HCAEC) injury and inflammatory response and the involvement of the PI3K/Akt/eNOS signalling pathway. Oxidized low‐density lipoprotein (OX‐LDL)‐induced HCAEC injury models were established and assigned to blank, miR‐138 mimic, miR‐138 inhibitor, LY294002 (an inhibitor of the PI3K/Akt/eNOS pathway), miR‐138 inhibitor + LY294002 and negative control (NC) groups. qRT‐PCR and Western blotting were performed to detect the miR‐138, PI3K, Akt and eNOS levels and the protein expressions of PI3K, Akt, eNOS, p‐Akt, p‐eNOS, Bcl‐2, Bax and caspase‐3. ELISAs were employed to measure the expressions of TNF‐α, IL‐4, IL‐6, IL‐8, IL‐10 and nitric oxide (NO) and the activities of lactate dehydrogenase (LDH) and eNOS. MTT and flow cytometry were performed to assess the proliferation and apoptosis of HCAECs. Compared to the blank group, PI3K, Akt and eNOS were down‐regulated in the miR‐138 mimic and LY294002 groups but were up‐regulated in the miR‐138 inhibitor group. The miR‐138 mimic and LY294002 groups showed decreased concentrations of TNF‐α, IL‐6, IL‐8 and NO and reduced activities of LDH and eNOS, while opposite trends were observed in the miR‐138 inhibitor group. The concentrations of IL‐4 and IL‐10 increased in the miR‐138 mimic and LY294002 groups but decreased in the miR‐138 inhibitor group. The miR‐138 mimic and LY294002 groups had significantly decreased cell proliferation and increased cell apoptosis compared to the blank group. These findings indicate that up‐regulation of miR‐138 alleviates HCAEC injury and inflammatory response by inhibiting the PI3K/Akt/eNOS signalling pathway.This study aimed to investigate the role of miR‐138 in human coronary artery endothelial cell ( Endothelial cells, a single layer of cells that line the inner surface of blood vessels in the human body, inhibit activation of pro‐inflammatory and clotting factors in vessels et al. et al. MicroRNAs (miRNAs) are a class of non‐coding RNAs that can result in mRNA degradation or suppress protein translation by binding to the 3′‐untranslated region of their target mRNAs 2 atmosphere at 37°C with 95% humidity), and foaming was avoided. After approximately 90% of cells adhered to the wall, subculturing was conducted. The culture solution was aspirated, and the cells were rinsed with phosphate‐buffered saline (PBS) twice and digested with pancreatic enzymes. As the intercellular gap increased, the enzyme solution was aspirated. The cells were resuspended again in the same culture medium mentioned above and routinely subcultured. Passaged cells in exponential growth were selected for further experiments. Injured HCAECs were selected for the construction of HCAEC injury models.HCAECs were obtained from Cell Applications, Inc. . Liquid nitrogen‐preserved cells were thawed in a 37°C water bath, and the cells were then transferred into 5 ml 10% modified serum‐free cell freezing medium (RPMI) 1640 . Then, the cells were centrifuged at 1000 r.p.m. for 5 min., and the supernatant was aspirated. After resuspension in 5 ml 10% modified RPMI 1640, the cells were cultured in a culture bottle  for 45 min. to establish OX‐LDL‐induced HCAEC injury models. Then, the cells were cultured in normal medium, and an optical microscope was used to observe and compare the morphological changes between the injured cells and normal cells. A LDH kit  was employed to detect LDH activity in the cells.−ΔΔCt method. All experiments were conducted three times.After the HCAECs were cultured, the medium was aspirated, and HCAECs were washed with PBS twice. Using the TRIzol reagent  instructions, total RNA was extracted using the TRIzol one‐step extraction method. Diethylpyrocarbonate (DEPC)‐treated ultrapure water was used to dissolve RNA, and a ND‐1000 UV‐Visible spectrophotometer  was used to measure absorbance at 260 nm and 280 nm to determine the RNA purity and concentration. The RNA concentration was adjusted for qRT‐PCR. TaqMan probes , along with shuttle vector pcDNA3.1 , negative control, miR‐138 mimic and miR‐138 inhibitor plasmids were constructed by Sangon Biotech Co., Ltd., . The sequences of miR‐138 mimic, negative control and miR‐138 inhibitor were 5′‐CCUGCUUGCUCAAAUCAAUTT‐3′, 5′‐CAGUACUUUUGUGUAGUACAA‐3′ and 5′‐GGCAUUCACCG CGUGCCUUA‐3′, respectively. The cells (second passage) were digested with pancreatic enzymes and inoculated into 24‐well plates, and the cells grew in a monolayer. The medium was aspirated, and the cells were assigned to five groups: the blank group (transfected with empty vector), the miR‐138 mimic group (transfected with miR‐138 mimic plasmid), the miR‐138 inhibitor group (transfected with miR‐138 inhibitor plasmid), the LY294002 group (treated with the inhibitor LY294002), the miR‐138 inhibitor + LY294002 group (transfected with miR‐138 inhibitor plasmid and treated with LY294002) and the NC group (transfected with negative control plasmid). The transfection procedures were as follows: Plasmid suspension solution (100 μl) was added to the corresponding plates with the injured cells in 200 μl serum‐free Dulbecco's modified Eagle's medium (DMEM) . After a 3‐h culture, 1 ml DMEM containing 10% serum  was added to the cells. In the LY294002 group, the injured cells were treated with LY294002, and in the miR‐138 inhibitor + LY294002 group, the injured cells were transfected with miR‐138 inhibitor and treated with LY294002.After a 48‐h transfection, the cells were collected. Then, 1× sodium dodecyl sulphate (SDS) lysis buffer  was added, and protein extracts were heated at 100°C for 5 min. Proteins (20 μl) were separated with 10% polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride (PVDF) membrane for 3.5 hrs with a voltage of 48 V. The membrane was sealed in 5% bovine serum albumin (BSA) for 2 hrs at room temperature and was rinsed with 1× Tris‐buffered saline plus Tween (TBST). The primary antibodies  , Beverly, MA, USA; diluted 1: 500) were separately added, and the membrane reacted at 4°C overnight. β‐Actin monoclonal antibody was used as an internal reference. The membrane was washed with 1× TBST and was incubated with the peroxidase‐labelled anti‐rabbit secondary antibody  for 1 hr at room temperature. After being washed with TBST, the membrane was incubated with Chemiluminescent Reagent  for 1 min. and developed by electrochemiluminescence (ECL). Photographs were taken with a gel imaging system . Optical density (OD) analysis of all Western blotting bands was performed. The / was regarded as the relative expression of protein.According to the ELISA kit  instructions, the concentrations of TNF‐α, IL‐4, IL‐6, IL‐8 and IL‐10 were detected after 48 hrs of transfection. Coating buffer was used to dilute the antibody to 1 μg/ml, and the diluted antibody was then added to 96‐well plates (100 μl/well) and cultured at 4°C overnight. Afterwards, the coating buffer was aspirated and the plate was washed three times. Each well was incubated with blocking buffer (150 μl) at room temperature for 1 hr. Then, the plate was washed again, followed by addition of 100 μl serum samples or standards. With the blank control set, the plate was cultured for 2 hr at room temperature. After being rinsed, each well was incubated for 1 hr at room temperature after addition of 100 μl horseradish peroxidase (HRP)‐labelled avidin. The wells were rinsed again, and then 100 μl tetramethylbenzidine (TMB) substrate was added to each well for a 15 min. reaction at room temperature, which was terminated by 2 M sulphuric acid (50 μl/well). The absorbance of each well was measured at a wavelength of 450 nm, and the standard curve was generated to determine the concentrations of TNF‐α, IL‐4, IL‐6, IL‐8 and IL‐10 (ng/ml).550. The eNOS activity was determined by the following processes. After 48 hrs of transfection, the cells were collected to detect the eNOS activity according to NOS kit instructions (Nanjing Jiancheng Bioengineering Institute). The cells were disrupted using PBS and centrifuged to obtain supernatant for the later use. According to the instructions, R1 and R2 were added and reacted for 5 min. at 37°C, followed by the addition of R4 and R5. The OD530 value was measured.The NO concentration was detected using the following protocol. After a 48‐h transfection, the cell supernatant was collected and then centrifuged at 3000 r.p.m. for 20 min. for analysis of the NO concentration following the NO kit instructions (Nanjing Jiancheng Bioengineering Institute). The sample and mixing reagents (R1 and R2) were combined and incubated in a water bath for 60 min. at 37°C, followed by the addition of R3 and R4. The mixture was incubated for 40 min. at room temperature and centrifuged at 3500 r.p.m. for 10 min. The supernatant was collected, mixed with colorimetric reagent and allowed to react for 10 min. prior to analysis of the OD3–6 × 103 cells/well; 200 μl/well). Each group was processed as described above, with three replicate wells. The cells were cultured in a 5% CO2 atmosphere at 37°C, and 20 μl 5 mg/ml 3‐‐dimethylthiahiazo(z‐y1)‐3,5‐di‐phenytetrazoliumromide (MTT)  was added to the cells for 4 hrs. Then, the supernatant was aspirated, and 150 μl dimethylsulphoxide (DMSO) was added to each well, followed by 10 min. of low‐speed shaking. At 24, 48 and 72 hrs, the OD value was detected at 490 nm. With time as the X‐axis and OD value as the Y‐axis, the cell activity curve was drawn.When the cell density reached 80%, the cells were rinsed with PBS twice and digested with 0.25% pancreatic enzymes to obtain single cell suspensions. After being counted, the cells were inoculated into 96‐well plates ‐free pancreatic enzymes. Then, the cells were centrifuged at 1000 r.p.m. for 5 min. with the supernatant aspirated. After washing with cold PBS three times, the cells were again centrifuged with the supernatant aspirated. In accordance with the Annexin‐V‐FITC apoptosis kit  instructions, 150 μl binding buffer and 5 μl Annexin‐V‐FITC were added to each tube and fully mixed. Then, the cells were cultured for 15 min. at room temperature in the dark. After that, 100 μl Binding buffer and 5 μl propidium iodide (PI)  were added and fully mixed. Finally, cell apoptosis was detected by flow cytometry.t‐tests and those among more than two groups using one‐way analysis of variance (anova). Pairwise comparison was performed by LSD t‐tests. P < 0.05 indicates significant differences.Statistical Analysis Commercial software was used for statistical analysis . Data are expressed as the mean ± standard deviation (S.D.). Comparisons between two groups were performed using P < 0.05; Fig. Under a microscope, the cells in the control group showed normal morphology, with a cobblestone‐like appearance Fig. A. The inP < 0.05). According to the Western blot analyses, PI3K, Akt and eNOS protein expressions were strongly up‐regulated in the HCAEC injury models . However, there were no significant differences in miR‐138 expression in the LY294002 and NC groups compared to the blank group (both P > 0.05). The blank group showed high expression of PI3K, Akt and eNOS mRNA. Compared with the blank group, PI3K, Akt and eNOS mRNA expressions were up‐regulated in the miR‐138 inhibitor group but were down‐regulated in the miR‐138 mimic and LY294002 groups , and there was no significant difference between the miR‐138 mimic and LY294002 groups (P > 0.05). There were no significant differences in PI3K, Akt and eNOS mRNA expressions among the blank, NC and miR‐138 inhibitor + LY294002 groups .The mRNA expressions of miR‐138, PI3K, Akt and eNOS in the groups after transfection were detected by qRT‐PCR and are shown in Figure P < 0.05) but were substantially down‐regulated in the miR‐138 mimic and LY294002 groups , while there were no significant differences in these protein expressions between the miR‐138 mimic and LY294002 groups . There were no significant differences in PI3K, Akt, eNOS, p‐PI3K, p‐Akt and p‐eNOS protein expressions among the blank, NC and miR‐138 inhibitor + LY294002 groups .The protein expressions of PI3K, Akt and eNOS in the groups after transfection were detected by Western blotting and are shown in Figure P < 0.05). IL‐4 and IL‐10 concentrations significantly decreased in the miR‐138 inhibitor group, but opposite trends were observed in the miR‐138 mimic and LY294002 groups compared to those in the blank group (both P < 0.05). There were no significant differences in the concentrations of TNF‐α, IL‐4, IL‐6, IL‐8 and IL‐10 between the miR‐138 mimic and LY294002 groups and among the blank, NC and miR‐138 inhibitor + LY294002 groups .ELISAs were performed to detect the concentrations of TNF‐α, IL‐4, IL‐6, IL‐8 and IL‐10 in HCAECs, and the results are shown in Figure P < 0.05). There were no significant differences in the LDH activity between the miR‐138 mimic and LY294002 groups and among the blank, NC and miR‐138 inhibitor + LY294002 groups .The LDH activity assays Fig. B showed P < 0.05). No significant differences in NO concentration were observed between the miR‐138 mimic and LY294002 groups and among the blank, NC and miR‐138 inhibitor + LY294002 groups .The NO concentration analysis indicated that the NO concentration significantly increased in the miR‐138 inhibitor group but decreased in the miR‐138 mimic and LY294002 groups compared to that of the blank group . There were no significant differences in the eNOS activity between the miR‐138 mimic and LY294002 groups and among the blank, NC and miR‐138 inhibitor + LY294002 groups . At 48 hrs and 72 hrs, compared with the blank group, cell proliferation was significantly decreased in the miR‐138 mimic and LY294002 groups, and the OD values were significantly different . No significant difference in OD values was observed between the miR‐138 mimic and LY294002 groups (P > 0.05). Compared to the blank group, the cell proliferation capacity in the miR‐138 inhibitor group significantly increased, and OD values showed significant difference (P < 0.05).The MTT assay indicated that no significant differences in cell proliferation were found among the groups at 24 hrs . There was no significant difference in the cell apoptosis rate between the miR‐138 mimic and LY294002 groups and among the blank, NC and miR‐138 inhibitor + LY294002 groups . Compared to the blank group, Bax and caspase‐3 proteins were significantly up‐regulated, but Bcl‐2 protein was significantly down‐regulated in the miR‐138 mimic and LY294002 groups . There was no significant difference in apoptosis‐related protein expressions between the miR‐138 mimic and LY294002 groups (P > 0.05). Compared with the blank group, Bax and caspase‐3 proteins were significantly down‐regulated, whereas Bcl‐2 protein was significantly up‐regulated in the miR‐138 inhibitor group  had significantly lower expression of circulating miRNAs compared with healthy individuals, and damaged endothelial cells can produce endothelial cell‐derived microparticles (EMP) with low miRNA expression We also found that compared to the blank group, the levels of TNF‐α, IL‐6, and IL‐8 were significantly decreased in the miR‐138 mimic and LY294002 groups but substantially increased in the miR‐138 inhibitor group, which indicated that up‐regulation of miR‐138 and inhibition of the PI3K/Akt/eNOS signalling pathway can suppress the inflammatory response in injured HCAECs. In addition, IL‐4 and IL‐10 were significantly increased in the miR‐138 mimic and LY294002 groups but decreased in the miR‐138 inhibitor group. Several reports have investigated the relationship between atherosclerosis and various factors, including endothelial cell injury, lipid infiltration, inflammatory response and thrombosis In conclusion, this study indicates that up‐regulation of miR‐138 alleviates HCAEC injury and inflammatory response by inhibiting the PI3K/Akt/eNOS signalling pathway, which elucidates the underlying mechanism of HCAEC repair and further suggests therapeutic strategies to preserve endothelial integrity and vascular health and inhibit inflammatory activation in cardiovascular disease.None.


Data on left ventricular (LV) function in patients with neurocardiogenic syncope (NS) is conflicting in adults and lacking in children. The aim of this study was to analyze LV myocardial performance in children with NS at rest and during head-up tilt-table (HUTT) testing.This is the first study to combine HUTT and speckle-tracking echocardiography (STE) in children with NS. 43 consecutive normotensive pediatric patients with NS  and 41 sex- and age-matched healthy controls were included in the study. The study groups consisted of 21 patients with a positive HUTT reaction (HUTT+) and 22 with a negative HUTT reaction (HUTT-). STE was used to analyze peak systolic LV myocardial strain and strain rate.p = 0.032) and significantly depressed longitudinal strain rate (p <  0.001) at rest. Interestingly, during HUTT testing LV global strain and strain rate were similar in both groups. LV strain rate was lowest in HUTT+ followed by HUTT- and control subjects both at rest and during HUTT.Conventional echocardiographic parameters were similar in all analyzed groups. When compared to healthy controls, children with NS had depressed levels of circumferential strain rate (Resting LV longitudinal strain rate is attenuated in children with NS, especially in those with a positive HUTT response. This is further evidence that NS patients feature altered cardiac mechanics rendering them prone to vasovagal perturbations that can ultimately result in collapse.Witten/Herdecke University ethics committee clinical study number: UWH-73-2014. Syncope is a sudden, brief loss of postural tone and consciousness followed by spontaneous recovery . Up to 1Advanced echocardiography techniques – including tissue-Doppler echocardiography (TDI) and speckle-tracking echocardiography (STE) – are promising modalities for the real-time quantitative evaluation of myocardial performance without the disadvantages of adverse effects or high costs. Based on TDI studies, cardiac mechanics such as left atrial and LV filling and contractility are postulated to be altered in neurocardiogenic syncope in adults , 10. STEclinical study number: 73/2014).In this prospective study, we altogether enrolled 84 children – 43 consecutive normotensive pediatric patients aged 13.9 ± 2.6 years  with a history of vasovagal syncope and 41 healthy sex- and age-matched controls  without medical problems and with a normal transthoracic echocardiogram. A priori, the study group was further sub-categorized according to their HUTT exam reaction. Inclusion criteria for the study group were a typical history of NS, age < 18 years and a written informed consent signed by the patient and their legal guardian. Moreover, the presence of any acute or chronic condition that might affect the cardiovascular system , ECG abnormalities, chronic medication use and the lack of cooperation to participate in this voluntary HUTT study were strict exclusion criteria. 60 patients were excluded from the study due to various reasons: 53 were incompliant regarding the HUTT challenge, 2 patients were obese (BMI > 30), 2 had insufficient image quality at baseline and in 3 patients the continuous ECG signal was corrupted due to technical difficulties which made consecutive speckle tracking post-processing impossible. The study was carried out in accordance with the declaration of Helsinki’s ethical principles for medical research involving human subjects and approved by the Witten/Herdecke University ethics committee  were utilized for all statistical tests.Demographic data, baseline clinical parameters, hemodynamics and echocardiographic aspects are presented as mean and standard deviation. Analysis of variance (ANOVA) testing was used to compare clinical, hemodynamic and echocardiographic data of the analyzed groups. Strain and strain rate are displayed as box-whisker-plots. Bonferroni correction was applied to exclude multiple testing bias. Hence, p = 0.318). Height (p = 0.375), body weight (p = 0.101), body mass index (p = 0.1) and body surface area (p = 0.165) were not statistically different between the three groups. All these values were within normal limits as evaluated by Z-scores. HUTT response was positive in 21 of the 43 patients with NS and 2 of the 41 healthy controls (p <  0.001). Hemodynamic parameters, e.g. heart rate and arterial blood pressure did not differ between the three groups neither at rest nor at the moment of STE assessment during HUTT testing (0.2 < p < 0.964).Baseline clinical characteristics are outlined in Table p = 0.480), fractional shortening (p = 0.760), LV dimensions (p ≥ 0.15) and LV mass (p = 0.578). LV stroke volume was lowest in the HUTT+ group (46.7 ± 17.3 vs. 53.5 ± 21.3 vs. 53.8 ± 17.6 ml) when compared to HUTT- and healthy control subjects. However, this difference was not statistically different (p = 0.796). Moreover, LV ejection fraction (EF) was 54.3 ± 2.9% in syncope HUTT+ patients, 54.7 ± 6.6% in HUTT- and 56.7 ± 4.4% in healthy controls (p = 0.263). Furthermore, diastolic function as reflected by E/A ratio and E/E’ did not differ significantly between the analyzed groups (p ≥ 0.352). Mitral deceleration time was longer in the HUTT- group  but similar in HUTT+ patients with NS and healthy controls (0.17 ± 0.07 vs. 0.16 ± 0.06 s). However, these values were still within normal limits as evaluated by population-based age-specific reference values [Standard 2D derived conventional echocardiographic parameters were similar in all groups  and median peak systolic longitudinal strain rate  were lowest in children with NS and a positive HUTT exam (HUTT+) as compared to the HUTT- group and healthy controls  than in the control group, but the difference was not statistically different (n = 4) or longitudinal strain rate values  at rest . Interestingly, all of these patients were in the HUTT- group. One of the four patients was female and one had a history of more than one syncopal episode. The average blood pressure of this subgroup was 121 ± 9 mmHg systolic and 68 ± 2 mmHg diastolic and the average heart rate was 74 ± 10 bpm at. None of the patients with NS and a positive tilt-table response had resting LV longitudinal or circumferential strain rate values ≥ the top quartile of the healthy control group.While the overall comparison to healthy controls revealed LV myocardial strain rate at rest to be significantly lower in patients with neurocardiogenic syncope, we also observed a heterogeneous distribution of LV deformation in those patients. Interestingly, a subset of patients with NS had high LV circumferential (This is the first study to simultaneously perform speckle-tracking echocardiography (STE) and head-up tilt-table (HUTT) testing in children with NS. Interestingly, we found LV resting longitudinal strain rate to be significantly lower in NS patients than in healthy controls with a more pronounced decrease in the HUTT+ group. During HUTT challenge we found a similar but not statistically significant tendency of lower longitudinal strain rate in patients with NS. This is in line with findings from Kojo et al., who utilized a Doppler flow meter during HUTT testing and found reduced carotid artery blood flow in pediatric patients with NS . Our finOn the other hand, our findings are counterintuitive in the light of the “ventricular theory” in that we did not measure an increase but a decrease in LV contractility in patients with NS and a positive HUTT response. This, however, apart from the fact that the ventricular hypothesis is far from being established conclusively, is in agreement with a similarly performed study by Goel et al., who report attenuated resting LV strain in HUTT+ patients and conclude that – other than previously believed – increased resting LV contractility is not a prerequisite for the development of NS . NotablyThe cardiac autonomic response pattern of patients with NS during HUTT challenge has been distinguished between HUTT+ and HUTT- patients in previous studies . In lineAnother unresolved issue regarding the enigmatic pathogenesis of NS is the factor age. Even though younger and older patients react differently on the HUTT, especially in the early phase , the rolInterestingly, based on the evidence of fear and threat bradycardia in animals and the beneficial vasovagal reflex during hemorrhagic shock in humans and animals, some researchers suggest NS not to be a recently evolved human-only disease but rather the remnant of an ancient defense mechanism that has once reduced myocardial oxygen consumption and supported hemostasis before the development of larger blood losses –44. UndeDespite the incremental value of these findings, this study design bares important limitations. Firstly, while STE has been implemented in specific clinical practice of pediatric cardiology, i.e. to monitor cardiotoxicity in chemotherapy patients , it is sThis study provides further evidence that decreased LV resting performance may be a relevant factor in the etiology of neurocardiogenic syncope in children. Further experimental animal and human studies should be designed to investigate LV contractility in children and adults with NS under different cardiac loading conditions at baseline and in the context of HUTT challenge.


Autism Res2018, 11: 509–518. © 2017 The Authors Autism Research published by International Society for Autism Research and Wiley Periodicals, Inc.Recent theoretical accounts propose that atypical predictive processing can explain the diverse cognitive and behavioral features associated with autism, and that difficulties in making predictions may be related to reduced contextual processing. In this pre‐registered study, 30 autistic children aged 6–14 years and 30 typically developing children matched in age and non‐verbal IQ completed visual extrapolation and false memory tasks to assess predictive abilities and contextual processing, respectively. In the visual extrapolation tasks, children were asked to predict when an occluded car would reach the end of a road and when an occluded set of lights would fill up a grid. Autistic children made predictions that were just as precise as those made by typically developing children, across a range of occlusion durations. In the false memory task, autistic and typically developing children did not differ significantly in their discrimination between items presented in a list and semantically related, non‐presented items, although the data were insensitive, suggesting the need for larger samples. Our findings help to refine theoretical accounts by challenging the notion that autism is caused by pervasively disordered prediction abilities. Further studies will be required to assess the relationship between predictive processing and context use in autism, and to establish the conditions under which predictive processing may be impaired. It has been suggested that autistic individuals have difficulties making predictions and perceiving the overall gist of things. Yet, here we found that autistic children made similar predictions about hidden objects as non‐autistic children. In a memory task, autistic children were slightly less confused about whether they had heard a word before, when words were closely related in meaning. We conclude that autistic children do not show difficulties with this type of prediction. Many theories have linked autism to cognitive impairments, including weak central coherence Frith, , impaireWhile varying in instantiation, these accounts all suggest that predictive processing is atypical in autism. Reports of reduced habituation or adaptation  > 1), although the strength of this evidence varied across conditions  and accumulation (BF01 = 3.99) tasks across occlusion durations.To determine whether there was sufficient evidence for the null hypothesis (of no group differences) relative to the alternative hypothesis of higher standard deviations in the autism group, we conducted one‐sided Bayesian independent samples P ≥ 0.45) nor interaction effects involving gender (P ≥ 0.06) when added as an additional factor in the ANOVA. Verbal IQ was negatively correlated with overall standard deviation in the position task , with higher IQ values linked with less variable RTs, r(58) = −.31, P = 0.02. We repeated the ANOVAs with verbal IQ as a covariate but found that this did not change the pattern of results. Additional exploratory analyses showed that overall standard deviation of RTs was not significantly correlated with age, SCQ or ADOS severity scores (P ≥ 0.08). Finally, we found no significant effects of group nor interactions between group and occlusion duration (P ≥ 0.53) in median RT error, but a significant effect of occlusion duration on all measures (P < 0.001).Further, exploratory analyses found neither significant main effects of gender , t(58) = 1.55, one‐sided P = 0.06, d = .40. A one‐sided Bayesian t‐test with a Cauchy prior width of .65 gave a BF01 of 0.73, meaning that the alternative hypothesis of group differences was 1.37 (1/0.73) times more likely than the null hypothesis, representing weak evidence [Jeffreys, Our second hypothesis was that autistic children would be less prone to false memories than typically developing children. A one‐sided independent samples t‐test on false memory d′ across all listed vs. non‐listed items. Again, the group difference was not significant , t(58) = 1.37, one‐sided P = 0.09, d = .35. The BF01 was 0.93, indicating little evidence for either the null or alternative hypothesis. Finally, Figure M = 0.51, SD = 0.26; autistic: M = 0.58, SD = .25), t(58) = 1.16, one‐sided P = 0.13, d = 0.30, corresponding to a BF01 of 1.20 suggesting weak evidence in favor of the null hypothesis.For a more general test of sensitivity, we also investigated P ≥ 0.42). We also tested whether gender differences were contributing to our results, finding no significant effect of gender or interactions between group and gender for any false memory measure (P ≥ 0.24). We next conducted exploratory analyses to investigate the effect of age, as it has been suggested that autistic and typically developing children may diverge in false memory susceptibility as they get older [Miller et al., P ≥ 0.16. Additionally, false memory measures were not significantly correlated with the SCQ or ADOS severity scores, P ≥ 0.28.Exploratory analyses confirmed that verbal IQ was not correlated significantly with performance  = −.29, P = 0.03, and correlated in the same direction, but not significantly, in the position task, r(56) = −.21, P = 0.11. The overall d′ measure was similarly correlated with standard deviation in both the accumulation, r(56) = −.38, P = 0.003, and position, r(56) = −.33, P = 0.01, tasks, but the proportion of lures rejected was not (accumulation: r(56) = −.18, P = 0.18; position: r(56) = −.09, P = 0.51).Our third and final hypothesis was that accurate discrimination would be associated with poor performance on the extrapolation tasks. We tested this with partial correlations between overall standard deviation of RT error  and false memory measures, controlling for age and performance IQ. Opposite to prediction, false memory We presented visual extrapolation and verbal false memory tasks to autistic and typically developing children and found no evidence for predictive impairments in the autistic children. Autistic children predicted the end‐point of occluded objects as well as typically developing children. The groups did not show clear differences in the false memory task, either, although there was weak evidence for autistic children being better at discriminating between presented and non‐presented items than typically developing children. Insofar as there were relationships between performance in the two tasks, they were opposite to our hypothesis, with children who were more sensitive in the false memory task showing less variable performance in the extrapolation tasks.We used visual extrapolation tasks to test Sinha et al. 's assertCould children succeed by using predictive eye movements in this task? These were shown to be unimpaired in a previous study of perception of moving targets in autistic individuals [Ego et al., Arguably, prediction abilities should not be considered as a single entity and instead theories should consider the underlying taxonomy of prediction abilities. Difficulties with prediction may become apparent when anticipating objects with more complex motion trajectories, or when making predictions between events that are more weakly associated, such as those involving social information [Balsters et al., d = 0.65 for a power of 80% and an alpha of .05 was 30 participants per group. Yet, most studies of false memory in autism have used far fewer than this. Moreover, the true effect size may be smaller than this [Anderson, Kelley, & Maxwell, d = 0.40), necessitating even larger samples. While the groups were not significantly different in our study, our Bayesian analyses showed that the data were insensitive to discriminate between the null and alternative hypotheses, with slightly more evidence in favor of the alternative hypothesis of group differences. Therefore, we do not rule out superior false memory performance in autistic individuals, but suggest that the effect may be relatively small. Moreover, while we used a recognition task, group differences may have become apparent in a less supported free recall task [Bowler, Matthews, & Gardiner, Our study also adds to the literature assessing false memory performance in autism. The false memory illusion is a robust effect in the general population [Zwaan et al., reduced performance in the visual extrapolation task would be related to increased sensitivity in the false memory task, but we found the opposite. This relationship could reflect domain‐general factors  or the involvement of a memory component. Future research with cross‐task comparisons will therefore be necessary to elucidate links between prediction abilities and contextual processing in autism.Our rationale for presenting these seemingly different tasks together was that theories proposing disordered prediction in autism are closely linked to theories proposing reduced use of context or ‘gist’. We predicted that In summary, we provide evidence of preserved prediction abilities in autistic children for dynamic objects, suggesting that prediction abilities are not generally impaired in autistic children. Future research will be needed to characterize the nature of predictive impairments in autism. Our results are compatible with superior false memory performance in autistic children, but larger samples will be needed to provide conclusive evidence. Our results therefore help to refine theories of both altered prediction and contextual processing in autism.Supporting InformationClick here for additional data file.


DDI) potential between the fixed‐dose combinations of ledipasvir/sofosbuvir 90/400 mg for hepatitis C virus and emtricitabine/rilpivirine/tenofovir alafenamide (TAF) 200/25/25 mg for HIV was evaluated in a randomized, open‐label, single‐center, multiple‐dose, 3‐way, 6‐sequence, crossover Phase 1 study in 42 healthy subjects. Emtricitabine/rilpivirine/TAF had no relevant effect on the pharmacokinetic parameters of maximum concentration [Cmax] and area under the concentration versus time curve over the dosing interval [AUCtau] for ledipasvir, sofosbuvir, and the metabolites GS‐566500 and GS‐331007. Ledipasvir/sofosbuvir had no effect on the Cmax and AUCtau for rilpivirine and emtricitabine. The Cmax and AUCtau of tenofovir, the major metabolite of TAF, were increased by 62% and 75%, respectively. However, the resulting absolute tenofovir exposures were markedly lower than the historical tenofovir exposures following tenofovir disoproxil fumarate (TDF) and, as such, were not considered to be clinically relevant. In contrast, additional adverse effect monitoring is recommended upon coadministration of ledipasvir and TDF due to elevated tenofovir exposures resulting from the DDI. This difference is explained by the fact that TAF 25 mg results in markedly lower (~90%) plasma tenofovir exposure compared to TDF 300 mg. Ledipasvir/sofosbuvir and emtricitabine/rilpivirine/TAF were generally well tolerated when administered alone or in combination. HIV/hepatitis C virus‐coinfected patients can coadminister ledipasvir/sofosbuvir and emtricitabine/rilpivirine/TAF without dosage adjustments.The drug–drug interaction ( AEadverse eventARTantiretroviral therapyARVantiretroviralAUCarea under the curveBCRPbreast cancer resistance proteinBLQbelow the limit of quantitationBMIbody mass indexCrClcreatinine clearanceCYPcytochrome P450DAAdirect‐acting antiviralsDDIdrug–drug interactionFDCfixed‐dose combinationGLSMgeometric least‐squares meanHCVhepatitis C virusHIVhuman immunodeficiency virusLDVledipasvirPKpharmacokineticRPVrilpivirineSOFsofosbuvirTAFtenofovir alafenamideTDFtenofovir disoproxil fumarateTFVtenofovirIn the United States, coinfection with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) is common, as approximately 25% of HIV‐infected persons are harboring HCV  with ledipasvir (LDV) has resulted in elevated tenofovir (TFV) exposure documented in pharmacokinetic (PK) studies and clinical trials , FTC, and TAF are available as a FDC R/F/TAF for the treatment of HIV‐1  as TDF and TAF are P‐gp substrates. Although FDCs have been shown to improve adherence in HIV or HCV treatment, they are limited by the inability to adjust doses of the individual components , and in general good health were enrolled into the study. Subjects were required to have a creatinine clearance (CrCl) ≥70 mL/min .HIV‐ and HCV‐negative male and non‐pregnant, non‐lactating female subjects between 18 and 45 years of age (inclusive), with a body mass index (BMI) between 19.0 and 30.0 kg/mAll screening laboratory evaluations  had to be within normal range, and subjects who had syncope, palpitations, or unexplained dizziness; who had an implanted defibrillator or pacemaker; or who had any serious or active medical or psychiatric illness were excluded. They were also excluded if they took any prescription medications or over‐the‐counter medications including herbal products within 28 days of commencing study drug dosing; exceptions were vitamins, acetaminophen, ibuprofen, and/or hormonal contraceptive medications. Additionally, subjects treated with systemic steroids, immunosuppressant therapies, or chemotherapeutic agents within 3 months of study screening were excluded. Subjects with current alcohol or substance abuse, judged by the investigator to potentially interfere with compliance or compromise safety, were excluded. Subjects were restricted, both before the first dose of study drug and through to discharge, from consuming alcohol‐containing products; using nicotine‐containing products; and consuming grapefruit juice, grapefruits, and Seville orange juice. Consuming caffeine and other methyl xanthines were prohibited on dosing days.Informed consent was obtained from each subject before initiation of study procedures. The study protocols and consent forms were reviewed and approved by a duly constituted institutional review board . The study was performed in accordance with the principles of Good Clinical Practice and the Declaration of Helsinki and was consistent with the requirements of the US Code of Federal Regulations Title 21, Part 312.This study was a randomized, open‐label, single‐center, multiple‐dose, 3‐way, 6‐sequence, crossover Phase 1 study in healthy adults under fed conditions . The treatments were LDV/SOF 90/400 mg (Treatment A), R/F/TAF 25/200/25 mg (Treatment B), and LDV/SOF+R/F/TAF (Treatment C). Subjects were randomized to 1 of 6 treatment sequences as described in Table Patients were administered the study drug within 5 minutes of completing the standardized breakfast. Serial blood samples for PK assessments were collected on the last day of each dosing sequence at the following time points: predose (≤5 min), 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 4.5, 5, 6, 8, 10, 12, 18, and 24 hours after the administration of the dose. Subjects were restricted from food intake until after the 4‐hour PK blood sampling time point.2H, 13C, and/or 15N) were used for each analyte. Response ratios of analyte:internal standard observed for calibration standards were subjected to linear regression with 1/2 weighting, and the concentration of an analyte in a sample was determined by interpolation of its response ratio in the standard curve equation. Concentrations below the calibrated ranges of the methods were reported as below the limit of quantitation (BLQ); samples with concentrations above the calibrated ranges of the methods were diluted with blank plasma prior to analysis and the appropriate dilution factor was applied to the result. For each method, the results of within‐run (intra‐assay) and between‐run (inter‐assay) precision assessments were reported as the coefficients of variation, each expressed as a percentage (%CV), and the results of accuracy assessments were reported as the relative error values expressed as percentages (%RE). Interference testing demonstrated that no analyte interfered in the quantification of any other analyte by any of the methods used for the study. The calibrated ranges of the method were 1–2000 ng/mL for LDV, 5–2500 ng/mL for SOF, 10–5000 ng/mL for GS‐566500 and GS‐331007, 1 to 500 ng/mL for RPV, 5 to 3000 ng/mL for FTC, 1 to 1000 ng/mL for TAF, and 0.3–300 ng/mL for TFV. For all eight analytes, all %CV values were <9.8% and all %RE values were within ±6.9% of 100%.Bioanalysis was conducted at QPS, LLC . Plasma concentrations of LDV, SOF, GS‐566500, GS‐331007, RPV, FTC, TAF, and TFV were determined by validated high‐performance liquid chromatography/tandem mass spectrometry (HPLC–MS/MS) methods with multiple reaction monitoring and electrospray ionization in the positive mode for LDV, RPV, FTC, TAF, and TFV, and in the negative mode for SOF, GS‐566500, and GS‐331007. Isotopically labeled internal standards  and concomitant medications, clinical laboratory analyses, vital sign measurements, and physical examinations. Treatment‐emergent AEs were defined as any event with an onset date of on or after the study drug start date and up to 30 days after the permanent discontinuation of study drug. Clinical and laboratory AEs were coded using the Medical Dictionary for Regulatory Activities (MedDRA) version 18. The severity of AEs was graded according to the Gilead Sciences, Inc. Grading Scale for Severity of Adverse Events and Laboratory Abnormalities (grades 1–4).The PK analysis sets included all randomized subjects who received at least 1 dose of study drug and had at least 1 plasma concentration data point for each analyte. Samples BLQ of bioanalytical assays occurring before the achievement of the first quantifiable concentration were assigned a value of zero to prevent overestimation of the initial area under the plasma concentration–time curve (AUC) and at all other time points were treated as missing data in WinNonlin. For summary statistics, samples BLQ at predose time were zero and postdose time points were assigned one‐half the value of the lower limit of quantitation.®, version 6.3; Certara USA, Inc., Princeton, NJ). The primary PK parameters were area under the plasma concentration versus time curve over the dosing interval (AUCtau), area under the plasma concentration versus time curve from zero to the last quantifiable concentration (AUClast), maximum observed plasma concentration (Cmax), and observed drug concentration at the end of the dosing interval (Ctau) depending on the analytes. The following PK parameters were also calculated: time of maximum observed plasma concentration (Tmax) and elimination half‐life of the drug in plasma (t1/2).Pharmacokinetic parameters were estimated using standard non‐compartmental methods in conjunction with the linear/log trapezoidal rule  for the geometric least‐squares mean (GLSM) ratios for the primary PK parameters of RPV were within 80% and 125% and of other analytes were within the boundaries of 70% and 143%.The primary endpoints were the PK parameters AUCAt least 36 evaluable subjects or 6 evaluable subjects per sequence were needed to achieve 90% CIs for the GLSM ratios of the test versus reference treatments within 80% and 125%, with regards to the primary PK parameters for RPV (85% chance if the estimated GLSM ratio was 100%), and within 70–143%, with regard to the primary PK parameters for FTC, TAF, TFV, SOF, GS‐566500, GS‐331007, and LDV (90% chance if the estimated GLSM ratio was 100%). SAS software  was used to perform the statistical summaries and analyses.® PROC MIXED; this model included treatment, sequence, and period as fixed effects and subject within sequence as a random effect. SAS® PROC MIXED  was used to conduct the treatment comparison analysis and the 90% CI calculations for the natural log‐transformed PK parameters. The safety analysis set included all randomized subjects who received at least 1 dose of study drug, and safety data were collected on the date of the first dose of study drug through 30 days after the last dose of study drug. Safety data including AEs, laboratory data, and vital signs were summarized by treatment, and the incidence of graded AEs and laboratory abnormalities was calculated.Subject demographic data and baseline characteristics were summarized by sequence using descriptive statistics. Plasma concentrations and PK parameters were summarized by treatment using descriptive statistics. For each analyte and PK parameter, a parametric mixed‐effects analysis of variance (ANOVA) model was fitted to the natural log‐transformed values of the PK parameter under evaluation using SAS2 (range: 22.8–29.9), and mean CrCl by Cockcroft–Gault method was 122.3 mL/min (standard deviation: 19.4). One subject discontinued due an AE of colitis.A total of 42 subjects were randomized and received at least 1 dose of study drug. The majority of subjects were male (71.4%) and white (61.9%). At baseline, the mean age was 34 (range: 18–45), mean BMI was 27.3 kg/mThe PK analysis sets included 42 subjects. Mean (SD) LDV, SOF, GS‐331007, RPV, FTC, TAF, and TFV plasma concentration–time profiles are presented in Figures tau 175% (169–181%), Cmax 162% (156–168%), and Ctau 185% (178–192%) outside the predefined boundaries.The coadministration of LDV/SOF and R/F/TAF did not noticeably increase the exposure of LDV, SOF, or GS‐331007; the 90% CIs for the GLSM ratios for the primary PK parameters were within the protocol predefined boundaries. LDV/SOF had no effect on RPV, FTC, or TAF; the 90% CIs for the GLSM ratios for the primary PK parameters were within the predefined boundaries. However, TFV exposure was increased, with the 90% CIs for the GLSM ratios for AUCLDV/SOF and R/F/TAF were generally well tolerated by the study subjects when administered alone or in combination. No Grades 3–4 AEs, serious AEs, or deaths were reported. One subject discontinued due to a Grade 2 nonserious AE of colitis, considered to be study drug related by the investigator. Commonly reported (in at least 2 subjects) treatment‐related AEs were only reported following treatment with LDV/SOF (Treatment A): nausea (5%) and vomiting (5%). The majority of the laboratory abnormalities were Grade 1 or 2 in severity. Three subjects had Grade 3 or 4 laboratory abnormalities, but no corresponding AEs were reported. The Grade 4 laboratory abnormality of increased creatine kinase was transient and consistent with physical exercise.Overall, LDV/SOF and R/F/TAF coadministration does not require dose adjustment since there is no clinically relevant interaction, despite elevations of TFV PK parameters outside of the protocol predefined lack of interaction boundaries. Notably, all the primary PK parameters of LDV, SOF, GS‐566500, GS‐331007, RPV, FTC, and TAF were within the protocol predefined lack of PK alteration boundaries.The AASLD and IDSA HCV guidance indicates that LDV/SOF can be used with most ARVs  and TDF (particularly with the PK boosters ritonavir and cobicistat) are concerns and may require ongoing renal monitoring (American Association for the Study of Liver Diseases, Coadministration of TDF with LDV/SOF has resulted in elevated TFV exposure documented in PK studies and clinical trials (German et al. When R/F/TDF is coadministered with LDV/SOF, TFV exposure was 4,780 ng·h/mL representing a 40% increase compared to R/F/TDF alone (German et al. The TFV PK data for R/F/TAF presented here and similar supportive data for the FDC E/C/F/TAF provide HIV health care providers with the option of switching TDF for TAF as a way to address the TDF drug interaction concern with LDV/SOF (Custodio et al. HIV/HCV‐coinfected patients on R/F/TAF can initiate LDV/SOF without concerns regarding potential drug interaction given the findings of this PK study. TAF has expanded the treatment options available to HIV/HCV‐coinfected patients and provides a safer option compared to TDF. Using a TAF‐based regimen, such as R/F/TAF, instead of a TDF‐based regimen in HIV/HCV‐coinfected patients will avoid the need for additional monitoring or modifying the ARV regimen to accommodate the HCV treatment.None declared.


LV) filling pressure (LVFP) and LV diastolic dysfunction. However, the evidence for it is not well summarized.Noninvasive echocardiographic tissue Doppler assessment (E/e′) in response to exercise or pharmacological intervention has been proposed as a useful parameter to assess left ventricular , patients with mixed/reduced LVEF, and patients with specific cardiac conditions. Overall, we found 28 studies with 9 studies for preserved LVEF, which was our primary interest. Studies had differing methodologies with limited data sets, which precluded quantitative meta‐analysis. We therefore descriptively summarized our findings. Only 2 small studies (N=12 and 10) directly or indirectly support use of E/e′ for assessing LVFP changes in preserved LVEF. In 7 other studies (cumulative N=429) of preserved LVEF, E/e′ was not useful for assessing LVFP changes. For mixed/reduced LVEF groups or specific cardiac conditions, results similar to preserved LVEF were found.Clinical studies that evaluated invasive LVFP changes in response to exercise or other interventions. We suggest that well‐designed prospective studies should be conducted for further evaluation.We find that there is insufficient evidence that E/e′ can reliably assess  Left ventricular diastolic dysfunction leading to heart failure with preserved ejection fraction (HFpEF) is a major clinical problem.Despite extensive use of E/e′, there continues to be ongoing debate about the usefulness of E/e′ in assessing LVFP.septal), lateral mitral annulus , and/or mean of septal and lateral values (E/e′mean) were selected.Original clinical studies that evaluated LVFP by using echocardiographic E/e′ and invasive techniques were screened from PubMed, Scopus, Embase, and Cochrane Library databases to September 2016 using a number of search strategies . SpecifiThe studies were included if they reported at least 1 of the following data sets: (1) E/e′ and LVFP values at baseline and after intervention; (2) changes in E/e′ and LVFP values because of intervention; (3) assessment of correlation between E/e′ and LVFP postintervention, alone, or combined with baseline; (4) assessment of correlation between changes in E/e′ and LVFP with intervention; and (5) the diagnostic accuracy of either postintervention E/e′ values or postintervention changes in E/e′ to predict elevated LVFP or LVFP changes.Included studies were grouped and analyzed based on patient cohorts. The first group was for studies that included patients with LVEF ≥50%, including HFpEF patients, but without a substantial number of moderate‐to‐severe valvular heart disease, hypertrophic or restrictive cardiomyopathy, congenital heart disease, acute coronary syndromes, septic shock, cardiac transplant, and atrial fibrillation. This group was our primary interest. Other groups were for studies that included patients with reduced/mixed LVEF, and for studies that included patients grouped with specific cardiac conditions . Overall, we found 28 studies: 9 studiesseptal >15 predicts PCWP ≥20 mm Hg with sensitivity of 83% and specificity of 100%. Maeder et alseptal with exercise and no correlation between poststress E/e′septal and PCWP. In the largest exercise study in patients with exertional dyspnea (N=181), Choi et alseptal despite a significant elevation of LV end diastolic pressure with passive and active leg raise.Table lateral in response to decreased LVFP for 10 HFpEF patients. However, this study did not provide any individual data for further analysis. Interestingly, in another publication from the same groupIn another set of studies, authors performed stress echocardiography using differing pharmacological interventionsIn 2 other studies, participants underwent preload changes leading to lower LVFP caused by low body negative pressure and increase in LVFP by saline infusion.r=0.59) between E/e′septal and LV mean diastolic pressure during single‐leg supine exercise. They reported high AUC value (0.89) for exercise E/e′septal to predict an elevation of LV mean diastolic pressure >15 mm Hg.septal >13 had sensitivity of ≈70% and specificity of ≈95% for estimating elevated LV mean diastolic pressure >15 mm Hg.lateral increased with preload stress. However, on detailed analysis, E/e′lateral increase was observed in only a small subset of patients (N=6). No correlation of E/e′ and LVFP or diagnostic value of E/e′lateral was reported.Table In 4 studies, investigators used different pharmacological agents to decrease LVFP and measured corresponding changes in E/e′ Table .30, 36, mean, E/e′lateral, or E/e′septal to predict the elevated mean left atrial pressure (≥15 mm Hg). In their study, E/e′lateral and E/e′mean had no correlation and E/e′septal had only modest correlation (r=0.46) with mean left atrial pressure on serial measurements.In 2 studies, the investigators performed serial measurements using implanted hemodynamic measurement devices  .septal increased whereas PCWP decreased after surgery.lateral.mean in 9 patients with differing LVEF in the intensive care unit following 2 days of treatment with diuretics and/or inotropes.mean and PCWP.In patients with severe aortic stenosis (N=28)The major findings of our study are that there is lack of robust clinical evidence to support the use of E/e′ in response to physiological and/or pharmacological intervention to estimate LVFP changes and LV diastolic dysfunction. Furthermore, most of the studies are single center with limited sample size with nonuniform study methodologies and data reporting that does not allow for quantitative meta‐analysis of the studies.Invasive LVFP measurements (primarily LV end diastolic pressure or PCWP as its surrogate) in response to altered physiological conditions provide incremental information about the LV function and stiffness.For evaluating the relationship of change in E/e′ to changes in LVFP in response to exercise or physiological/pharmacological intervention, we find that there are only 2 studies with limited sample size that directlyA number of guidelines/tools such as STARDIn summary, our review indicates that there is inadequate evidence for using E/e′ for estimating LVFP changes in response to exercise/other physiological interventions. Well‐designed prospective multicenter studies are required for evaluation and validation before recommending it for clinical and research purposes.The study was supported by a National Institutes of Health National Heart, Lung, and Blood Institute grant R01‐HL104018. The funding organizations did not have any role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.None.Table S1. Data Sources and Search StrategyTable S2. Full‐Text Studies Excluded After Evaluation (No Data of Interest)Table S3. Detailed Summary of Studies With Subjects With LVEF ≥50%Table S4. Detailed Summary of Studies With Subjects With Mixed or Reduced LVEFTable S5. Detailed Summary of Studies With Subjects With Specific Cardiac ConditionsClick here for additional data file.


The prepared water‐soluble DC‐CNDs@PSMA can be successfully applied to in vitro and in vivo dual‐color bioimaging and optogenetics. In vivo optical imaging can visualize the biodistribution of intravenously injected DC‐CNDs@PSMA. In addition, the light‐triggered activation of ion channel, channelrhodopsin‐2, for optogenetic applications is demonstrated. As a new type of fluorophore, DC‐CNDs offer a big insight into the design of charge‐transfer complexes for various optical and biomedical applications.The development of intrinsically multicolor‐emitting carbon nanodots (CNDs) has been one of the great challenges for their various fields of applications. Here, the controlled electronic structure engineering of CNDs is performed to emit two distinct colors via the facile surface modification with 4‐octyloxyaniline. The so‐called dual‐color‐emitting CNDs (DC‐CNDs) can be stably encapsulated within poly(styrene‐ Carbon nanodots (CNDs) have been widely investigated as new multifunctional nanoparticles for various applications ranging from optoelectronic devices to theranostic agents.Recently, multicolor PL nanomaterials have become greatly important in cell counting, cell sorting, diagnostics, and other therapeutic applications.Figureco‐maleic anhydride) (PSMA), enabling biomedical applications to in vitro and in vivo dual‐color bioimaging and light‐triggered activation of ion channel for optogenetic applications  by controlling the electronic structure of CNDs through a facile surface modification with 4‐octyloxyaniline Figure1a. DC‐Figure13C nuclear magnetic resonance (NMR) analyses. The deconvoluted C1s XPS spectra indicated the presence of C—C/C=C (284.5 eV), C—N (285.9 eV), and C=O (288 eV) bondings on the surface of DC‐CNDs, likely due to oxidative carbonization . In Figure S3a (Supporting Information), pristine CNDs showed chemical shifts of 14.1, 22.7, 29.6, 31.9, 32.6  and 129.7, 129.9 ppm  for the oleyl group, and 33.8 ppm for amido carbon, indicating that oleylamine was passivated on the surface of CNDs. After modification with 4‐octyloxyaniline, DC‐CNDs showed peaks at around 68.3, 132.3, and 158.6 ppm for methoxy, anilino, and phenoxy groups, together with the oleyl peaks, respectively . The results revealed the presence of partially substituted 4‐octyloxyanilines on the surface of DC‐CNDs. The 1H NMR spectrum of DC‐CNDs@PSMA in Figure S3c (Supporting Information) showed the chemical shifts at 1.2, 1.8 , 2.2 (arene‐CH), 3.1 (HO—CH), 3.3 (O=C—NH), and 7.1 ppm (arene‐H), indicating the presence of PSMA on the surface of DC‐CNDs. The surface functionalities of DC‐CNDs have been further identified by the Fourier‐transform infrared spectroscopy . Both pristine CNDs and DC‐CNDs exhibited the C=O stretching (1690 cm−1) and N—H bending (1500 cm−1) peaks, confirming the presence of carbonyl and amide (from oleylamine) groups on the surface of CNDs. After surface modification, the N—H stretching (3500–3300 cm−1) and C—O stretching (1260 cm−1) peaks were developed and the N—H bending and C=C stretching (1600–1450 cm−1) bands were intensified due to the chemical bonding of 4‐octyloxyaniline to the surface of CNDs.The pristine CNDs were prepared by the solvothermal carbonization of citric acid. C—C/C=C 84.5 eV, The electronic structure and optical property of DC‐CNDs were investigated by UV–vis absorption spectroscopy and PL spectroscopy. Figure A > 0) on all timescales. The photoexcited electrons from intrinsic states were consumed to other accessible states, but were not allowed to access extrinsic states, indicating that the intrinsic states were localized in the polyaromatic domains and isolated from the 4‐octyloxyaniline‐modified polyaromatic domains. By the excitation at 540 nm, ∆A values showed strong stimulated emission (∆A < 0) even after 1 ns time delay. The photoexcited electrons from extrinsic states might be directly related to the red emission (580 nm). The TA spectra were fitted using biexponential functions . The relatively long decay time by the excitation at 540 nm might be attributed to the molecule‐like, defectless characteristics of extrinsic states.In order to explore the energy structure of DC‐CNDs, we conducted time‐resolved absorption (TA) spectroscopy and time‐resolved PL (TRPL) spectroscopy. TA spectra were recorded to show the electronic transition in intrinsic and extrinsic states at the probe wavelength of 580 nm under the excitation wavelengths of 350 and 540 nm, respectively. Each of them corresponds to the transition energy of intrinsic and extrinsic states . By the excitation at 350 nm, DC‐CNDs exhibited excited state absorption . The TRPL spectra were well fitted to single exponential functions, reflecting that each emission state had one dominant radiative recombination pathway. The PL lifetime of extrinsic emission (3.1 ns) was longer than that of intrinsic emission (1.9 ns) in consistent with the TA decay time data. Furthermore, the PL lifetime of intrinsic emission of DC‐CNDs 2.0 ns) was analogous to that of the pristine CNDs (2.4 ns) , reflecting that there was no relaxation pathway of photoexcited electrons from intrinsic energy states to extrinsic energy states. From TA and TRPL spectra analysis, the electronic structure of DC‐CNDs was schematically represented in Figure .0 ns was−1. DC‐CNDs showed high cytocompatibility with a cell viability over 88% even at the high concentration of 2 mg mL−1. As a control group, we used commercially available CdSe/ZnS quantum dots coated with a polymer bearing carboxyl groups, which showed high toxicity at the same concentrations. The low cytotoxicity of DC‐CNDs@PSMA was also confirmed in several different kinds of normal cells like embryonic kidney cells (HEK293) as well as cancer cells like hepatocellular carcinoma cells (HepG2) and skin melanoma cells (B16F10) .For further biomedical applications, the cytotoxicity of DC‐CNDs was evaluated in the normal hepatocyte of FL83B cells Figure i. The he−1 for 1 h, DC‐CNDs@PSMA appeared to be internalized into the cells, showing the bright and robust fluorescence emission . Figure Inspired by two different excitation and emission centers Figure , we carrFigureFinally, we assessed in vivo acute toxicity of DC‐CNDs using the collected blood samples and the dissected major organs 48 h after intravenous injection of DC‐CNDs@PSMA. According to the blood biochemistry study, the important hepatic indicators of total protein, ALB, total bilirubin, alkaline phosphate did not show the sign of liver injury as shown in In conclusion, water‐soluble DC‐CNDs with two distinct excitation and emission centers have been successfully developed via the facile surface modification with 4‐octyloxyaniline. The two emission centers showed no spatial overlap and the quantum yield of the red emission center showed the highest value among red‐emissive CNDs. The DC‐CNDs showed the minimal cytotoxicity and demonstrated the great potential as a bioimaging agent for multicolor bioimaging, two‐photon microscopy, and in vivo optical imaging. Moreover, DC‐CNDs could be successfully exploited for optogenetic applications via the wavelength‐dependent activation of ion channels. Taken together, we could confirm the feasibility of DC‐CNDs with the unique characteristics of multicolor‐emitting, facile and robust preparation, and biocompatibility for various biomedical applications.The authors declare no conflict of interest.SupplementaryClick here for additional data file.


Background: People with Parkinson's disease (PD) who develop visuo‐perceptual deficits are at higher risk of dementia, but we lack tests that detect subtle visuo‐perceptual deficits and can be performed by untrained personnel. Hallucinations are associated with cognitive impairment and typically involve perception of complex objects. Changes in object perception may therefore be a sensitive marker of visuo‐perceptual deficits in PD.Objective: We developed an online platform to test visuo‐perceptual function. We hypothesised that (1) visuo‐perceptual deficits in PD could be detected using online tests, (2) object perception would be preferentially affected, and (3) these deficits would be caused by changes in perception rather than response bias.Methods: We assessed 91 people with PD and 275 controls. Performance was compared using classical frequentist statistics. We then fitted a hierarchical Bayesian signal detection theory model to a subset of tasks.Results: People with PD were worse than controls at object recognition, showing no deficits in other visuo‐perceptual tests. Specifically, they were worse at identifying skewed images (P < .0001); at detecting hidden objects (P = .0039); at identifying objects in peripheral vision (P < .0001); and at detecting biological motion (P = .0065). In contrast, people with PD were not worse at mental rotation or subjective size perception. Using signal detection modelling, we found this effect was driven by change in perceptual sensitivity rather than response bias.Conclusions: Online tests can detect visuo‐perceptual deficits in people with PD, with object recognition particularly affected. Ultimately, visuo‐perceptual tests may be developed to identify at‐risk patients for clinical trials to slow PD dementia. © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society. Dementia affects up to 50% of people with Parkinson's disease (PD) within 10 years of diagnosisVisual hallucinations affect 40% to 50% of people with PDd'), and bias to report signal presence, described as criterion, c.Deficits in visuo‐perceptual processing could potentially be caused by bottom‐up sensory deficits, or top‐down factors, such as response bias, a tendency to report signals as present or absent. The framework of signal detection theory allows us to test which is most affected in PD. The theory proposes that perceptual performance is determined by sensitivity of a system to signal relative to background noise, known as d‐prime (We therefore developed an online test of visuo‐perception in PD. We hypothesised that (1) visuo‐perceptual deficits in PD could be detected using online tests, (2) object perception tasks would be preferentially affected, and (3) differences in performance would be caused by changes in perception rather than response bias.Participants accessed the study in 2 ways: a local group accessed the website at our study center, and a web‐based group accessed the website independently Fig. A. Patienhttp://www.sussex-vision.co.uk). Cognition was assessed using the Mini‐Mental State Examination and Montreal Cognitive Assessment.The severity of symptoms was assessed using the MDS‐UPDRS. Visual acuity was measured using a 6‐m chart and converted to decimal acuity. Contrast sensitivity was measured using a Pelli‐Robson chart  using a Dell latitude 3340 laptop )  or their own devices. Participants completed a clinical questionnaire on the website, providing demographic information and disease status and indicating the presence or absence of visual hallucinations  encouraged central fixation. Short presentation times prevented eye movement confounds.1This probes ability to recognise objects at extreme angles and may be linked with infero‐temporal cortex activity.https://uk.mathworks.com/).http://www.kaggle.com) were converted to grayscale and Fourier transformed. The phase matrix of each image was combined with white noise and the average magnitude matrix of the whole stimulus set using an inverse Fourier transform.Stimuli were prepared using MATLAB 2014a . Participants scoring ≤ 50% at lowest difficulty level were excluded .1A central circle surrounded by smaller circles appears larger than an identically sized circle surrounded by larger circles.The stimulus was adapted from the classic illusion to maximise comparisons with fewer trials. A central small circle , was surrounded by 4 large inducers . This was surrounded by 8 peripheral “test” circles, with varying diameter, each surrounded by 12 small inducers . The ratio of test circle to target circle indicated the magnitude of illusion for that participant. Geometric mean ratio of selected circles was calculated and transformed back into log space.2This is the ability to match rotated objects and may involve mid‐level visual processing regions such as V3.Stimuli consisted of a reference 5 × 5 grid, above 3 test grids. Within the reference grid were 4 blue circles (pseudorandom positions). Test grids were rotated ±90° or ± 180°. In 2 test grids, a blue circle was moved to an adjacent position. Participants selected the grid that was identical to the reference grid, but rotated .A Taylor's‐E optotype was presented, surrounded on 4 sides by outward‐pointing triangles . The participants clicked the triangle beside the open side of the “E.” The size of the next optotype was determined using an adaptive staircase converging onto the participant's acuity .An online tapping test . We accounted for confounding influences of age by adjusting for age and separately examining differences between groups when the oldest PD patients were removed until the groups were age matched.Performance was compared between groups using 2‐tailed Welch's P < .05, Bonferroni corrected for multiple comparisons , was accepted as the threshold of statistical significance. Statistical analysis was performed using R (https://www.r-project.org/).We used linear regression to examine effects of factors on task performance. We then accounted for differences in age and gender by including these variables as covariates in additional regression models for effect of PD on task performance. Where tasks included more than 1 difficulty level, the most discriminatory level was used, defined as greatest effect size, followed by highest significance level. To assess whether perceptual deficits in PD are reflected either by changes in perception and/or a bias in decisions and reporting, we used the signal detection theory framework. Robust estimation of parameters of the signal detection model typically requires large numbers of trials per participant, which would not be feasible using an online testing system. Hierarchical models allow robust estimates of group‐level parameters in cases with large samples, but few trials per participant, as here. Unlike fixed‐effects models that collapse data across participants, a hierarchical Bayesian approach is the correct way to combine information about within‐ and between‐subject uncertainty, such that group‐level parameters are less influenced by single‐subject fits that have high degree of uncertainty. In turn, hierarchical modelling mutually constrains subject‐level parameter estimation because of the shared dependence on group‐level parameters.d') and criterion (c) were calculated separately:z indicates inverse of the cumulative normal distribution, H hit rate, and FA false alarm rate. We additionally fitted a hierarchical Bayesian signal detection modeld') and bias (c) parameters. We used Markov Chain Monte Carlo using Gibbs sampling implemented in JAGS (Just Another Gibbs Sampler) in Rλ):We fit the hierarchical Bayesian signal detection theory model to tasks that employed a 2‐alternative‐forced‐choice design. Responses were sorted into hits, false alarms, misses, and correct rejections. For each participant, detection sensitivity , where high probability indicates strong evidence in favor of a difference. We denote these probabilities as Pθ to distinguish them from classic P values.JAGS was called with 2000 adaptation steps, 5000 burn‐in samples, and 50,000 effective samples; 3 chains for each parameter were run. Convergence of chains was assessed visually and using the potential scale‐reduction statistic A total of 428 participants accessed the website across the 2 groups. We excluded 62 participants based on exclusion criteria of age, screen width, and atypical PD Fig. A. The reF1,327 = 24.8, P < .0001; main effect difficulty: F2,654 = 433, P < .0001). The interaction between presence of PD and difficulty tended to significance at a Bonferroni‐corrected level: F2,654 = 4.1, P = .017 . Hidden figures performance was also worse with age, but the effects of PD were no longer significant when adjusted for age. There was no effect of age on biological motion, subjective size perception, or mental rotation.Object invariance and peripheral object recognition was worse in the men than in the women, especially in people with PD. When we adjusted for gender, we continued to see a strong effect of PD.In biological motion, female controls tended to perform worse than male controls, an effect not seen in PD. When we examined the overall relationship between gender and performance, no significant relationship was seen. Performance in hidden figures tended to be worse in men (with and without PD), and the effect of PD was no longer significant when adjusted for gender. The men also showed a slightly lower effect for subjective size perception. There was no effect of gender on mental rotation .We found no relationship between visuo‐perceptual performance and disease duration or presence of hallucinations .P < .0001), showing differences were not solely caused by acuity deficits.There was no relationship between performance in visuo‐perceptual tasks and visual acuity, with the exception of object invariance, which showed a trend (that disappeared when adjusted for age) and peripheral object recognition . Effects of PD on peripheral object recognition persisted when adjusted for acuity , indicating that worse performance was not driven by higher number of missed trials.R2 = 0.017, P = .013), and this was not significant when adjusted for age (estimate = 0.049 ± 0.030), t = 1.7, P = .098).The presence of PD trended to correlation with visual acuity  and bias (c) separately for controls and PD patients. Trials with no responses were excluded from this analysis . This approach revealed significant effects of PD on perceptual sensitivity (d') in all 3 tasks. Conversely, there were no group differences in detection criterion, a measure of general bias in reporting presence or absence of signal, regardless of sensory input  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.R.S.W.: 1A, 1B, 1C, 2A, 2B, 3AD.S.S.: 1A, 3BB.B.: 1AS.M.F.: 2A, 3BB.M.J.: 1CT.J.C.G.: 1CA.P.S.: 1BL.E.M.: 1BK.P.: 1CI.P.: 1CR.N.S.: 1CA.J.N.: 3BS.J.C.: 1B, 3BA.O.K.: 2A, 2B, 2CA.E.S.: 2C, 3BH.R.M.: 1A, 3BRSW: No disclosures. DSS: No disclosures. BB: No disclosures. SMF: No disclosures. BMJ: No disclosures. TJCG: No disclosures. APS: No disclosures. LEM: No disclosures. KP: No disclosures. IP: No disclosures. RNS: No disclosures. AJN: Reports support from Prothena pharmaceuticals. SJC: No disclosures. AOK: No disclosures. AES: Reports personal fees from Medtronic and AstraZeneca. HRM: Reports personal fees from Teva, AbbVie, Boehringer Ingelheim, and GlaxoSmithKline.Additional Supporting Information may be found in the online version of this article at the publisher's website.Supplementary InformationClick here for additional data file.Supplementary InformationClick here for additional data file.


SBP) in obese adults without diabetes.To explore the effects of dual therapy with dapagliflozin and exenatide on body weight, body composition, glycaemic variables and systolic blood pressure (2) to oral dapagliflozin 10 mg once daily plus subcutaneous long‐acting exenatide 2 mg once weekly or placebo. MRI was used to assess change in body composition. Participants were instructed to follow a balanced diet and exercise moderately.In this single‐centre, double‐blind trial, we randomized 50 obese adults without diabetes , which was mostly attributable to adipose tissue reduction without lean tissue change; 36.0% versus 4.2% of participants achieved ≥5% body weight loss, respectively; and prediabetes was less frequent with active treatment . The difference in SBP change for dapagliflozin/exenatide versus placebo was −6.7 mm Hg. As expected, nausea and injection‐site reactions were more frequent with dapagliflozin/exenatide than with placebo. Only two and three participants, respectively, discontinued because of adverse events.Of 25 dapagliflozin/exenatide‐ and 25 placebo‐treated participants, 23 (92.0%) and 20 (80.0%) completed 24 weeks of treatment, respectively. At baseline, the mean participant age was 52 years, 61% were female, the mean body weight was 104.6 kg, and 73.5% of participants had prediabetes (impaired fasting glucose or impaired glucose tolerance). After 24 weeks, for dapagliflozin/exenatide versus placebo: the difference in body weight change was −4.13 kg (95% confidence interval −6.44, −1.81; SBP over 24 weeks and was well tolerated in obese adults without diabetes.Compared with placebo, dapagliflozin/exenatide dual therapy reduced body weight, frequency of prediabetes and  In patients with T2D, mean body weight reduction with exenatide 2 mg once weekly versus placebo, estimated from a mixed treatment comparison meta‐analysis, is about −1.6 kg over 24 weeks,Combination therapy with agents acting via different mechanisms may be the most effective pharmacological approach to treating obesity and addressing evolutionary counter‐regulatory mechanisms that maintain body weight.The present proof‐of‐concept study examined body weight loss during dual therapy with oral dapagliflozin 10 mg once daily and subcutaneous exenatide 2 mg once weekly in obese participants without diabetes. Body composition, glycaemic control, and blood pressure were also examined. To the best of our knowledge, this is the first report of a randomized controlled trial evaluating initiation of dual therapy with an SGLT2 inhibitor and a GLP1‐RA.22.1ClinicalTrials.gov identifier: NCT02313220). The study was conducted at a single centre in Sweden from December 2014 until August 2015. An optional 28‐week, open‐label extension followed, to be reported separately. Obese men and women aged 18 to 70 years, without diabetes and with a BMI of 30 to 45 kg/m2, were eligible for enrolment .In this collaborative, investigator‐sponsored, 24‐week, single‐centre, randomized, parallel‐group, double‐blind, placebo‐controlled phase IIa study Figure A, we evaAll participants provided written informed consent before enrolment. The protocol was approved by the Independent Regional Ethics Committee in Uppsala, Sweden, and by the Swedish Medical Products Agency. The study was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonisation/Good Clinical Practice guidelines.Participants were enrolled at a screening visit 1 to 2 weeks before randomization at week 0. Thereafter, follow‐up visits occurred at weeks 4, 8, 12 and 24.2.2Eligible participants were randomized 1 : 1 to receive active treatment or matching placebo using computer codes independently generated by PCG Clinical Services AB  and sequentially assigned by the study investigator. Product identity was concealed using sequentially numbered containers. Random assignment to study treatment was stratified by sex and predefined to generate a study population comprising 60% women and 40% men so as to avoid the >80% female enrolment rate commonly observed in obesity trials.2.3All participants received training in self‐administration of a subcutaneously injected placebo microsphere preparation (no active ingredient). At randomization, participants assigned to active treatment (dapagliflozin/exenatide) received dapagliflozin 10 mg once daily taken orally each morning and exenatide 2 mg once weekly extended‐release formulation administered subcutaneously on the same day each week, at any time of day  without changing dosages and to take no new prescription or non‐prescription medications unless advised by a physician. Participants were requested to report changes in medication or dosages to study investigators.At each study visit, participants were given a standard leaflet and instructed to follow a balanced diet as per national guidelines. Participants were also advised to exercise moderately ; however, diet and exercise modification was not mandated or documented. To evaluate pharmacological effects, major lifestyle changes were not an aim of the study.2.42.4.1The primary efficacy endpoint was change in body weight (kg) from baseline (day of randomization) to 24 weeks with dapagliflozin/exenatide versus placebo. Body weight was measured using the same electronic scale (TANITA) at baseline, 4, 8, 12 and 24 weeks, with participants in light indoor clothing without shoes. The secondary efficacy endpoint was the percentage of body weight change from baseline to 24 weeks.Exploratory efficacy endpoints measured at baseline and 24 weeks included proportions of participants with ≥5% and ≥10% body weight reduction, body composition measures, glycaemic measures, SBP and waist/hip circumference.At weeks 0 and 24 MRI was conducted (File S1) to assess changes in volumes of abdominal visceral adipose tissue, abdominal subcutaneous adipose tissue, total adipose tissue, total lean tissue and liver fat content.Glycaemic measures included changes from baseline in glycated haemoglobin (HbA1c) and fasting plasma glucose (FPG), derived from blood samples taken between 07:30 and 10:30 hours after an overnight fast. A standard oral glucose tolerance test (OGTT), conducted at screening and at 24 weeks (File S1), assessed 2‐hour plasma glucose, and proportions with impaired fasting glucose , impaired glucose tolerance , and prediabetes (any IFG or IGT). A dapagliflozin/placebo tablet was taken orally 30 minutes before the 24‐week OGTT.Urine dipstick tests were performed at baseline and showed no overt glucosuria. No further urinary glucose testing was undertaken until 3‐hour urine collection during the 24‐week OGTT; 24‐week urinary glucose was analysed after study completion to maintain blinding until database lock.Cardiovascular measures included change from baseline in seated SBP, diastolic blood pressure (DBP), which were both measured as the average of two readings and rounded to the nearest 1 mm Hg, and heart rate.2.4.2Safety and tolerability were assessed over 24 weeks by evaluating the changes from baseline in estimated glomerular filtration rate ,All blood and urine analyses were performed using standard assays at the Clinical Chemistry Laboratory of the Uppsala University Hospital.2.5The primary, secondary and exploratory efficacy endpoints were analysed in the full analysis set (FAS), defined as participants who received ≥1 dose of study medication during the 24‐week double‐blind treatment period, who had a baseline value and ≥1 post‐baseline value for ≥1 efficacy measure during the double‐blind period, and who were not found to have serious protocol non‐compliance. The analysis of safety used the safety analysis set, defined as all randomized participants who received ≥1 dose of study medication.For primary and secondary endpoints, least‐squares (LS) mean changes from baseline to 24 weeks and associated 95% confidence intervals (CIs) and p values for dapagliflozin/exenatide versus placebo differences were derived from a mixed model for repeated measures, with treatment, week, treatment‐by‐week interaction and sex as categorical fixed covariates, and baseline value as a continuous fixed covariate. An unstructured matrix for the within‐participant error variance‐covariance was used. A similar approach was used for changes in HbA1c, blood pressure, heart rate, eGFR and serum lipids. For MRI, FPG and fasting ketones, similar model terms were employed, but analysis of covariance was used. The significance of changes in proportions from baseline to 24 weeks for IFG and IGT was evaluated with paired McNemar tests for within‐treatment group changes and a Cochran–Mantel–Haenszel test, adjusted for treatment, for between‐treatment group changes.For sample‐size calculations, see File S1. All statistical analyses were performed using SAS version 9.4 . CIs and p values were unadjusted for multiple comparisons.33.1Of 25 participants randomized to receive dapagliflozin/exenatide and 25 randomized to receive placebo, 23 (92.0%) and 20 (80.0%) completed 24 weeks of double‐blind treatment, respectively Figure B. Two daGlycaemic variables, vital signs and renal function were balanced across treatment groups at baseline; however, mean age, body weight, duration of obesity, waist circumference and body fat measures were numerically higher at baseline in the dapagliflozin/exenatide group than in the placebo group Table . Overall3.23.2.1Dapagliflozin/exenatide reduced body weight significantly more than placebo Figure , with a 3.2.2Dapagliflozin/exenatide reduced visceral, subcutaneous and total adipose tissue volume, but not lean tissue, significantly more than placebo. Total adipose tissue reduction versus placebo was −4.09 L after 24 weeks , and with IGT was significantly reduced from 48.0% to 17.4% (p = .020). Combined, the proportion of participants with prediabetes (any IFG or IGT) significantly decreased from 68.0% to 34.8% (p = .005). In contrast, the corresponding proportions among placebo‐treated participants did not change significantly from baseline to 24 weeks  than with placebo (0.3 mmol/3 h).3.2.4In this predominantly normotensive population (mean baseline SBP of 134 mm Hg), dapagliflozin/exenatide reduced SBP significantly more than placebo Figure H, with a3.3No meaningful differences between dapagliflozin/exenatide and placebo were observed for eGFR, fasting ketones or serum lipids Table , or otheAll participants reported at least one AE Table . SeriousUrinary tract infections or genital infections were reported in three participants receiving dapagliflozin/exenatide and one participant receiving placebo; no participants reported AEs potentially related to renal impairment/failure Table .Gastrointestinal and injection‐site‐related AEs were more common with dapagliflozin/exenatide than placebo. Gastrointestinal AEs occurred in 16 (64.0%) and 10 (40.0%) of dapagliflozin/exenatide‐ and placebo‐treated participants, respectively; nausea was the most frequent, occurring in seven (28.0%) and three (12.0%) participants, respectively. Injection‐site‐related AEs occurred in 11 (44.0%) and eight 32.0%) of dapagliflozin/exenatide‐ and placebo‐treated participants, respectively  received the SGLT2 inhibitor canagliflozin or placebo.Ongoing clinical trials investigating the SGLT2 inhibitor/GLP‐1RA combination initiated together in patients with T2D will provide important information on body weight loss in T2D. These include DURATION‐8 (NCT02229396), evaluating exenatide 2 mg once weekly and dapagliflozin 10 mg once daily, administered as single agents and as dual therapy in patients withT2D with inadequate glycaemic control on metformin, and AWARD‐10 (NCT02597049), evaluating empagliflozin added sequentially to dulaglutide.Identification of the type of tissue lost during treatment with dapagliflozin/exenatide is important because lean tissue loss would be less beneficial to obese adults than adipose tissue loss, in particular visceral or ectopic fat deposits. In the present study, compared with placebo, dapagliflozin/exenatide treatment significantly reduced subcutaneous and visceral abdominal adipose tissue (but not lean tissue). An earlier study indicated that the body weight lost with dapagliflozin 10 mg once daily added to metformin in patients with T2D was two‐thirds fat mass and one‐third lean mass.Hypertension is a frequent comorbidity of obesity and diabetes and an important part of metabolic syndrome. Improving blood pressure control is a common clinical goal for obese patients. Individually, dapagliflozin 10 mg once daily has been associated with SBP reductions ranging from −2.6 to −3.6 mm HgThis study was too small to identify uncommon AEs. Nevertheless, no new safety findings, and no marked increase in discontinuations or expected AEs, were observed with SGLT2 inhibitor/GLP‐1RA dual therapy. Urinary tract infections , genital infections , and renal AEs (0%) were infrequent with dapagliflozin/exenatide but, as anticipated, nausea, injection‐site mass, and injection‐site pruritus appeared more frequent with dapagliflozin/exenatide  than with placebo .Use of a high‐dose GLP‐1RA has been suggested as an alternative to combination pharmacotherapy for body weight loss, but the incidence of AEs and need for additional injections may limit its use. Liraglutide has been approved as a body weight loss therapy at a higher dose (3 mg once daily) than used to treat T2D (1.8 mg once daily). In a placebo‐controlled study in obese participants without diabetes receiving liraglutide 3 mg once daily, body weight loss and relative reductions in prediabetes prevalence with liraglutide appeared to be similar to those observed in the present study, but nausea occurred more frequently.The present study has some limitations. It was a small, short‐term, single‐centre, proof‐of‐concept study with no monotherapy comparator groups, which are needed to formally evaluate whether effects with dapagliflozin/exenatide on body weight loss, glycaemic control and SBP are additive. Body weight responses to dapagliflozin/exenatide varied between individuals and further research is required to identify behaviours or biomarkers predicting response to dapagliflozin/exenatide. Moreover, different results might be observed in patients undergoing intensive lifestyle intervention. The study was conducted in participants without diabetes, and results cannot be directly extrapolated to patients with T2D.In summary, this proof‐of‐concept study shows that dapagliflozin/exenatide reduces body weight, prediabetes occurrence and SBP compared with placebo and is well tolerated in an obese population without diabetes. These findings, together with the convenience of once‐daily oral dosing and once‐weekly injection, support further investigation to assess dapagliflozin/exenatide for body weight loss and prevention of diabetes in obese individuals.File S1. Inclusion/Exclusion Criteria; MRI methods; OGTT methods; sample size calculations; Table S1 (other laboratory parameters).Click here for additional data file.