium (orientation relative to water surface: 90°). The voltage applied to the bender (Vact) determined the speed of the capillary’s lateral displacements, or stimulus strength (in % relative to maximum voltage). Groups of 30 larvae were exposed to 6 stimulation units delivered with an inter-stimulation-interval of 250 ms. Each unit consisted of 99 repetitions of 40 ms lateral displacements. We used a Vact of 3, 4.5 or 6 V. Stimulations were carried out under white illumination at 28 °C. After stimulation, larvae were kept in Petri dishes for cortisol measurement (9.5 min later), or transferred to a swimming chamber for behavioural testing, where they remained without perturbation for 10 min before recordings. Light pulse: larvae were dark-adapted for 3 min and then exposed to a 180 s squared pulse of flashing blue light of varying light power, as described elsewhere. Custom-made drivers, amplifiers, pulse generators and the USB-IO box (see above) allowed computer control of the squared pulse of light, delivered through a custom-made array of LEDs placed inside a light-proof enclosure (see below). The LEDs were positioned at a fixed distance above the Petri dish. The incident angle of the LEDs allowed for homogeneous illumination of the dish. Each squared pulse of light consisted of 100 ms flashes delivered at 5 Hz. Light power was measured through a hand-held light power meter . After light exposure, larvae were kept in the dish for cortisol measurement (6 min later), or transferred to a swimming chamber for behavioural testing, where they remained without perturbation for 12 min before recordings.Groups of thirty larvae in 30 mm Petri dishes were exposed to known stress protocolsCortisol and behavioural measurements were made on different groups of equally treated larvae and therefore constitute fully independent samples. For the behavioural measurements, each replicate involved a single larva. Yet, these individual measurements were made on larvae that had also been kept in wells containing a total of thirty larvae per well. Thus, the number of single larvae matched the number of independent wells. In this manner, the density of larvae per well during stressor exposure remained a constant factor for both the cortisol and behavioural measurements. In sum, for each cortisol measurement, all thirty larvae in a well were used, whereas each behavioural measurement involved only one larva - the remaining twenty-nine larvae in the well were used elsewhere outside the study. In sum, each replication was fully independent from the others thus avoiding pseudo-replication.11.Groups of thirty larvae were immobilized in ice water after being exposed to HCl, NaCl, strong flows or the light pulse. Unexposed larvae (control samples) were collected after equal handling, omitting stressor exposure. Samples were then frozen in an ethanol/dry-ice bath and stored at −20 °C for subsequent extraction. Each replicate consisted of a well with 30 larvae. Cortisol extraction and detection were carried out using a home-made cortisol ELISA protocol, as described elsewhere−1  with a lens  positioned above rectangular  or cylindrical (hydrodynamic input) custom made swimming chambers. EthoVision XT 7 software (Noldus Information Technology) was used to monitor the movements of individually swimming larvae. Motion values were expressed as distance swum every second (mm per 1 s) or 40 ms (mm per 40 ms). All experiments involved single larvae moving freely within the swimming chamber. Before tests, each larva was given an initial time period of several minutes to adapt to the chamber’s conditions. Experiments were conducted at 28 ± 1 °C, unless otherwise stated. A thermocouple  connected to a temperature control system  monitored the temperature inside the swimming chamber. All the experiments were performed in a blind fashion as to group identity. Control animals for each group were handled in the same fashion, but omitting stressor presentation.Behavioural experiments using visual, hydrodynamic and thermal inputs were conducted under both white and infrared illumination, delivered through a custom-made array of white- and infrared-LEDs mounted inside a light-proof enclosure. Tests involving hydrodynamic and thermal inputs were conducted under infrared light only. The complete setup was placed on a vibration-free platform . Larvae were imaged at 25 frames s−2) of fixed contrast, diameter (in degrees), velocity (in degrees s−1) and number, as previously defined13. In each session, a larva was first presented with stationary dots and allowed to swim freely for 60 s. Next, once the test started, as soon as the larva reached any of the chamber’s far ends, the dots automatically began to move in parallel towards the opposite end of the chamber. As a result, due to the optomotor reflex, the larva would align itself along the direction of the large visual field displacement and begin to swim consistently with the moving dots towards the opposite far end of the chamber. To quantify response strength, we measured ‘latency’, in seconds, as the time interval in-between when the larva started to move with the dots and when it reached the opposite end of the chamber.Control and treated larvae were placed individually in a rectangular chamber  filled with 3 ml of E2 medium and mounted above a horizontally oriented computer screen. The screen displayed visual stimuli created via an algorithm written in MATLAB 2009b . Visual stimuli consisted of grey dots . The chamber also had two cylindrical side channels  opposite to each other opening 200 μm above the transparent glass bottom, with their longest axis oriented at an angle of 30° relative to horizontal. One such channel held the thermocouple monitoring the temperature inside the chamber, while the other allowed passage of the end of a rigid silica capillary tube, or stimulus source , submerged ~400 μm into the chamber’s inner medium (depth: 5 mm). The opposite end of the capillary tube was fixed to a multilayer bender actuator  GmbH + Co. KG, Karlsruhe, Germany) with an operating voltage of 0–60 V, a maximum displacement of ±1,000 μm and an unloaded resonant frequency of 160 Hz. The bender, coupled to a pulse generator, a dual piezo amplifier and a TTL control system, produced unidirectional lateral displacements (of 50 μm and controllable speed) of the capillary’s submerged end, creating minute, non-stressful WMs within the chamber. The input voltage applied to the actuator (0.5 V) determined the speed of the capillary’s lateral displacements. Single larvae in the swimming chamber were video-recorded for 120 s under infrared light and constant temperature. They were then presented with 1 ms lateral displacements of the silica capillary tube delivered at 1 Hz (input voltage: 0.5 V) for 120 s. Motion before and during stimulation was calculated using the integrals of motion over 120 s.Computer control of minute water motions was achieved through custom-made drivers, amplifiers, pulse generators and the USB-IO box, as described below; for details, see also ref. −1 with the aid of the pump . In between the end-walls and the overtures (inlet and outlet), thermocouples  monitored the temperature of the flowing medium. One such thermocouple provided feedback to a control system  that either kept the flowing medium at 28 °C (±0.1 °C) or increased its temperature in a highly controlled manner until a stable temperature gradient was established inside the chamber and confirmed via independent measurements. In each recording session, a single larva, which had been kept for 10 minutes at 28 °C (±0.1 °C) in a second, parallel chamber offering exactly the same medium and flow conditions, was moved into the test chamber and video-recorded for 300 s under infrared light. To assess avoidance of above preferred  water temperature (28 °C), the chamber was divided in four virtual quadrants equal in size and the percentage of time spent in each of these quadrants over the entire recording session was used as a measure of space use. The resulting ‘differential space use’ values (in %) were then calculated as the difference between the percentage of time spent in the low and high temperature quadrant for each larva.We used a custom-made, rectangular test chamber  with two vertically-oriented walls  at its opposite ends. These two end-walls had contiguous rows of equidistant 100 µm openings allowing the E2 medium to diffuse. On each side of the chamber, beyond the end-walls, two opposite overtures  allowed the medium to flow at 150 μl minpost hoc tests). Normality was tested using Kolmogorov–Smirnov, Shapiro–Wilk and D’Agostino tests. Analyses were made with MS-Excel  and Prism 5 .All data are shown as boxplots (median and whiskers: min to max) except indicated otherwise. We used a random experimental design and ANOVAs for multiple group comparisons (followed by Bonferroni’s The data that support the findings of this study are available from the authors on request.


OXT‐R) polymorphisms (rs53576 and rs2254298), their interaction with maternal care (GxE), and ED behaviours in a community sample. We studied 3698 women from the Avon Longitudinal Study of Parents and Children (ALSPAC) who participated in a two‐phase prevalence study of lifetime ED and had genotype data. The GG rs53576 genotype was associated with binge eating and purging, and the rs2254298 AG/AA genotype with restrictive eating lifetime. In addition, the rs2254298 AG/AA genotype interacted with poor maternal care to increase the odds of binge eating and purging ). This study replicates previous findings of an association between OXT‐R polymorphisms and ED, and it is the first to show an interaction between OXT‐R genotype and poor maternal care. As such, these findings highlight the important role of oxytocin in understanding the pathophysiology of ED. © 2016 The Authors European Eating Disorders Review published by Eating Disorders Association and John Wiley & Sons LtdThis study aimed to investigate associations between oxytocin receptor gene ( There is increasing evidence that variations in levels and response to the neuropeptide oxytocin are relevant to several psychiatric disorders. In particular, research on the role of oxytocin on cognition and behaviour has focused on autism spectrum disorders, social behaviour, and emotional responses both in animals and humans Brune, .OXT‐R) gene. Genetic polymorphisms of the OXT‐R have received much attention in relation to prosocial behaviour, empathy, and neuropsychiatric disorders including autism spectrum disorders; in particular, the most studied single nucleotide polymorphisms (SNPs) of the OXT‐R gene are rs53576 and rs2254298. For example, the rs53576 GG genotype has been associated with higher levels of empathy, lower levels of depression, and pro‐social behaviour   in a clinical sample of 90 women with BN compared to controls (n = 103) (n = 124), subjects with rs53576 and rs2254298 GG/GG haplotype had lower scores on overall ED psychopathology measures, less body shape and food preoccupation, fewer obsessive compulsive behaviours, and lower anxiety  . AmongstGiven a recent data collection of lifetime interview data on ED amongst ALSPAC women (about 20 years after enrollment), hence more precise phenotyping, and lack of availability of information on other ED behaviours  in the previous study, we aimed to extend previous findings by focusing on lifetime interview‐collected ED behaviours. This approach allowed us to investigate narrow ED behavioural phenotypes that might be more closely associated with specific genotypes, compared to heterogeneous diagnostic syndromes, and the limited ED variables investigated in Connelly et al. .OXT‐R SNPs and poor parenting. We therefore aimed to determine: (i) whether polymorphisms in the OXT‐R system genes were associated with ED behaviours amongst adult women from a community sample, and (ii) whether these polymorphisms showed an interaction with poor maternal care in childhood. We hypothesized that OXT‐R gene polymorphisms, previously shown to be associated with ED and ED behaviours, would be associated with ED behaviours, in particular binge/purge type behaviours in this community sample of women. Given the role of OXT in affiliation and bonding, we expected an interaction between OXT‐R genotype and poor maternal care in increasing the risk for ED behaviours.To our knowledge, despite the well‐known role of early childhood adversity in increasing the risk for ED, no previous studies have investigated gene–environment interactions between OXT‐R SNPs and specific ED behaviours (i.e. food approach and compensatory behaviours vs. restrictive type behaviours); and (ii) if any associations became evident, taking into account the overlap between ED behaviours under study.Because of the known overlap and crossover of syndromes and behaviours in ED, and the potential anorexigenic effect of OXT, we were particularly interested in: (i) determining the associations between initial pregnancies, there was a total of 14 676 fetuses, resulting in 14 062 live births and 13 988 children who were alive at 1 year of age. Women and their children have been followed up since. Please note that the study website contains details of all the data that is available through a fully searchable data dictionary: <http://www.bris.ac.uk/alspac/researchers/data‐access/data‐dictionary/>ALSPAC is a longitudinal, population‐based, extensive prospective study of women and their children, set up in the 1990s to investigate the effects of environment, genetics, and other factors on child health and development   for a period of at least 3 months), and had not engaged at any point in their life in binge eating. Purging: women who had, at any time in their life, engaged in purging behaviours  at a frequency of at least once a week for a period of at least 3 months, but had never engaged in restrictive eating. Binge‐eating: women who had, at any time in their life, engaged in binge eating behaviours (overeating with loss of control) at a frequency of at least once a week for a period of at least 3 months, but had never engaged in restrictive eating.n = 394) women reported any of the ED behaviours above.Three hundred and ninety‐four (n = 4708). Women who participated to the ED sub‐study were overall more likely to have received secondary education and less likely to have had prior pregnancies (Given the high sensitivity (97.3%) of the Phase 1 screening , all womgnancies .Maternal care score was obtained from the maternal care subscale of the Parental Bonding Instrument (PBI) , and genotypes were called with Illumina GenomeStudio. PLINK (v1.07) was used to carry out quality control measures on an initial set of 10 015 subjects and 557 124 directly genotyped SNPs. SNPs were removed if they displayed more than 5% missingness or a Hardy–Weinberg equilibrium OXT‐R gene, rs2254298 and rs53576; both have three levels that represent genotypes GG, AG, and AA. The OXT‐R gene is located on chromosome 3 p25. It spans 19 206 base‐pairs and contains four exons and three introns. Within this gene rs53576 and rs2254298 are the two genetic variants , situated in the third intron, that have often been investigated. Although these variants have not been found to have any clear functional impact on the gene they may be in linkage disequilibrium with a yet unidentified functional SNP , A/G , and A/A (MAF = 0.41). This distribution does not deviate from the Hardy–Weinberg equilibrium, χ2(1) = 0.369, p = 0.54. Genotype distribution (and frequency) obtained for rs2254298 was: G/G , A/G , and A/A (MAF = 0.214). This distribution does not deviate from the Hardy–Weinberg equilibrium, χ2(1) = 0.369, p = 0.54. The genotype of rs2254298 was imputed using results from the GWAS analysis. Validity of the imputation was shown in Connelly et al.  [weight (kg)/height(m)Ethical approval for the study was obtained from the ALSPAC Ethics and Law Committee and the Local Research Ethics Committees.SNP and haplotype association analyses were performed with a logistic regression model using the SNPstats programme  were the outcomes, and BMI was included as a covariate. Post‐hoc analyses investigated the effect of binge eating, purging, and their overlap (binge eating and purging). Interaction analyses between OXT‐R genotypes and low maternal care were carried out including an interaction term (GxE) as a predictor. Univariable, multivariable, and interaction analyses were carried out in STATA 13.We also tested the effect of the In total, data on ED behaviours was available on 5102 women, and 3698 women had complete data on ED behaviours and had valid genotype data. Missing data on BMI and maternal care restricted the sample for multivariable analyses to 3135 women. Women who had complete data on ED behaviours and genotype were slightly older at enrollment (mean age = 29.6 (SD = 4.4)), were more likely to have completed secondary education or higher (50.4%) compared to those women who had missing data on genotype , completed A‐levels or degree (40.3%)).OXT‐R rs53576 A allele was negatively associated with binge eating and purging (respectively: odds ratio (OR) = 0.52 : 0.36–0.76), OR = 0.66 (95%CI: 0.47–0.92)), suggesting a protective effect. In contrast, the rs2254298 A allele was positively associated with purging behaviours (OR = 1.50 (95%CI: 1.03–2.19), p = 0.03) and restrictive eating (OR = 2.14 (95%CI: 1.42–3.23), p = 5.1 × 10−4).The p = 2 × 10−4) and purging (OR = 1.92 (1.06–3.47), p = 0.03) , .41–2.96,p = 0.001) and purging (OR = 1.55 (1.09–2.22), p = 0.01). Restrictive eating was not associated with rs53576 genotype (Table 3). The rs2254298 AG/AA genotype carriers had increased odds of restrictive eating (OR = 2.23 (1.42–3.49), p < 0.0001), but not of binge eating or purging.In BMI‐adjusted multivariable analyses, the rs53576 GG genotype carriers had increased odds of binge eating (OR = 1.91 (1.31–2.78), p = 0.01; OR = 1.67 (1.02–2.72), p = 0.04).Poor maternal care was independently associated with both binge eating and purging , OXT‐R genotype for either rs53576 and rs2254298 and poor maternal care in respect to all three outcomes . As shown in Table p = 0.03), and women with both the rs2254298 AG/AA genotype and poor maternal care had a four‐fold increased odds of binge eating and purging (OR = 4.40 (1.11–17.4)) (see Table Given the observed association between OXT‐R), well‐studied in the context of social behaviour, were associated with ED behaviours and interacted with poor maternal care in increasing the risk for ED behaviours in a large population‐based study of women (ALSPAC). We focused on the two most studied SNPs in the oxytocin receptor gene (rs2254298 and rs53576). Both rs2254298 and rs53576 showed associations with ED behaviours. Women who were homozygotes for the G allele in the rs53576 SNP were more likely to have had lifetime binge eating and purging behaviours; once we accounted for the co‐occurrence of these behaviours, this association became stronger. These findings are in line with findings from Kim et al  in increasing the risk for bulimic type behaviours. Given our increased understanding of the role of the oxytonergic system in appetite and eating behaviour, these findings lay an important foundation to further its study in the context of ED and eating behaviours.This study replicates and extends previous findings of an association between OXTR gene polymorphisms and ED behaviours. It is the first study to demonstrate an interaction between poor maternal care and an


Levodopa‐induced dyskinesias are a common side effect of dopaminergic therapy in PD, but their neural correlates remain poorly understood.This study examines whether dyskinesias are associated with abnormal dopaminergic modulation of resting‐state cortico‐striatal connectivity.Twelve PD patients with peak‐of‐dose dyskinesias and 12 patients without dyskinesias were withdrawn from dopaminergic medication. All patients received a single dose of fast‐acting soluble levodopa and then underwent resting‐state functional magnetic resonance imaging before any dyskinesias emerged. Levodopa‐induced modulation of cortico‐striatal resting‐state connectivity was assessed between the putamen and the following 3 cortical regions of interest: supplementary motor area, primary sensorimotor cortex, and right inferior frontal gyrus. These functional connectivity measures were entered into a linear support vector classifier to predict whether an individual patient would develop dyskinesias after levodopa intake. Linear regression analysis was applied to test which connectivity measures would predict dyskinesia severity.P < .0001). Modulation of resting‐state connectivity between the supplementary motor area and putamen predicted interindividual differences in dyskinesia severity . Resting‐state connectivity between the right inferior frontal gyrus and putamen neither predicted dyskinesia status nor dyskinesia severity.Dopaminergic modulation of resting‐state connectivity between the putamen and primary sensorimotor cortex in the most affected hemisphere predicted whether patients would develop dyskinesias with a specificity of 100% and a sensitivity of 91% (The results corroborate the notion that altered dopaminergic modulation of cortico‐striatal connectivity plays a key role in the pathophysiology of dyskinesias in PD. © 2016 International Parkinson and Movement Disorder Society The main risk factors are disease duration, levodopa dose, and age at onset.A total of 40 patients with a clinical diagnosis of PD were enrolled in the study. Exclusion criteria comprised (i) tremor‐dominant PD, (ii) psychiatric illness, (iii) dementia, (iv) contraindications to MRI, (v) sedatives or serotonergic medication, and (vi) off‐ or biphasic dyskinesias. Ten patients were excluded because they did not tolerate withdrawal of medication or developed claustrophobia inside the scanner. Fourteen of the remaining 30 patients had choreiform peak‐of‐dose dyskinesias (LID group). All 14 LID patients underwent structural T1‐weighted MRI of the brain. Two of the 14 patients had to be excluded from the analysis of resting‐state fMRI because they developed dyskinesias before the first postlevodopa resting‐state fMRI scan (see below). A total of 10 PD patients without LID (No‐LID) participated in both structural MRI and resting‐state fMRI. Of the No‐LID patients, 4 underwent only structural MRI, and in 2 No‐LID patients only resting‐state fMRI could be acquired due to technical problems.Puncorrected = .031, structural MRI study: Puncorrected = .045). We did not assess blood levels of levodopa because previous studies have shown no differences in the pharmacokinetics of levodopa between patients with and without LID.The LID and No‐LID group were matched regarding multiple clinical measures Table . LevodopAn MRI was acquired using a 3 T Verio scanner  with a 32‐channel head coil. Prior to MRI acquisition, dopaminergic medication was withdrawn according to 6 half‐lives as described previously.1 (BA 1‐4) as well as the SMA  based on previous work.1, (iii) the SMA, and (iv) the rIFG using the first eigenvariate from the unthresholded SPM of all voxels corresponding to the respective region.Analyses were conducted using Statistical Parametric Mapping . Standard preprocessing procedures comprised accounting for T1‐equilibrium effects, realignment, correction for nuisance variables , normalization to stereotactic space using the Montreal Neurological Institute template, and smoothing with an isotropic Gaussian kernel at 8 mm full‐width at half maximum. The following steps were then conducted using a priori defined regions of interest (ROIs) using anatomical masks1 and putamen, we extracted parameters from the most and less affected hemisphere, respectively, for each patient. Importantly, lateralization of predominant motor symptoms was equally distributed in the LID and No‐LID group . Because previously reported SMA activation in LID patients comprised a midline cluster extending into both hemispheresP < .001, Pearson correlation). We computed dopaminergic modulation of resting‐state connectivity by subtracting the baseline measure in the off‐medication state from the postlevodopa measure. These parameters were transformed into z scores for further analyses (see below). Z‐scoring was done for each resting‐state connectivity measure  for the whole group  by subtracting the mean and dividing by the standard deviation, that is, it normalized the data to a mean of 0 and a standard deviation of 1. The results did not change when data were not z‐transformed (not shown).For resting‐state connectivity between the SMP = .05 family wise error (FWE)‐corrected at the cluster level using a cluster‐building threshold of Puncorrected = 0.001.To test whether regions outside the considered ROIs showed an abnormal levodopa‐induced modulation of connectivity with the putamen, we also conducted a 2 × 2 analysis of variance using Group (LID and No‐LID) and Dopamine (postlevodopa and OFF) as main effects and tested for a Group × Dopamine interaction. The results were thresholded at http://surfer.nmr.mgh.harvard.edu) using a standard processing pipeline. The technical details of these procedures have been previously described in detail.1 (BA 1‐4), SMA , rIFG (BA 44 and 45), and bilateral putamen. The extracted volume measures were transformed into z scores for further analyses.Cortical reconstruction and volumetric segmentation were performed with Freesurfer software  and gray matter volume (n = 6) were tested regarding their accuracy in classifying patients as LID or No‐LID patients Fig. A. We appr = –0.608, P = .036, Pearson correlation), we additionally conducted partial regression analyses using time to LID onset as a covariate to account for individual differences in the time between intake of levodopa and development of LID. We conducted the same regression analyses with gray matter volumes of ROIs. To assess whether putative predictions were specific for the development of dyskinesias, we repeated the analyses using the individual UPDRS OFF scores as a dependent variable.We applied linear regression analyses to test whether measures reflecting dopaminergic modulation of resting‐state connectivity predicted dyskinesia severity of the LID patients  scores as dependent variable). The Bonferroni method was applied to correct for multiple comparisons. One LID patient was detected as a significant outlier (Grubbs test) and thus excluded. Because patients with more severe LID also developed dyskinesias more quickly (P > .1). Furthermore, we measured respiration and pulsation during resting‐state fMRI acquisition because drug‐induced changes in these physiological parameters can affect measures of resting‐state connectivity.P > .1).Because head movements can affect the analysis of resting‐state connectivity, we calculated the number of images moved more than 1 mm in relation to the previous image for each group and session.P >.1; independent‐sample t tests).All LID patients developed dyskinesias after an intake of 200 mg levodopa either inside the scanner room or shortly after completion of MRI acquisition. None of the No‐LID patients developed dyskinesias. In 7 LID patients, dyskinesias already emerged between the first and second postlevodopa resting‐state fMRI. Therefore, only the resting‐state fMRI scan in the off state and the first postlevodopa resting‐state fMRI were used for analysis of resting‐state connectivity because they were available in all participants. The first postlevodopa resting‐state fMRI was acquired approximately 25 min after levodopa intake  significantly predicted whether an individual patient would develop dyskinesias  between SM1 and the putamen increased after levodopa intake in No‐LID patients, it decreased in LID patients. The classifier yielded 95.8% accuracy corresponding to 1 false negative prediction (91.7% sensitivity) and no false positive predictions (100% specificity), which is illustrated in Figure 1–putamen resting‐state connectivity in the most affected hemisphere before intake of levodopa (OFF) predicted LID with an accuracy of 70.9% , but this result did not survive Bonferroni correction for multiple comparisons . Other connectivity parameters before or after levodopa intake did not predict emergence of LID (Supplementary Table 1).Dopaminergic modulation of resting‐state connectivity between the SMR2 = 0.627, P = .004). The more levodopa decreased resting‐state connectivity between the SMA and putamen, the more pronounced were peak‐of‐dose LID . There were no other significant predictions of UDysRS‐ or UPDRS‐scores . In contrast to resting‐state fMRI, regression analyses based on the volumetric MRI data failed to predict LID severity after correction for multiple comparisons.Acute dopaminergic modulation of resting‐state connectivity between the SMA and putamen predicted the severity of LID  or postlevodopa session (58.3%) separately was clearly inferior to classification accuracy using dopaminergic modulation of SM1–putamen connectivity (95.8%). This finding highlights the usefulness of using pharmacological fMRI in LID patients. It also speaks against the possibility that subclinical movements accounted for the high classification accuracy because this should also lead to high classification accuracy in the postlevodopa session where LID patients were on the verge of developing dyskinesias. Although the physiological significance of BOLD fluctuations during resting‐state is not entirely understood, it has been proposed that they encode coordination and synchronization of activity in interconnected neural areas.1–putamen network in LID patients. An abnormal dopaminergic modulation of SM1 in LID patients has also been shown with transcranial magnetic stimulation (TMS). TMS studies targeting the SM1 demonstrated aberrant changes in cortical plasticity after levodopa intake in LID patientsIn this study, abnormal dopaminergic modulation of resting‐state connectivity between the SMssion 58.% separatP > .5), but it predicted LID severity (P = .004). The more levodopa reduced resting‐state connectivity between the SMA and putamen, the more severely patients expressed peak‐of‐dose dyskinesias. This finding is in good agreement with our previous results obtained with task‐related fMRI in the same cohort.1‐putamen resting‐state connectivity is related to the all‐or‐nothing response to levodopa,Levodopa‐induced modulation of resting‐state connectivity between the SMA and putamen did not distinguish patients with and without LID (P > .1) nor predicted LID severity. In contrast, a recent study reported that rIFG resting‐state connectivity with the putamen and SM1 was abnormally modulated by dopamine in LID patients using rIFG as a seed region.Although the rIFG is a key region for inhibitory motor control,Limitations of the current study comprise the small sample size and the absence of an independent validation cohort. Furthermore, in this cross‐sectional study, patients had already developed LID. Yet it would be clinically highly relevant to assess whether a given PD patient will develop LID in the future. In contrast to task‐related fMRI, resting‐state fMRI does not require patients to engage in a specific task and can be easily standardized across imaging centers. Therefore, levodopa‐induced changes in resting‐state connectivity may serve as a powerful neuroimaging marker for emerging LID in PD. Future studies in independent validation cohorts and prospective studies are needed to test whether resting‐state fMRI can be used to identify patients at risk to develop LID. Different approaches, such as seed‐based(1) Research Project: A. Conception, B. Organization, C. Execution; (2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique; (3) Manuscript: A. Writing of the First Draft, B. Review and Critique.D.M.H: 1A, 1B, 1C, 2A, 2B, 3AB.N.H.: 1C, 2A, 3BS.H.N.: 2A, 2B, 3BK.H.M: 1B, 1C, 2A, 3BA.L.: 1B, 1C, 2A, 3BH.R.S.: 1A, 1B,, 2A, 3BD.M.H. received funding from the European Union's Horizon 2020 research innovation programme under the Marie Sklodowska‐Curie grant agreement 655605 (which is not related to the present work). B.N.H., S.H.N., and K.H.M. have no disclosures to report. A.L. reports congress participation with Desitin and honoraria from Ipsen. H.R.S. has received honoraria as senior editor from Elsevier Publishers and research funds from Biogen‐idec not related to the present work.Additional Supporting Information may be found in the online version of this article at the publisher's web‐site.Supplementary Information Table 1Click here for additional data file.


P < .05). Moreover, remarkably up‐regulated ciRS‐7 and RELA expressions, as along with down‐regulated miR‐7 expressions, were found within NSCLC tissues and cells in comparison with normal ones (P < .05). Besides, overexpressed ciRS‐7 and underexpressed miR‐7 were correlated with increased proliferation, migration and invasion, yet reduced apoptosis rate of NSCLC cells (P < .05). More than that, ciRS‐7 specifically targeted miR‐7 to reduce its expressions (P < .05). Ultimately, the NSCLC cells within miR‐7 + RELA group were observed with superior proliferative, migratory and invasive capabilities than those within miR‐7 group (P < .05), and RELA expression was also significantly modified by both ciRS‐7 and miR‐7 (P < .05). In conclusion, the ciRS‐7/miR‐7/NF‐kB axis could exert pronounced impacts on the proliferation, migration, invasion and apoptosis of NSCLC cells.The purpose of this study was to figure out the effect of ciRS‐7/miR‐7/NF‐κB axis on the development of non‐small cell lung cancer (NSCLC). In response, the expressions of ciRS‐7, miR‐7 and NF‐κB subunit (ie RELA) within NSCLC tissues and cell lines were determined with real‐time polymerase chain reaction (RT‐PCR) and Western blot. Moreover, the NSCLC cells were transfected with pcDNA3‐ciRS‐7‐ir, pcDNA3‐ciRS‐7, miR‐NC and miR‐7 mimic. Furthermore, the targeted relationships between ciRS‐7 and miR‐7, as well as between miR‐7 and RELA, were confirmed by luciferase reporter assay. The proliferation, migration and apoptosis of NSCLC cells were, successively, measured using CCK‐8 assay, wound‐healing assay and flow cytometry test. Consequently, ciRS‐7, miR‐7, histopathological grade, lymph node metastasis and histopathological stage could independently predict the prognosis of patients with NSCLC (all  The fresh specimens were cryopreserved at −80°C within 30 minutes after exairesis. All cases were pathologically confirmed by >2 senior professionals, and none of them had received chemotherapy or radiation before surgery. The histopathological grading of NSCLC was evaluated in light of World Health Organization (WHO),2.22 and saturated humidity at 37°C.Human NSCLC cell lines  and the normal human embryonic lung fibroblast cell line (ie MRC5) were purchased from Shanghai Institute of Biochemistry and Cell biology, Chinese Academy of Sciences. All the cell strains were inoculated within cell medium that contained 10% bovine foetal serum, 2 μmol/L glutamine, 100 IU/mL penicillin and 100 μg/mL streptomycin sulphate. Subsequently, the cells were managed to grow in 5% CO2.3−▵▵Ct method was used to calculate relative quantification (RQ) values, and each experiment was repeated for at least 3 times.Total RNA was extracted by consulting the operational manual of routine TR1201 reagent , and the extracted RNAs were preserved at −80°C. Then, the purity and concentration of RNA were measured by applying spectrophotometry. Moreover, the expressions of miR‐7 and ciRS‐7 were measured through qRT‐PCR (Invitrogen Corporation), and their primers were designed and composed by Sangon Biotech (Shanghai) Corporation, China .2.5The cells at the exponential phase were digested with pancreatins, and they were processed into cell suspension after low‐speed centrifugation. Subsequently, after being mixed with the trypan blue saline solution (percentage: 0.4%), they were dropped upon the blood counting chamber, and cell counting was completed within 3 minutes. Finally, under the microscopic observation, the cells dyed to blue were determined as dead, while the colourless and transparent ones were judged as live.2.63 cells, and the cells were cultured in 5% CO2 at 37°C. After each hole was added with 50 μg MTT, the cells were again cultured for another 4 hours. Then, the supernatants were sucked out before the addition of 200 μl DMSO. Ultimately, the optical densities  of each hole at the wavelength of 570 nm were measured with utilization of microplate reader at the 24th, 48th, 72nd and 96th hours, respectively. The inhibition rate (IR) of cell proliferation was calculated according to the following formula: Each hole of 96‐well culture plates was added with 100 μL cell suspension that contained 2 × 102.7Transwell chambers were placed within 24‐well plates, and the bottom membranes were coated with the diluted Matrigel. Serum‐free DEME nutrient solution was used to dilute cell sediments, and the concentration was adjusted to 5 × 10³/mL. The upper and lower Transwell chambers were, respectively, added with 200 μL cell suspension and 500 μL DEME solution that included 10% foetal bovine serum. After being cultured for another 12 hours, cells were washed with pre‐cooled PBS for twice. Methyl alcohol was prepared to immobilize membranes and cells for 15 minutes, which were then dyed with 1% crystal violet for 30 minutes. Under the high‐power microscope, the invasive cells below the chamber membrane were quantified, and 5 views were randomly observed to get the average results.Besides, the migration experiment was conducted mostly in accordance with the above procedures, except that Matrigel glue was hardly applied.2.85/mL. Flow cytometry was employed to detect the cell apoptotic conditions, following the instructions of Annexin V‐ fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit. The resultant scatter diagram was explained in the following way: (1) the left‐lower quadrant indicated live cells marked with FITC‐/PI−; (2) the left‐upper quadrant indicated live cells marked with FITC‐/PI+; (3) the right‐upper quadrant indicated dead cells marked with FITC+/PI+; and (4) the right‐lower quadrant indicated the early apoptotic cells marked with FITC+/PI−.The cells at the logarithmic phase were seeded within the culture vessels at the density of 1.25 × 102.9PCR was performed to amplify the miR‐7 fragment that embraced the binding site for ciRS‐7. Subsequently, the products were cloned into the pmirGLO Dual‐luciferase miRNA Target Expression Vector, deriving wild‐type miR‐7 (ie miR‐7‐wt). Besides, the same putative binding site was mutated, and miR‐7‐Mut was drawn. With assistance of Lipofectamine 2000 , the cells were, respectively, transfected with pcDNA‐ciRS‐7 and pcDNA‐ciRS‐7‐ir and pRL‐TK reporter gene carrier. As for the targeted relationship between miR‐7 and RELA, the RELA fragment that covered the binding site of miR‐7 was also amplified by PCR before being cloned into the pmirGLO Dual‐luciferase Expression Vector , in which way the reporter vector RELA‐wild‐type (ie RELA‐wt) was constructed. Similarly, the RELA with the mutated binding site of miR‐7 was called RELA‐mutated‐type (ie RELA‐Mut). Subsequently, the cells were, respectively, transfected with miR‐7 mimics and miR‐NC, and each group was then transfected with RELA‐Wt 3′UTR‐PGL3 plasmid/RELA‐Mut 3′UTR‐PGL3 plasmid and pRL‐TK reporter gene carrier, respectively. The activities of Firefly and Renilla luciferase were evaluated successively by applying dual‐luciferase reporter assay system (Promega) in 48 hours after transfection. All the assays were repeated for at least 3 times.2.10By means of the 10% SDS‐PAGE electrophoresis, the extracted total protein was transferred onto nitrocellulose membranes for 60 minutes. Then, PVDF membranes were sealed at room temperature for 2 hours, and rabbit anti‐human RELA primary antibody  was incubated overnight. Subsequently, the rat anti‐rabbit second antibody  was added and incubated for another 2 hours at room temperature. Then, enhanced chemiluminescence (ECL) was applied for exposure and development, and gel image analysis system  was employed to analyse the electrophoresis strips. The β‐actin was regarded as the control of ciRS‐7 regarding calculation of the intensity values. All the assays were repeated for at least 3 times.2.112 test, while measurement data (mean ± SD) were contrasted using t‐test. The method of Kaplan‐Meier was applied to fit overall survival (OS) curves of the included subjects. It would be considered statistically significant when P value was <.05.All the statistical analyses were conducted with SPSS 20.0 software. The enumeration data were compared with χ33.1P < .05), whereas the miR‐7 expression followed an inverse trend (P < .05). The different trends also rendered a significantly negative correlation between ciRS‐7 and miR‐7 expressions within NSCLC tissues (P < .05)   Figure B. Analog) Figure C.3.2P < .05). Moreover, Kaplan‐Meier analysis also manifested that NSCLC subjects with highly expressed ciRS‐7 or lowly expressed miR‐7 possessed inferior OS to ones with lowly expressed ciRS‐7 or highly expressed miR‐7 (P < .05)  (Table The incorporated patients with NSCLC were divided into higher ciRS‐7 expression (>average ciRS‐7 expression) group and lower ciRS‐7 expression (<average ciRS‐7 expression) group Table . Simulta) Figure D,E. Furt5) Table .3.3P < .05)     group displayed a better multiplicative capacity than NC group  Figure A,B. Mean) Figure C,D. In a) Figure E,F. Corr) Figure G.3.4http://starbase.sysu.edu.cn/seedTargetInfo.php?type=circRNA&database=hg19&name=hsa-miR-7-5p&geneName=CDR1-AS_hsa-circRNA8162&autoId=94962&orgTable=mirCircRNAInteractionsAll), altogether 45 targeted sites were shared by ciRS‐7 and miR‐7 , yet the cells cotransfected with pcDNA3‐ciRS‐7 and miR‐7‐mut hardly generated this change , yet down‐regulated miR‐7 expression could barely modify the expression level of ciRS‐7 , while ciRS‐7 expression went up with the rising of RELA expression (P < .05)  Figure F,G. Conc) Figure K,L; none) Figure A,B. In s3.6P < .05), suggesting that RELA suppressed the lowered effect of miR‐7 on NSCLC cells’ activity    value of miR‐7 + RELA group was dramatically higher than that of miR‐7 group  Figure D,E. Fina) Figure F.4It was generally accepted as neoplastic invasion and migration when tumour cells aggressively grew from primary lesions to surrounding tissues, blood circulation and lymphatic circulation.As a matter of fact, competitive endogenous RNAs (ceRNAs), which mainly incorporated lncRNAs and circRNAs, were revealed to compete with miRNA for the miRNA recognition elements (MREs) of target mRNAs, so as to reduce the control over target genes and to affect the development of diseases.It was also documented that miR‐7, which was positioned on chromosome 15, held up the growth of lung cancer cells (ie A549) through down‐regulating Bcl‐2 expression.Correspondingly, ciRS‐7 and miR‐7 expressions were also detected within NSCLC tissues and cells here, validating that they could be widely and stably expressed within biological cells. Also, the apparent differences of ciRS‐7 and miR‐7 expressions between NSCLC and adjacent normal tissues suggested that they might play a role in the occurrence and development of NSCLC. In addition, the negative association of ciRS‐7 with miR‐7 further provided hints that ciRS‐7 might function as the suppressor of miR‐7 or ceRNA that constrained miR‐7 expression. Furthermore, relevant experiments of trypan blue staining cell counting, MTT, Transwell and flow cytometry indicated that ciRS‐7 promoted migration, invasion and proliferation of NSCLC cells, and inhibited apoptosis of the cells. Nonetheless, the role of miR‐7 was the opposite to ciRS‐7 regarding the above end indicators.Moreover, the Western blot experiments in this study Figure K,L demonAll in all, the current investigation logically linked ciRS‐7 with miR‐7 and NF‐kB signalling in accelerating proliferation, invasion and migration of NSCLC cells, yet several shortcomings were still evident. Firstly, recruiting a larger population of different ethnicities could make the study result be generalized to a wider range of crowds. Secondly, additional molecules, besides NF‐kB‐p65, should be explored to validate the role of NF‐kB signalling in modulating NSCLC development. Finally, animal models should further be established to confirm the study result, so that ciRS‐7 might become crucially diagnostic and treatment biomarkers for NSCLC.The authors declare that no conflict interest existed.


Septic cardiomyopathy is defined as a reversible left ventricular systolic dysfunction. Patients with severe septic cardiomyopathy have a high mortality rate, even if they receive conventional therapy. For those patients, previous reports showed intra‐aortic balloon pump (IABP) efficacy. We report two rare cases with IABP introduction leading them to drastic improvement, and survival from severe septic cardiomyopathy. Case 1 is a 78‐year‐old woman diagnosed with renal calculus pyelonephritis, septic shock, and septic cardiomyopathy. Case 2 is a 62‐year‐old man diagnosed with pneumonia, septic shock, and septic cardiomyopathy.In both cases, despite conventional therapy for cardiomyopathy, including high‐dose catecholamine therapy, shock was not reversed, and the IABP was inserted. Circulatory status was improved after the introduction of the IABP.Our findings suggest that an IABP can be useful for salvaging patients with septic cardiomyopathy who do not respond to conventional therapy. Septic cardiomyopathy is defined as a reversible left ventricular systolic dysfunction that occurs in patients with sepsis. Most patients improve with conventional therapy after 7–10 daysThe strategy of treatment for septic cardiomyopathy is generally the same as that for cardiomyopathy without sepsis, such as by fluid replacement and administration of catecholamine. Additional therapy is controversial for patients who do not respond to this conventional therapy. Although the intra‐aortic balloon pump (IABP) has been used in some cases, its efficacy remains controversial,We report two cases with IABP introduction leading to drastic improvement, and survival from severe septic cardiomyopathy when conventional therapy was not effective.This study was approved by the ethics committee of Yokohama City University . Written informed consent was obtained from the subjects and/or guardians for publication of this report.The patient was a 78‐year‐old woman with shock who was transferred to our hospital on the day after ureterolithotomy.2 of 89% on oxygen at 6 L/min and heart rate was 115 b.p.m. A diagnosis of septic shock was made. The patient underwent tracheal intubation for mechanical ventilation and noradrenaline was given.Glasgow Coma Scale was E1V1M4, indicating disturbance of consciousness. She had tachypnea with a respiration rate of 26 breaths/min. Oxygenation was impaired, with SpO2, 63.6 mmHg; HCO3−, 21.1 mmol/L; lactate, 3.9 mmol/L. Cardiomegaly and pulmonary edema were observed on the chest X‐ray. Abdominal computed tomography showed a right renal calculi and pyelonephritis. The electrocardiogram revealed ST segment depression in lead aVR and ST segment elevation in leads V3–6. Echocardiography showed that the left ventricular ejection fraction (LVEF) was 25% with diffuse LV systolic dysfunction. Coronary angiography did not identify any significant coronary artery stenosis.The results of laboratory tests showed: increased leucocytes  and troponin‐I (1.1 ng/mL); blood urea nitrogen, 36 mg/dL; creatinine, 3.7 mg/dL; prothrombin time – international normalized ratio, 1.5; activated partial thromboplastin time, 43.3 s. Blood gas analysis revealed: pH 7.14; PaCOThe patient was admitted to the intensive care unit (ICU) with renal calculus pyelonephritis, septic shock, and septic cardiomyopathy. Her course after admission is shown in Figure The patient was a 62‐year‐old man who presented with high fever and impaired oxygenation. He was referred to our hospital for treatment of pneumonia.2 98.4 mmHg). The LVEF was 20% on ECG, and diffuse LV systolic dysfunction was observed. His chest X‐ray showed infiltration in the left lower lung field. Chest computed tomography revealed bilateral lower lobe pneumonia.The results of laboratory tests showed: decreased leucocytes, 2590/μL; elevated troponin‐I, 0.13 ng/mL; blood urea nitrogen, 35 mg/dL; creatinine, 1.7 mg/dL; prothrombin time – international normalized ratio, 1.3; activated partial thromboplastin time, 46.4 s. Blood gas analysis under 10 L/min oxygen inhalation revealed oxygenation failure  or IABP was useful as the last rescue therapy for unresponsive sepsis cardiomyopathy patients.However, it has been reported that complications can occur, such as thrombocytopenia and bleeding, so long‐term use of an IABP should be avoided. Septic cardiomyopathy is a reversible condition, so an IABP may only be required for short‐term circulatory assistance until cardiac function improves spontaneously.As a differential diagnosis, Takotsubo cardiomyopathy was not ruled out by coronary angiography. We need to accumulate additional patients with septic cardiomyopathy who receive treatment with the IABP in the future to evaluate its usefulness in more detail.Our findings suggested that the IABP could be useful for salvaging patients with septic cardiomyopathy who do not respond to conventional therapy.Conflict of Interest: None declared.