NO) transmission and preventing inflammatory reaction. Although previous study showed the myricitrin possesses antibone loss effects via reducing the expression of IL‐6 and partially suppressing reactive oxygen species (ROS) production. However, the effects of myricitrin on nuclear factor‐kappaB ligand (RANKL)‐stimulated osteoclastogenesis have not yet been further investigated. The current study was aimed to demonstrating the inhibitory effects of myricitrin on RANKL‐stimulated osteoclastogenesis and relevant mechanisms. We found myricitrin significantly suppressed osteoclastogenesis suggesting that it may acts on RANKL/RANK induced downstream signal cross cascading in osteoclast precursors. In that, our Western blotting results showed myricitrin significantly attenuated RNAKL/MAPKs  and AKT signal cascading. Complementing previous study, our results suggesting as a natural inhibitor, myricitrin possesses the potential therapeutic effects on inflammatory osteolysis.Myricitrin is a natural occurring flavonoid glycoside that possesses effects on inhibiting nitric oxide ( Osteoclastogenesis is primarily governed by 2 key cytokines, receptor activators of the nuclear factor‐κB (NF‐κB) ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF).Natural product myricitrin Figure  is a bot22.1Myricitrin was obtained from ChemFaces. Recombinant human macrophage colony‐stimulating factor (M‐CSF) and human RANKL were obtained from PeproTech EC, Ltd. . Rabbit antibody against NFATc1 was purchased from Cell Signaling Technology, Inc. . Rabbit antibody against c‑Fos was purchased from Santa Cruz Biotechnology, Inc. . The XTT assay kit was obtained from Roche . Western blot antibodies for phosphor‐AKT, phosphor‐ERK, ERK, phosphor‐JNK, JNK, phosphor‐p38 and p38 were from Santa Cruz Biotechnology Inc. ; β‐actin antibody was purchased from Sigma‐Aldrich, Inc. .2.24 cells/well in α‐MEM/10% FBS and were cultured in the presence of M‐CSF (20 ng/mL) and RANKL (40 ng/mL) for 4 days in the presence or absence of myricitrin . TRAP‐positive multinucleated cells with greater than three nuclei were counted as OCs.All animal care and experimental protocols were approved by the “Medical Ethics Committee of Hong‐Hui Hospital, Xi'an Jiaotong University School of Medicine” No.1002016011) and performed strictly according to the “Guidelines of Jiaotong university institutes of Health for the care and use of laboratory animals.” Bone marrow macrophage cells (BMMs) culture, osteoclast differentiation methods and myricitrin dose treating were following previous studies.011 and p2.34 cells with different concentrations of myricitrin. XTT solution was added to each well and incubated for 4 hours. The plate was read at 450 nm. Meanwhile, after the myricitrin‐treated BMMs for 48 hours, the cells then stained by Annexin V‐PE and 7 AAD for 15 minutes. Subsequently, cell apoptosis were assessed by excited at 488 nm and signals from 10 000 cells acquired at 585/42 (564‐606 nm) and 702/64 (670‐735 nm) in a FACS Canto II (BD). Results were analysed by the FACSDiva (BD) software and expressed as the percentage of apoptosis cells within each population.Cell viability and apoptosis of BMMs after myricitrin  treatments was studied by XTT  and flow cytometry as previous studies reported, respectively.2.4Bone absorption assay and filamentous actin immunofluorescence staining method were following previously described.2.52), 100 mmol/L DTT, RNase inhibitor, and Superscript II reverse transcriptase (Invitrogen).The mouse GAPDH gene was used as internal control. The amplification parameters consisted of an initial denaturation step at 95°C for 5 minutes followed by 40 cycles of denaturation at 95°C for 1 minute, annealing at 60°C for 30 seconds and extension at 72°C for 1 minute. The specificity of the SYBR green assays was confirmed by melting‐point analysis. Expression data were calculated from the cycle threshold (Ct) value using the Ct. Primers employed for amplification are shown in Table Total RNA was isolated with QIAzol reagent  according to the manufacturer's instructions RNA 1 μg) was reverse transcribed using oligo dT primers (10 μg) and dNTPs (10 mmol/L). The mixture was incubated at 65°C for 5 minutes, and cDNA was produced by incubating at 42°C for 50 minutes with first strand buffer . Thirty micrograms of total cell proteins was mixed with loading buffer and separated on 10% SDS‐PAGE gels, and the proteins in the gels were electro‐transferred onto nitrocellulose membranes . Horseradish peroxidase (HRP) conjugated secondary antibodies were used to visualize bands under an ECL‐based imaging system. The membranes were blocked with 5% nonfat milk in Tris‐buffered saline contacting 0.1% Tween‐20 (TBST) for 1 hour, before blotting with the primary antibodies for 2 hours at room temperature. The membranes were washed in TBST and incubated for 1 hour with HRP‐conjugated immunoglobulin antibodies. Signals were analysed in ImageJ and compared to controls after normalization.2.7P < .05).Experiments were conducted separately at least 3 times, and all data are presented as the mean ± standard deviation (SD). All data analysis was performed using SPSS software package ver. 14.0 ; one‐way ANOVA was used for comparison among the different groups. Post hoc testing of differences between groups was performed by using Duncan's test when the ANOVA was significant. All results were considered to be significant at the 5% critical level . Complemented to above bone resorption pit study, otherwise, the ring‐liked sealing zone of filamentous actin ring formation is a key indicator of osteoclast formation. Consist with resorption pits electron microscopy scanning results, after treatment with myricitrin, both number and morphology of the ring‐liked sealing zone of filamentous actin were down‐regulated by myricitrin treatment . As results shown, after myricitrin treatment, the pit's number and size of bone resorption were significantly down‐regulated.  screening, which is initial step for drug discovery.Documents studies showed myricitrin regulates several extracellular signalling pathways, such as STAT3, PI3K/Akt/eNOSRANKL interacts to its receptor RANK is a pivotal upstreaming signal pathway involved in osteoclastogenesis, further triggers various downstreaming signalling cascades, such as MAPKs  and Akt, which leading to stimulate the activation of critical genes for osteoclastogenesis.In that, as previous study reported, phosphorylation‐ERK is the crucial regulator of AP‐1 (c‐Fos and c‐JUN) activation in BMMs.In sum, our current study demonstrated the inhibitory effects of myricitrin on osteoclastogenesis in vitro and therapeutic effects in pathological osteoporosis in vivo, respectively. Moreover, our study explored myricitrin consequently attenuates the activation of the OCs‐specific transcription factors c‐Fos/NFATc1. Further, we identified that myricitrin functioned by attenuating the RANKL/MAPKs signalling pathway cross‐talking RANKL/AKT signalling cascades. Combined with previous studies, our current results indicate myricitrin are promising candidates for the treatment of pathological bone loss diseases.The authors declare that they have no competing interests.The manuscript is approved by all authors for publication.All data and materials were included in the manuscript.


The aim of this study was to examine the association between height and plasma glucose level, as well as risk of GDM among Chinese women.  A total of 6941 pregnant Chinese women were recruited from the Healthy Baby Cohort study in Hubei Province, China, in 2012–2014. Measured height was categorized into four groups according to the quartile distribution . GDM was defined based on the International Association of the Diabetes in Pregnancy Study Group criteria. Linear regression was used to estimate the association between height and plasma glucose levels. Logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for the association between height and GDM. P value for trend < 0.05), but not with fasting plasma glucose levels. A significant negative trend was found between height and risk of GDM , and each centimeter increase in height was associated with 2%  lower risk of GDM. Women in the highest quartile of height (>164.0 cm) had 23%  lower risk of developing GDM than those in the shortest quartile of height (≤158.0 cm), after adjusting for potential confounders.  The prevalence of GDM was 14.7% in our study. Height was inversely associated with the 1 h and 2h plasma glucose levels (all  Our findings suggested that height was negatively associated with risk of GDM among Chinese women. The difference in plasma glucose levels is present in the 1 h and 2 h plasma glucose, but not with fasting plasma glucose. Gestational diabetes mellitus (GDM) is characterized by glucose intolerance with onset or first recognition during pregnancy . The preAdult height is determined by the combination of genetic and environmental factors . StudiesPrevious studies have shown an inverse relationship between height and risk of GDM in several countries , 12–15, Therefore, we examined the association between height and plasma glucose level, as well as risk of GDM among pregnant Chinese women based on data from the Healthy Baby Cohort (HBC) study.n = 6), those with missing information on height or other important covariates (n = 70), or those with missing values for fasting, 1 h, and 2 h plasma glucose (n = 4294). The final analytic sample included 6941 women. Except for alcohol consumption before pregnancy, the main characteristics of the women with OGTT results (6941) and the women without OGTT results (4294) on height (161.26 ± 4.52 cm versus 161.06 ± 4.54 cm), age at delivery (28.48 ± 3.46 years versus 27.77 ± 4.01 years), prepregnancy BMI (20.69 ± 2.75 kg/m2 versus 20.27 ± 2.59 kg/m2), education level (college or above rate: 73.6% versus 76.5%), employment status (employed rate: 84.4% versus 74.7%), exposure to passive smoking during pregnancy (88.3% versus 86.2%), smoking before pregnancy (0.5% versus 1.1%), and taking physical activity (yes: 87.7% versus 89.4%) were statistically different.The HBC study was conducted at the Women and Children Medical and Healthcare Center of Wuhan City in the Hubei Province, China. This ongoing prospective cohort study is designed to assess the environmental and genetic factors that affect child health and development. Briefly, a total of 11,311 pregnant women who gave birth at this hospital were recruited between September 2012 and October 2014. Each participant was required to provide blood and urine samples and complete a standard questionnaire by a face-to-face interview at the time of institutional delivery. In the present study, we excluded participants with a history of diabetes  during the first antenatal care visit in the hospital. We categorized height into four groups according to the quartile distribution .In this study, all pregnant women underwent a 75 g 2 h oral glucose tolerance test (OGTT) in the morning after overnight fasting of at least 8 hours at 24–28 gestational weeks. Fasting, 1 h, and 2 h plasma glucose levels were measured at the Women and Children Medical and Healthcare Center of Wuhan City using the Roche Modular P800 Automatic Biochemistry Analyzer. According o the IADPSG criteria , women wData on demographic characteristics  and lifestyle factors  were collected by questionnaires. Passive smoking was defined as exposure second-hand smoking more than once per week and for >15 min per time. Information on parity was obtained from medical records. Prepregnancy weight was self-reported at the first antenatal care visit . Prepregnancy body mass index (BMI) was calculated as self-reported prepregnancy weight in kilograms divided by the measured height in square meters.t-test was used for continuous variables, and chi-square test was used for categorical variables.Continuous variables were described as mean ± standard deviation (SD), and categorical variables were presented as a number and percentage. To compare the differences between the GDM group and non-GDM group, Student's Differences in mean fasting, 1 h, and 2 h plasma glucose levels among height quartiles were compared by analysis of covariance. Dunnett's test was used for post hoc analysis. Linear regression models were used to estimate the association of fasting, 1 h, and 2 h plasma glucose levels with height. Logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the association between height and risk of GDM. Models were fit using height as a categorical variable, based on the quartile distribution of height, and the lowest quartile was used as the reference group. Covariates was selected based on established or potential associations with GDM , 5, incl2, the cutoff value of overweight for Chinese adults [To assess a potential effect modification, we performed subgroup analyses to examine the association between height and risk of GDM stratified by maternal age , prepregnancy BMI (<24 or ≥ 24 kg/me adults ), educatP value < 0.05 was considered statistically significant.All analyses were performed using SAS 9.4 software . A two-tailed P values < 0.05).Among the 6941 women, the mean age at delivery was 28.5 (SD: 3.5) years, the mean height was 161.3 (SD: 4.5) cm, and 89.6% were primiparous. In total, 1017 (14.7%) women were diagnosed with GDM based on the IADPSG criteria used in the present study. Differences in distribution of the characteristics between GDM group and non-GDM group are presented in P values for trend < 0.001) without adjustment for confounders. After adjustment for potential confounders, the height was still inversely associated with the 1 h and 2 h plasma glucose levels . Post hoc analyses suggested that women with taller height had lower 1 h and 2 h plasma glucose levels than those with shorter height .P values < 0.001). After controlling for potential confounders, every centimeter increase in height was associated with a linear change in 1 h plasma glucose levels with −0.0157 mmol/l  and in 2 h plasma glucose levels with −0.0231 mmol/l . No significant association was observed between height and fasting plasma glucose levels before and after adjusting for potential confounding variables.P value for trend < 0.05).The unadjusted and adjusted prevalence of GDM according to height quartiles are shown in P value for trend < 0.05), and each centimeter increase in height was associated with 2%  lower risk of GDM. After adjustment for potential confounders, women in the highest quartile of height (>164.0 cm) had lower risk of GDM with an adjusted OR of 0.77 , compared with those in the shortest quartile height (≤158.0 cm).P values for interaction > 0.05)  .In this study, we investigated the association between height and risk of GDM among Chinese women. We observed height was negatively associated with risk of GDM after adjustment for potential confounders. In addition, we found that height was inversely associated with the 1 h and 2 h plasma glucose levels, but not with fasting plasma glucose levels.Several studies have investigated the association between height and GDM, and the findings were inconsistent. The Omega Cohort Study including 1644 American women suggested an inverse relationship between self-reported height and risk of GDM based on the National Diabetes Data Group (NDDG) diagnostic criteria independent of age, race/ethnicity, education, and prepregnancy BMI . Similarβ-cell dysfunction and insulin resistance [β-cell function and insulin resistance [The mechanisms underlying the relationship between height and risk of GDM are not clear. GDM is characterized by pancreatic sistance . Shortersistance , 20, whisistance , 21. Inasistance –24. Our Low socioeconomic status during childhood was reported to be related with shorter adult height . StudiesInterestingly, we observed that height was inversely correlated with 1 h and 2 h plasma glucose levels, but not with fasting plasma glucose levels. Our findings were in agreement with a study from Brazil, reporting a negative association of height with 1 h and 2 h plasma glucose levels but not with fasting glucose levels without adjusting any . SimilarWe found height was negatively associated with 1 h and 2 h plasma glucose, but not with fasting plasma glucose. A possible reason for this may be the different metabolisms of a standard dose of glucose during OGTT among taller and shorter women. Women with shorter stature have a lower mass of metabolically active tissues in response to a 75 g OGTT compared with women with taller stature .Our study had several strengths, including a large sample size and a wide range of potential confounders. In addition, the diagnosis of GDM in our study was based on the measure of OGTT, performed with unified instruments at one hospital, which minimized the misclassification of GDM and non-GDM cases.P value < 0.05. Moreover, the participants did not know the research purpose at the stage of data collecting; it was unlikely that women with shorter height tended to take or not take the OGTT test in this study. Second, although a variety of known risk factors of GDM were taken into account in the analyses of our study, it is not possible to rule out the residual confounding attributable to unmeasured factors, such as early-life nutrition factors, history of GDM, family history of diabetes, and maternal dietary intakes during pregnancy. Third, height and GDM might be jointly determined by genetic factors, but we are unable to examine their association.However, some limitations should be considered. First, some characteristics of the women with OGTT results and without OGTT results were statistically different, which might introduce selection bias, potentially reducing the generalization of this analysis. However, the differences were relatively small. Due to the large sample of the women with OGTT results (6941) and the women without OGTT results (4294), a slight difference would lead to a Our result suggested that height was negatively associated with risk of GDM in pregnant Chinese women, independent of potential confounders. The difference in plasma glucose levels is present in the 1 h and 2 h plasma glucose, but not with fasting plasma glucose.


Candida albicans, an important yeast pathogen of humans, prior studies were not able to generate circular plasmids that were autonomous (duplicated without inserting themselves into the chromosome). Here, we found that linearizing circular plasmids with sequences from telomeres, the chromosome ends, allows the plasmids to duplicate and segregate in C. albicans. We used this system to identify chromosomal sequences that facilitate the initiation of plasmid replication (origins) and to show that an ∼100-bp fragment of a C. albicans origin and an origin sequence from a distantly related yeast can both function as origins in C. albicans. Thus, the requirements for plasmid geometry, but not necessarily for origin sequences, differ between C. albicans and baker’s yeast.Circular plasmids are important tools for molecular manipulation in model fungi such as baker’s yeast, yet, in  Candida albicans, a prevalent human fungal commensal and pathogen. Instead, plasmids generally integrate into the genome. Here, we assessed plasmid and transformant properties, including plasmid geometry, transformant colony size, four selectable markers, and potential origins of replication, for their ability to drive autonomous plasmid maintenance. Importantly, linear plasmids with terminal telomere repeats yielded many more autonomous transformants than circular plasmids with the identical sequences. Furthermore, we could distinguish (by colony size) transient, autonomously replicating, and chromosomally integrated transformants . Candida albicansURA3 and a heterologous marker, ARG4, yielded many transient transformants indicative of weak origin activity; the replication of the plasmid carrying the heterologous LEU2 marker was highly dependent upon the addition of a bona fide origin sequence. Several bona fide chromosomal origins, with an origin fragment of ∼100 bp as well as a heterologous origin, panARS, from Kluyveromyces lactis, drove autonomous replication, yielding moderate transformation efficiency and plasmid stability. Thus, C. albicans maintains linear plasmids that yield high transformation efficiency and are maintained autonomously in an origin-dependent manner.The ability to generate autonomously replicating plasmids has been elusive in IMPORTANCE Circular plasmids are important tools for molecular manipulation in model fungi such as baker’s yeast, yet, in Candida albicans, an important yeast pathogen of humans, prior studies were not able to generate circular plasmids that were autonomous (duplicated without inserting themselves into the chromosome). Here, we found that linearizing circular plasmids with sequences from telomeres, the chromosome ends, allows the plasmids to duplicate and segregate in C. albicans. We used this system to identify chromosomal sequences that facilitate the initiation of plasmid replication (origins) and to show that an ∼100-bp fragment of a C. albicans origin and an origin sequence from a distantly related yeast can both function as origins in C. albicans. Thus, the requirements for plasmid geometry, but not necessarily for origin sequences, differ between C. albicans and baker’s yeast. Saccharomyces cerevisiae  × 100.Yeast transformants grown overnight in SDC(−AA) for mitotic stability assay were diluted 100-fold into SDC medium and grown overnight at 30°C. The cultures were 10-fold serially diluted, and 5 µl of each dilution was spotted on both SDC(−AA) and SDC plates. The plates were incubated at 30°C for 2 days, and the number of colonies was counted from the highest dilution at which they were well separated. The proportion of cells that retained the plasmid without selection was calculated as [no. of colonies on SDC(−AA)/no. of colonies on SDC] from the same dilution. The plasmid loss rate was then determined as described in the work of Longtine et al. .RAMP gene was used to probe the plasmids on the blot.The genomic DNA was extracted from 10-ml overnight-grown cultures in SDC(−AA) as described previously . FifteenRAMP gene was used to determine plasmid copy number, and CEN of chromosome 1 was used as a reference gene. The two primer sets used had similar efficiencies in the reaction; therefore, fold change in the copy number of plasmids was determined relative to the genomic control. Copy number of plasmids was calculated as copy number = (fold change × 2)/mitotic stability.qPCR was carried out with the genomic DNA from autonomous transformants using SYBR green master mix  per the manufacturer’s protocol in a Bio-Rad CFX96 Touch real-time PCR detection system. Cycling conditions were as follows: 95°C (3 min), 40 cycles of 95°C (5 s) and 60°C (30 s), and melt curve from 65.0 to 95.0 for 5 s. The 2O and resuspended in 1 ml SDC(−AA); 10 µl was inoculated in 100 µl SDC(−AA) in a 96-well round-bottom sterile polystyrene plate (Corning). For tiny colonies that could not be propagated in liquid medium, three independent colonies were directly inoculated from the plate into 100 µl SDC(−AA). The plate was subsequently incubated at 30°C in a Tecan Infinite F200 Pro  microplate incubator/spectrometer with a shaking duration of 900 s, and the OD600 was collected every 15 min over a 24-h period. OD versus time was plotted to generate growth curves.From fresh transformation plate, three independent colonies per colony size were inoculated into 2 ml SDC(−AA) and grown overnight at 30°C and 250 rpm. Fifty microliters of cell culture was washed with ddHE. coli  to cut the linear plasmid at both of the ends, resulting in removal of telomere repeats. The digested DNA was ligated overnight with T4 DNA ligase . The ligation product was transformed into electrocompetent E. coli , and the


Alcohol labeling provides a relatively low-cost, population-level approach to providing information about alcohol’s content and harms.We conducted an online between-subjects experiment with two tasks to examine the impact of alcohol labels (n = 1884). In one task, participants were randomized to view one of four different unit labels . We assessed participants’ accuracy of estimating weekly serving limits of alcohol. In a second task, participants were randomized to view one of eight health warnings . We assessed the motivation to quit after viewing the health warning.Accuracy of estimating weekly serving limits of alcohol was greater for participants who viewed novel unit labels compared to the industry standard labels. Motivation to drink less was higher amongst participants who had viewed both cancer and negatively framed messages, compared to mental health and positively framed messages.Existing unit labels used by the alcohol industry can be improved; the inclusion of unit information per serving and how these relate to low-risk drinking guidelines may be important for facilitating consumer understanding. Health warning labels should be included alongside units to provide consumers with information about the harms associated with alcohol and discourage riskier drinking behavior. More thAlcohol units  are a method of providing information about the strength of an alcoholic drink in relation to its serving size. A similar method used in Australia, known as ‘standard drink’ labeling, has been shown to effectively improve drinkers’ estimates of their consumption and help them monitor their intake . HoweverThis highlights the important role of designing health warnings on alcohol labels that can increase public awareness of the potential harms of excessive alcohol consumption . CurrentSome effort has been made to improve understanding of alcohol’s content and risks. In 2011, the alcohol industry agreed to a voluntary Responsibility Deal with the UK Government, which included labeling at least 80% of their products with unit content, low-risk guidelines, pregnancy warnings and responsibility statements . HoweverHere we examine the influence of unit labels and health warnings on drinkers’ understanding, attitudes and behavioral intentions regarding drinking and examine optimal methods of delivering this information on labels. This is important given the current political interest in alcohol labeling and can be used to guide the development of label designs that can effectively support consumers.22.1http://doi.org/10.17605/OSF.IO/JBY5X).This was an online between-subjects experimental study with two tasks. In the first task, participants were pseudo-randomized to view one of the four-unit labels where the primary outcome measure was the accuracy of estimating weekly serving limits of alcohol. Two of the unit labels displayed industry standard information typically seen on alcohol products and the other two displayed novel methods of presenting unit information (see 2.3.3 for details). In the second task, participants were randomized to view one of eight health warnings . Motivation to drink less was the primary outcome measure. Support for alcohol labeling policies before and after the experiments was also assessed. The study protocol was published on the Open Science Framework prior to testing, where more details about the methods are described (2.2https://www.prolific.ac/). Participants read an information statement before giving their consent to participate. Participants were required to be at least 18 years of age, live in the UK and report drinking alcohol. Prolific Academic was the ideal platform for conducting this study based on the large (n ≈ 8000) number of individuals who regularly use the site who were eligible to participate based on our inclusion criteria. The study was approved by the Faculty of Science Research Ethics Committee at the University of Bristol (reference: 23051753685).We recruited alcohol consumers using the crowdsourcing platform Prolific Academic  .2.3.2Participants were asked to what extent they agree with the following statements: 1) ‘Alcoholic beverages should include more information about alcohol strength ’, 2) ‘Alcoholic beverages should have information about the health impact of drinking ’, and 3) ‘Alcoholic beverages should include more nutritional information ’. These questions were answered using a 100-point visual analog scale with the anchors ‘STRONGLY DISAGREE’ and ‘STRONGLY AGREE’.2.3.32.3.3.1Participants were randomly assigned to one of the four-unit label conditions: 1) Basic ABV (Alcohol by Volume refers to the strength of the drink), 2) Responsibility Deal, 3) Food Label Equivalent or 4) Pie Chart. The ‘Basic ABV’ label displayed alcohol strength information, which is legally required on all alcohol labels in the UK. This information was also included in the other conditions. The ‘Responsibility Deal’ label showed the total units per bottle, which is used on many existing labels that adhere to the Responsibility Deal requirements. The novel ‘Food Label Equivalent’ label provided the number of units per serving as a percentage of the low-risk amount, which follows the example of voluntary food labeling schemes in the UK. The novel ‘Pie Chart’ label was designed by the research team and displayed the number of units per serving as a visual proportion of the low-risk amount (14 units per week). The unit label was presented four times to each participant with information corresponding to one of four different alcoholic drinks on each presentation  beer brands, simultaneously on screen, each alongside the unit label as per their randomly assigned condition see . The thr2.3.42.3.4.1Participants were asked ‘Does this health warning make you feel motivated to drink less?’ This question was answered on a five-point scale from ‘STRONGLY DISAGREE’ (coded 1) to ‘STRONGLY AGREE’ (coded 5) (adapted from ). This m2.3.4.2The Brief Reactance to Health Warnings Scale was administered . This re2.3.4.3Avoidance was measured with three items, preceded by the text ‘Imagine that all alcohol containers had this warning’ 1) ‘How likely is it that you would try to avoid thinking about the warning?’ 2) ‘How likely is it that you would try to avoid looking at the warning on your drink?’, and 3) ‘How likely is it that you would keep the drink out of sight to avoid looking at the warning?’ Questions were answered on a five-point scale from ‘NOT AT ALL LIKELY’ (coded 1) to ‘EXTREMELY LIKELY’ (coded 5).2.3.4.4Participants were asked ‘How believable is this health warning?’ This question was answered on a five-point scale from ‘NOT AT ALL BELIEVABLE’ (coded 1) to ‘EXTREMELY BELIEVABLE’ (coded 5).2.3.5To assess self-efficacy, participants were asked ‘For me cutting down on the number of alcohol units that I drink in the next week would be…’ ‘VERY DIFFICULT’ (coded 1) to ‘VERY EASY’ (coded 5). To assess response-efficacy, participants were asked, ‘To what extent do you think that cutting down on your drinking would reduce your risk of alcohol-related disease?’ Questions were answered on a five-point scale from ‘NOT AT ALL LIKELY’ (coded 1) to ‘EXTREMELY LIKELY’ (coded 5). To assess alcohol craving, participants completed the Alcohol Urges Questionnaire (AUQ) .2.4http://www.qualtrics.com/). Participants were first shown an information statement and then completed a tick-box consent page. Participants then completed the demographic screening questions and reported their support for alcohol labeling policies. Participants were pseudo-randomized into the different experimental conditions  such that an equal number of participants were in each condition. They then completed the unit information and health warning tasks in that order.The study was designed and hosted on the Qualtrics online survey platform . Finally, participants reported their support for alcohol labeling policies, completed the AUDIT, and provided their educational attainment and student status. The experiment lasted approximately seven minutes, and at the end, participants were debriefed and reimbursed £1.2.5The sample size was calculated based on the primary outcome measure for the health warning task, motivation to drink less. To detect the main effect in motivation to drink less across participants in health warning conditions of 0.5 , at 95% power and an alpha level of 1%, a sample of 1786 participants was required. We aimed to recruit 1900 participants to account for exclusions based on participants failing attention checks.Estimates of how many servings of each of the four drinks an individual could have in a week before reaching the limit of 14 units were subtracted from the correct answer for each of the four different beverages. These four scores were then averaged to create an overall accuracy variable. Data were analyzed in IMB SPPS Statistics. The full data analysis plan is presented in the study protocol (http://doi.org/10.17605/OSF.IO/JBY5X).3https://doi.org/10.5523/bris.2l19s5lmdoh7k2jc5n8l2ta95i.The data that form the basis of the results are available from the Bristol Research Data Repository, data.bris, at 3.1SD = 12) and 83% lived in England, 10% in Scotland, 5% in Wales and 2% in Ireland. 58% of participants’ highest level of education was from a higher education institution, and 13% were currently students. Of these, 71% were studying for undergraduate degrees. The mean AUDIT score was 7 (SD = 5).Data were collected from 1908 participants. Of these, 24 participants  were excluded from all analyses for failing the attention check question. Remaining participants (n = 1884) were 50% female, had a mean age of 35  = 22.16 p < 0.001, η2 = 0.03). Bonferroni corrected post-hoc t-tests indicated that those in the Food Label Equivalent and Pie Chart conditions were more accurate than those in the Basic ABV and Responsibility Deal conditions  = 4.61, p < 0.001). There were no differences between the estimates for the Basic ABV and Responsibility Deal conditions and between the Food Label Equivalent and Pie Chart conditions. As shown in 3.2.2A series of one-way ANOVAs were conducted with the unit condition on each of the secondary outcome measures ). A series of Shapiro-Wilk tests indicated that the data were not normally distributed (ps<0.001) and visual inspection confirmed this. We performed Kruskal-Wallis tests on these data, but these did not meaningfully change the findings. As a result, here we report only the results of the ANOVAs, with the means presented in 3.2.2.1 = 39.30 p < 0.001, η2 = 0.06). Participants in the Basic ABV condition completed the unit task in the shortest time followed by the Pie Chart condition and Food Label Equivalent condition. Participants in the Responsibility Deal condition completed the unit task in the slowest time, and post-hoc Bonferroni corrected t-tests indicated that the only evidence of a difference was between the Responsibility Deal condition and the other three conditions  = 6.93, p < 0.001).There was a main effect of time taken to complete the task  = 1.15 p = 0.33, η2 = 0.002).There was relatively high self-efficacy across participants , and there was no evidence of a difference between those in the four conditions  = 2.73 p = 0.04, η2 = 0.004) with the highest response-efficacy among those in the Basic ABV condition.Overall there was relatively high response-efficacy across participants . There was weak evidence of a difference between those in the four conditions  = 2.14 p = 0.09, η2 = 0.003).There was weak evidence of a difference in alcohol craving between those in the four conditions . There was no evidence that any of the four conditions increased the ordered log odds of choosing a higher strength beverage =−0.08–0.07, p = 0.86; see 3.3A series of 2 (content) × 2 (specificity) × 2 (framing) factorial ANOVAs were conducted on each of the outcome measures. Visual inspection indicated that the believability, self-efficacy, and response-efficacy data were negatively skewed and the alcohol craving (AUQ) data were positively skewed (Shapiro-Wilk tests p < 0.001). Log-transforming these data and re-running the ANOVAs did not meaningfully change the results. As ANOVA is robust to non-normality, particularly when the sample size is large, we report here only the ANOVA results for non-transformed data. Means are reported in 3.3.1 = 6.45, p = 0.01, η2 = 0.003). Avoidance of warnings was also higher among those in this condition  = 21.93, p < 0.001, η2 = 0.012).There was evidence that motivation to drink less was higher among those participants randomized to the cancer warning versus mental health warning  = 7.04, p = 0.008, η2 = 0.004), found the warning more believable  = 16.75, p < 0.001, η2 = 0.009) and had higher response-efficacy  = 6.94, p = 0.008, η2 = 0.004) versus those who viewed a general warning.General Versus Specific Warnings. There was evidence that those randomized to view the specific warning reported lower levels of reactance  = 7.01, p = 0.008, η2 = 0.004), reactance  = 5.87, p = 0.015, η2 = 0.003) and avoidance  = 6.10, p = 0.014, η2 = 0.003) than the positively framed warnings.Negative Versus Positive Warnings. The negatively framed warnings received higher scores for motivation to drink less  = 15.07, p < 0.001, η2 = 0.008) characterized by an increase in support for strength information  = 8.52, p < 0.001) and calorie information  = 4.43 p < 0.001) after the experiment, but not health warning information  = 0.91, p = 0.36). A main effect of label type  = 56.74 p < 0.001, η2 = 0.029) indicated that support for strength information was marginally greater than for the calorie information (t(3766) = 1.89 p = 0.06), which in turn was greater than for the health warning information (t(3766) = 7.21, p < 0.001), which had the lowest levels of support.A 2 (pre-study vs.post-study) × 3  ANOVA was conducted to examine change in support for labeling policies. This revealed an interaction  and avoidance  and higher levels of warning believability , motivation to drink less , self-efficacy  and response efficacy .4Alcohol labeling provides a relatively low-cost, population-level approach to providing consumers with information about the content and harms related to alcohol consumption. The presentation and format of health information can impact on the effectiveness of labels to communicate accurate information and encourage healthier behavior. Overall, participants underestimated the number of drink servings they could have within the low-risk weekly amount of 14 units, which demonstrates the difficulty drinkers have estimating alcohol consumption in units and across multiple drinks. The absence of a difference in accuracy between the UK industry standard Basic ABV and Responsibility Deal conditions and the increased accuracy shown in the novel Food Label Equivalent and Pie Chart conditions demonstrates that current unit labels could be improved. Accuracy of estimating weekly serving limits of alcohol was the least accurate amongst participants in the Responsibility Deal unit condition, as well as the slowest, which suggests that these labels are particularly difficult for consumers to use, despite the intention to provide ‘labels with clear unit content’ . The infIn comparison, information in the Food Label Equivalent and Pie Chart conditions was always per serving. Portman Group guidance recommends that alcohol labels should include units per container as a minimum key element, while units per typical serving are optional . Our finincrease their drinking. Martin-Moreno and colleagues considered this issue in a review of the labeling literature and concluded that the potential for misuse of information was not an adequate reason to withhold it from the public, but presents a strong case for the inclusion of health information , in which only a small proportion of participants suggested that they would drink less in response to the unit information. However, it is also possible that if consumers were previously underestimating how many drinks constituted 14-units , then they could feel encouraged to ormation . Unit laormation .Our findings provide suggestions for the type of warnings that could be used. Participants reported higher motivation to drink less after viewing both cancer messages and negatively framed messages. The latter is consistent with previous research that found negatively framed messages could support a reduction in alcohol consumption  and reseOur study has a number of important strengths, including a large sample recruited using Prolific Academic, a crowdsourcing site which has been shown in recent research to produce high-quality data . There a5Alcohol labeling can improve understanding of the content of drinks and harms related to consumption. Current unit labels implemented as part of voluntary industry schemes can be improved. Further work is needed to identify the features of optimum labels, but this study suggests that including unit information per serving, as well as in the context of the low-risk drinking guidelines, may be key. Clear and consistent unit labeling could help consumers monitor their drinking and understand the extent to which this differs from the recommended guidelines. This study also supports previous claims that unit information should be presented alongside health messages to discourage riskier drinking behavior. Health messages should aim to maximize population reach and expand on the single pregnancy messages currently used in many industry labels.O.M.M., K.D., A.K.M.B., A.S.A. and M.M. contributed to the conception and design of the study and contributed to the study protocol. K.D. managed the day-to-day running of the study and testing of participants. O.M.M. performed the data analysis and all authors helped with data interpretation. This manuscript was written by A.K.M.B. and O.M.M. with input from all co-authors. All authors read and approved the final version of the manuscript.Alcohol Research UK grant (SG 15/16 222) and an ESRC New Investigator Grant (ES/R003424/1), both awarded to O.M.M. and by the Medical Research Council Integrative Epidemiology Unit at the University of Bristol, which is supported by the Medical Research Council and the University of Bristol (MC_UU_12013/6). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This work was supported by an No conflict declared.


Intraperitoneal (IP) insulin administration is a last‐resort treatment option for selected patients with type 1 diabetes mellitus (T1DM). As the IP route of insulin administration mimics the physiology more closely than the subcutaneous (SC) route, we hypothesized that IP insulin would result in less oxidative stress (expressed as systemic level of free sulphydryl (R‐SH) content) compared to SC insulin in subjects with T1DM.Prospective, observational case‐control study. Serum thiol measurements were performed at baseline and at 26 weeks in age‐ and gender‐matched patients with T1DM. Serum‐free thiols, compounds with a R‐SH group that are readily oxidized by reactive oxygen species, are considered to be a marker of systemic redox status.A total of 176 patients, 39 of which used IP and 141 SC insulin therapy were analysed. Mean baseline R‐SH concentration was 248 (31) μmol/L. In multivariable analysis, the route of insulin therapy had no impact on baseline R‐SH levels. The estimated geometric mean concentrations of R‐SH did not differ significantly between both groups: 264  for the IP group and 258  for the SC group with a difference of 6  μmol/L.Based on R‐SH as a marker of systemic oxidative stress, these findings demonstrate that the route of insulin administration, IP or SC, does not influence systemic redox status in patients with T1DM. ALTalanine aminotransferaseASTaspartate aminotransferaseBMIbody mass indexCGMcontinuous glucose measurement(s)CIPIIcontinuous intraperitoneal insulin infusionCSIIcontinuous intraperitoneal insulin infusionCVcoefficient of variationeGFRestimated glomerular filtration rateGamma‐GTGamma‐glutamyl transpeptidaseHbA1cglycated haemoglobinHDLhigh‐density lipoproteinIGFinsulin‐like growth factorIPintraperitonealIQRinterquartile rangeLDLhigh‐density lipoproteinMAGEmean average glucose excursionsMODDmean of daily differences MDI, multiple daily injectionsR‐SHfree sulphydrylSCsubcutaneousSDstandard deviationT1DMtype 1 diabetes mellitus1Currently, Continuous intraperitoneal insulin infusion (CIPII) is used as a last‐resort treatment option for selected patients with type 1 diabetes mellitus (T1DM) who fail to reach glycaemic control despite intensive subcutaneous (SC) insulin therapy. With CIPII, insulin is infused directly in the intraperitoneal (IP) space resulting in higher concentrations of insulin in the portal vein catchment area, higher hepatic insulin extraction and lower peripheral plasma insulin concentrations compared with SC insulin administration.The aberrant production of reactive oxygen species (ROS), due to hyperglycaemia, is considered to be the central element of oxidative stress and, ultimately, plays an important role in the pathogenesis of T1DM, its progression and ultimately micro‐ and macrovascular complications.Indeed, in the animal model, delivering the same dose of insulin IP resulted in lower hepatic oxidative stress and inflammation as compared to continuous SC insulin delivery.22.1This multicentre study was investigator‐initiated and had a prospective, observational matched case‐control design. Inclusion took place at Isala hospital  and Diaconessenhuis hospital . Primary aim of this study was to compare the effects of long‐term IP insulin delivery to SC insulin delivery, with respect to glycaemic control. Aim of the present analysis was to test the hypothesis that IP insulin therapy would result in a more favourable redox status compared with SC insulin therapy. Serum‐free thiols, compounds with a free sulphydryl (R‐SH) group that are readily oxidized by reactive oxygen species, were used as marker systemic redox status. From the measures available to measure ROS, we considered R‐SH as an appropriate measure for oxidative stress in the current study: R‐SH are a robust and powerful read‐out of the systemic in vivo reduction‐oxidation (redox) status.2.2Cases were subjects on IP insulin therapy using an implantable insulin pump  for the past 4 years without interruptions of >30 days, in order to avoid effects related to initiating therapy. Inclusion criteria for cases were have been described in detail previously.2.3®, Sanofi‐Aventis) was administered with an implantable pump for IP insulin users and patients using CSII or MDI continued their own insulin regime consisting of fast‐acting insulin analogues and for MDI patients also long‐acting insulin analogues or NPH insulin. All patients received standard care. The implantable insulin pump used during this study and related procedures has been described in more detail previously.There were four study visits. During the first visit, baseline characteristics were collected using a standardized case record form. During the second visit (5‐7 days later), laboratory measurements were performed. During the third visit, 26 weeks after visit 1, clinical parameters were collected. During the fourth visit, 5‐7 days after the third visit, laboratory measurements were performed. Throughout the study period, insulin . Blood pressure was measured using a blood pressure monitor  using the highest mean of 4 measurements (2 on each arm). Patients were instructed to visit the laboratory in a fasting state. Laboratory measurements included creatinine, c‐peptide, total cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), y‐glutamyl transpeptidase (gamma‐GT), alkaline phosphatase and urine albumin/creatinine ratio and HbA1c. HbA1c was measured with a Primus Ultra2 system using high‐performance liquid chromatography .Systemic redox status was assessed using measurements of thiols. Thiols are compounds with a free sulphydryl (R‐SH) moiety. These R‐SH groups are readily oxidized by ROS and other reactive species. The circulating concentrations of total R‐SH have recently been proposed to directly reflect the whole‐body redox status: a decrease in circulating R‐SH concentration represents increased oxidative tone and thus indicates a state of high oxidative stress.TM serum tubes, centrifuged and directly stored in aliquots at −80°C without thawing until measurement of R‐SH. R‐SH were measured as previously described, with minor modifications.Venous blood samples were collected in BD vacutainerThe 24‐hours interstitial glucose profiles were recorded using a blinded CGM device . The CGM device was inserted in the periumbilical area, and in pump users contralateral to the (implanted) insulin pump. Patients were instructed to perform a minimum of 4 blood glucose self‐measurements daily during the CGM period, using a blood glucose metre  to calibrate the sensor. All procedures related to the CGM were performed by one, trained physician (PRvD).2.5Results were expressed as mean (with standard deviation (SD)) or median (with interquartile range [IQR]) for normally distributed and non‐normally distributed data, respectively. A significance level of 5% (two‐sided) was used. Normality was examined with Q‐Q plots. Differences between the IP and SC groups averaged over the study period and in time were estimated using the general linear model.P < .05. The quantity of the b‐coefficient, with a 95% CI, gives the difference between both treatment modalities over the study period adjusted for baseline differences.A regression model based on covariate analysis (ANCOVA) was applied in order to adjust for possible baseline imbalances. In the model, the fixed factors CIPII and SC insulin therapy were used as determinants. The difference in scores was determined based on the b‐coefficient of the particular (CIPII or SC) group. Significance of the b‐coefficient was investigated with the Wald test based on a P‐value of <.1 were included in the multivariable linear regression using backward selection. The quality of the model was described using the accuracy of the prediction by the adjusted R2 value. In order to avoid collinearity, only the coefficient of variation (CV) of the CGM measurements was used. The CV measures intraday variation in glucose patterns, is defined as the SD divided by the mean of blood glucose values and is advocated to be the most optimal measure of glycaemic variability.Furthermore, to evaluate the independent impact of several variables, including the route of insulin administration, on R‐SH concentrations, a multivariate regression model with R‐SH score as primary outcome variable was constructed. For this model, the baseline values were used since the most extensive characterization of the population  was performed at baseline. First, univariable linear regression analyses were applied to identify variables that are independently associated with R‐SH. Subsequently, all variables that associated with R‐SH with a Statistical analyses were performed using SPSS . The study protocol was registered prior to the start of the study (NCT01621308 and NL41037.075.12) and approved by the local medical ethics committee. All patients gave informed consent.32 (n = 2): 184 patients were followed up during the 26‐week study period. Due to insufficient serum, R‐SH could not be measured in 4 patients. Consequently, 180 patients were included in the present analyses. At baseline, 39 patients were treated with IP insulin and 141 with SC insulin (67 with MDI and 74 CSII). Mean age of the population was 49.8 (12.5) years, diabetes duration 26.1 (12.3) years and HbA1c 63.8 (10.5) mmol/mol  or an estimated glomerular filtration rate of <40 mL/min/1.73mBaseline R‐SH concentrations were normally distributed with a mean concentration of 248 (31) μmol/L  for the IP group and 257.7  for the SC group with a difference of 6.0  μmol/L. After adjustment for the total insulin dose, the difference between groups remained nonsignificant: 263.7  for the IP group and 257.7  for the SC group with a difference of 6.0  μmol/L. During the study period, R‐SH concentrations increased among all patients with 20.8  μmol/L  that investigated the influence of the route of insulin administration on the redox status, an identical dose of insulin was administered for 4 weeks via the IP and SC route to STZ‐induced diabetic rodents.The previous observations that IP insulin administration in human T1DM patients results in lower Hba1c, less glycaemic variability and higher IGF‐1 levels as compared to SC insulin treatment This may suggest that the pathways  that influence redox status and are known to be differently influenced by IP and SC insulin, counteract each other resulting in a stable redox status. Despite the fact that no differences in lipid metabolism and high‐sensitive CRP concentrations were present in the present study, it cannot be ruled out that these or other (unmeasured) pathways influenced redox status in the present study.Obviously, differences in treatment duration, species and the parameters of oxidative stress used could also account for the different findings of this study as compared to Dal et al In the current study, patients were stable on their mode of therapy for at least 4 years while in the study of Dahl et al rats were treated for 4 weeks.As expected, obesity and ageing were inversely associated with increased oxidative stress.Strengths of the present study include the inclusion of patients who have been using their current route of therapy for at least 4 years, thus creating a stable situation, and measurements made on two points in time. During the study period, there was an increase of R‐SH concentrations in both treatment groups. To explore this increase in more detail, we post hoc repeated this analysis for R‐SH using total insulin dose, glucose or albumin as covariates. However, the R‐SH increase over the study period remained significant. This may indicate that other nonmeasured variables  were involved here. Other limitations should be mentioned. Major limitation of the present study is the nonrandomized design. Ideally, R‐SH measurements should be performed prior and after initiation of IP insulin therapy to compare the changes in R‐SH status from baseline (with SC insulin therapy). However, the global shortage of implantable insulin pumps precludes such a study design. Taken together, no conclusions can be made regarding causality of our findings. And although we prespecified redox status as a secondary outcome in the study protocol, no separate power calculation was performed to detect potential relevant differences in R‐SH. By using the directions of the 95% confidence intervals, one could hypothesize that there are undetected differences in R‐SH between the IP and SC group. Finally, the lack of information with regard to other plasma antioxidant species such as ascorbate, uric acid and small‐molecular‐weight thiols and markers of inflammation (due to cost constraints) should be mentioned.In conclusion, the findings in this study demonstrate that the route of insulin administration, IP or SC, does not influence systemic redox status in subjects with T1DM, at least, measured as per R‐SH group detection.The authors declare that they have no financial or other relationships that might lead to a conflict of interest. PRv.D. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.PRv.D. involved in design, inclusion of patients, measurements, statistical analysis and writing manuscript. KG involved in design, statistical analysis and critically reviewing manuscript. TV involved in critically reviewing manuscript. H.v.G. involved in design and measurements. NK, FW, SJJL and HJGB involved in design and critically reviewing manuscript.The study protocol was in compliance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. The study protocol was registered prior to the start of the study (NCT01621308 and NL41037.075.12) and approved by the local medical ethics committee. All patients gave informed consent. Click here for additional data file.


Despite the great potential of numerous antioxidants for pharmacotherapy of diseases associated with inflammation and oxidative stress, many challenges remain for their clinical translation. Herein, a superoxidase dismutase/catalase‐mimetic material based on Tempol and phenylboronic acid pinacol ester simultaneously conjugated β‐cyclodextrin (abbreviated as TPCD), which is capable of eliminating a broad spectrum of reactive oxygen species (ROS), is reported. TPCD can be easily synthesized by sequentially conjugating two functional moieties onto a β‐cyclodextrin scaffold. The thus developed pharmacologically active material may be easily produced into antioxidant and anti‐inflammatory nanoparticles, with tunable size. TPCD nanoparticles (TPCD NP) effectively protect macrophages from oxidative stress‐induced apoptosis in vitro. Consistently, TPCD NP shows superior efficacies in three murine models of inflammatory diseases, with respect to attenuating inflammatory responses and mitigating oxidative stress. TPCD NP can also protect mice from drug‐induced organ toxicity. Besides the passive targeting effect, the broad spectrum ROS‐scavenging capability contributes to the therapeutic benefits of TPCD NP. Importantly, in vitro and in vivo preliminary experiments demonstrate the good safety profile of TPCD NP. Consequently, TPCD in its native and nanoparticle forms can be further developed as efficacious and safe therapies for treatment of inflammation and oxidative stress‐associated diseases. According to these findings, we designed a pharmacologically functional material that can scavenge a broad spectrum of reactive species ‐mimetic agent that can effectively scavenge superoxide anion and oxygen radicals.es Figure1a. Thi2O2, regardless of the number of PBAP. In the case of Tpl‐conjugated β‐CD (TCD) materials, they were initially synthesized using excess amount of Tpl. Measurement by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry revealed that TCDs are water‐soluble only when the conjugated Tpl unit is less than 4 . In addition, TCDs cannot be effectively hydrolyzed in the presence of physiological levels of H2O2. Consequently, TCD with ≈2 Tpl units was used to synthesize TPCD that can be hydrolyzed into water‐soluble products in the presence of low levels of H2O2, which is beneficial for excretion from the body.TPCD was synthesized by sequentially conjugating Tpl and PBAP onto β‐CD, which was achieved by activation of Tpl and PBAP with 1,1‐carbonyldiimidazole (CDI), followed by chemical conjugation of both moieties with hydroxyl groups of β‐CD , C—N stretch vibration (at 1430 cm−1), and phenyl groups (at 1680–1580 cm−1) in the Fourier transform infrared (FTIR) spectrum . Consistently, 1H NMR spectroscopy confirmed the presence of Tpl and PBAP moieties in the resulting β‐CD material, in which proton signals from methyl of Tpl and phenyl groups of PBAP were clearly observed phenol (HMP) in water. We analyzed hydrolysis products of TPCD by HPLC. At low concentrations of H2O2, hydrolysis of TPCD produced HMP after 24 h of incubation . In this case, only a small amount of Tpl was released . By contrast, exposure to high concentrations of H2O2 resulted in the generation of a considerable amount of both HMP and Tpl . This result indicated that hydrolysis of carbonate ester‐linked PBAP was more sensitive to H2O2 than that of Tpl. Measurements by 1H NMR spectroscopy and MALDI‐TOF mass spectrometry validated the production of β‐CD, Tpl, HMP, and pinacol boronate ester after complete hydrolysis of TPCD  was 0.85 mg mL−1. It was also found that the radical eliminating capacity was proportional to the dose of TPCD  and H2O2 by TPCD. As expected, TPCD was able to effectively scavenge superoxide anion and H2O2, in a dose‐response pattern after incubation for 40 min and 24 h, respectively  elimination capacity of TPCD, we used a luminescent nanoprobe developed in our previous study .The ROS‐scavenging capacity of TPCD was then examined in vitro. First, we evaluated the free radical eliminating capability of TPCD by 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) assay.2.3TPCD NPs were produced by a modified nanoprecipitation/self‐assembly method in the existence of a small amount of lecithin and DSPE‐PEG.2O2 concentration , with the decrease degree positively correlated to the H2O2 concentration. Concomitantly, the zeta‐potential value was evidently reduced after hydrolysis . Further observation by TEM also confirmed the H2O2‐dependent hydrolysis profile of TPCD NP . The smallest particle size was observed for TPCD NP incubated at 1.0 × 10−3m H2O2, suggesting dramatic hydrolysis in this case, which well agrees with the DLS result. Since the maximal extracellular H2O2 level in normal mammalian tissues is within the range of 2–4 × 10−6m,Subsequently, in vitro hydrolysis tests were performed to examine ROS‐sensitivity of TPCD NP. It was found that the hydrolysis rate and degree of TPCD NP were positively correlated to the H2.4The generation of intracellular ROS is one of the key features of inflammatory cells.FigureObservation by confocal microscopy showed time‐dependent internalization of Cy5/TPCD NP in RAW264.7 cells Figure3a. Eve−6m H2O2 resulted in significant apoptosis when compared with the control cells treated with fresh medium, as illustrated by flow cytometric analysis  and typical pro‐inflammatory cytokines (TNF‐α and IL‐1β) in cell free peritoneal lavage fluid were significantly increased in the model group treated with the saline  in peritoneal lavage fluid revealed varied degrees of efficacies after peritonitis‐bearing mice were treated with PCD NP, TCD, tenfold Tpl, and Tpl/HMP , an inflammatory pulmonary disease with the most severe manifestation of acute respiratory distress syndrome, which remains a significant source of morbidity and mortality in critically ill patients.−1 by i.v. injection. By contrast, treatment with free Tpl at a dose equals to 1.0 mg kg−1 TPCD NP showed no significant effects. Also, the pro‐inflammatory cytokines TNF‐α and IL‐β in bronchoalveolar lavage fluid were remarkably reduced after treatment with TPCD NP, particularly at 1.0 mg kg−1 ‐stained sections. The model group displayed significant infiltration of inflammatory cells and alveolar hemorrhage , no beneficial outcomes were found in inhibition of H2O2 and MPO (P > 0.05). Likewise, limited effects were obtained for TCD, 10‐fold Tpl, and Tpl/HMP. This was further affirmed by the significantly reduced neutrophil count and notably improved lung tissue structure as manifested by H&E sections , which is intimately related to oxidative stress due to the overproduction of ROS.−1. By contrast, free Tpl at a dose corresponding to 1.0 mg kg−1 TPCD NP did not significantly reduce ALT and AST. Consistently, therapy with 1.0 mg kg−1 of TPCD NP notably inhibited the abnormally increased expression of pro‐inflammatory cytokines TNF‐α and IL‐1β . In this case, the percentage of viable cells remained above 80% at 1000 µg mL−1. Likewise, when a HepG2 human liver cancer cell line was employed, almost no cytotoxicity was found after 6 h of incubation with TPCD NP at different doses . Subsequently, the potential risk of hemolysis was examined using fresh erythrocytes isolated from whole blood of Sprague Dawley rats. After incubation with various concentrations of TPCD NP for 2 h, direct observation showed negligible hemolysis , although severe hemolysis was observed in the positive control group treated with deionized water. Further quantification revealed that the degree of hemolysis was less than 1% even at a relative high concentration of 2 mg mL−1 TPCD NP .In view of the above desirable therapeutic results, both in vitro and in vivo experiments were performed to interrogate the safety profile of TPCD NP. Cytotoxicity evaluation was initially examined in RAW264.7 mouse macrophages. After incubation for 6 h, high cell viability was detected regardless of various doses of TPCD NP . Even at the dose as high as 1000 µg mL−1 in Kunming mice, all animals remained healthy and survived, without any behavioral abnormalities such as convulsions, vomiting, and diarrhea. In addition, TPCD NP‐treated mice displayed gradually gained body weight , which was similar to the control healthy mice. At day 30 after administration, mice were euthanized. All the treated mice showed the comparable organ index for major organs, including heart, liver, spleen, lung, and kidney . Also, complete blood count revealed no distinct variations in typical hematological parameters, such as red blood cell (RBC), hemoglobin (HGB), white blood cell (WBC), and platelet (PLT) . This in vivo result is in line with the finding on in vitro hemolysis tests . Furthermore, with the exception of ALT level for the i.v. injection group, we did not find abnormally increased serum levels of typical biomarkers associated with hepatic and renal functions including AST, CREA, and UREA in mice treated with TPCD NP . Likewise, examination on H&E‐stained sections of major organs from TPCD NP‐administered mice indicated that there were no distinguishable injuries and pathological patterns such as cellular edema, infiltration of inflammatory cells, necrosis, hyperemia, or changes in the morphology of vessels . Consequently, these preliminary data indicated that TPCD NP did not cause obvious in vitro cytotoxicity, in vivo systemic toxicity, and inflammatory reactions, and therefore it can serve as a safe nanotherapy.Since TPCD NP is expected to be delivered via injection, we performed safety studies after administration via i.p. and i.v. routes. After either i.p. or i.v. injection of TPCD NP at 1000 mg kg3In summary, we developed a SOD/catalase‐mimetic material capable of eliminating a broad spectrum of ROS, which can be facilely synthesized by conjugating two functional moieties onto a cyclodextrin scaffold. This pharmacologically active material TPCD can be easily processed into effective antioxidant and anti‐inflammatory nanotherapies. Cellularly, a TPCD‐derived nanotherapy protected macrophages from oxidative stress‐induced apoptosis, after internalization into cells and efficiently scavenging ROS. In several murine models of inflammatory diseases including paw edema, peritonitis, and acute lung injury, the nanotherapy TPCD NP effectively attenuated oxidative stress and inhibited inflammatory responses in diseased tissues or organs. In addition, TPCD NP was able to serve as a nano‐antidote to significantly inhibit drug‐induced liver and/or kidney injury, by eliminating overproduced ROS. In all these cases, treatment with TPCD NP afforded more beneficial outcomes than the corresponding control small‐molecule drug, mainly resulting from its passive targeting capability due to impaired biological barriers at diseased sites of inflammation or injury. Importantly, the broad‐spectrum ROS‐scavenging capacity contributed to the superior efficacy of TPCD NP. In addition, preliminary in vivo studies demonstrated that TPCD NP was safe after delivery via i.p. and i.v. routes at a dose dramatically higher than that used for in vivo therapy. Consequently, TPCD NP deserves further development as an efficacious and safe nanotherapy for treatment of numerous diseases associated with inflammation and oxidative stress. The pharmacological activities of TPCD NP may be further enhanced by surface engineering with functional moieties targeting diseased tissues or mitochondrial oxidative stress, which can also be achieved by packaging with other anti‐inflammatory therapeutics.The authors declare no conflict of interest.SupplementaryClick here for additional data file.


While several recent studies have found that a post-encoding period of quiet, eyes-closed waking rest benefits memory consolidation, others have reported null effects. To more precisely estimate this effect, we conducted a quasi-exact behavioural replication of a recent study from our lab, which found that post-training eyes-closed waking rest improved declarative memory relative to a distractor task. Contrary to our hypothesis, the observed effect was not significant; however, it did fall within the 95% confidence interval of our previous finding. Furthermore, a meta-analytic effect summarizing n = 10 similar studies indicates a moderately sized and significant benefit of waking rest for verbal memory . We argue that the apparently conflicting results in this literature are most parsimoniously explained by variability due to sampling and/or measurement error, in a group of studies often underpowered to detect a smaller-than-expected effect of rest. Additionally, exploratory analyses revealed that increased trait daydreaming frequency negatively correlated with memory retention during eyes-closed rest. Together with our replication and meta-analysis, these studies suggest that waking rest confers a small but significant benefit on memory consolidation, and that this benefit requires the mind to be free from attention to either external tasks or spontaneous thought. In several studies using declarative12, spatial8, or motor memory2 tasks, participants perform better on a delayed test when learning is followed by a short period of waking rest, rather than by an active wake control condition. This benefit may arise from reduced demand on hippocampal and other memory resources while participants are in an “offline” eyes-closed state of rest, providing an opportunity to consolidate newly learned information7. This offline resting state is distinct from active, “online” wake in that participants are not explicitly attending to any external stimuli13, nor to any internally focused cognitive task15, both of which have been shown to impede consolidation. Of note, disengagement from the external environment and the absence of directed cognition are also key features of sleep, which has long been shown to benefit memory consolidation22.Following learning, newly formed memory traces undergo a period of “consolidation”, during which memory is strengthened, stabilized against interference, and in some cases, reorganized in the brain. A nascent body of research suggests that resting with one’s eyes closed following learning may improve memory consolidation, relative to an equivalent period of active wake25. First and most obviously, both sleep and waking rest are characterized by reduced sensory input, thereby decreasing encoding demands on the hippocampus, which has been proposed as a prerequisite for declarative memory consolidation to take place7. Albeit to a lesser extent, like sleep, waking rest is also characterized by slower and more synchronous EEG rhythms relative to active wake, with increasing amounts of alpha (8–12 Hz) and theta (4–7 Hz) predominating over the faster beta (>13 Hz) rhythms of active wake. Neurobiologically, sleep and waking rest are also both marked by decreased acetylcholine levels relative to active wakefulness, and by the presence of hippocampal sharp-wave ripple complexes, both of which are proposed to facilitate memory consolidation27. Thus while sleep is a neurobiologically distinct state, key features thought to account for sleep’s memory benefit are also present during quiet waking rest. These features are absent from the typical active wake controls in studies examining the effect of sleep on memory, in which participants may watch videos28, listen to music30, or even leave the laboratory34.Indeed, offline periods of quiet rest manifest many of the features thought to account for the memory benefits of sleep13 (a feature not present in the above-cited studies9). In two other studies, waking rest did not lead to a significant improvement over the control condition for the details of a text story, word-picture pairings, or aurally presented words36. Our own laboratory conducted a study manipulating cognitive load and sensory stimulation during a 15 min period of post-training waking rest, and we found that memory was not improved by periods of low cognitive load or reduced sensory stimulation37. These non-significant effects may simply be due to sampling or measurement error, which would be expected to lead to some non-significant findings, even when testing a true effect. They might also be impacted by the relatively low sample sizes across the studies, which have lower power to detect real effects.However, waking rest has not consistently been found to benefit memory, with some recent investigations reporting null effects. For example, in one study, post-training waking rest improved memory for a word list after a delay of seven days, but unexpectedly, this occurred only when an immediate memory test was conducted at the end of the first (training) sessionet al.1) that reported a benefit of waking rest for verbal declarative memory1, using the same short story task as Dewar et al.4. Our 2016 study found that 15 min of eyes-closed rest following learning led to better memory for the short story than 15 min spent completing a distractor task1. This behavioural observation was the same as that reported by Dewar et al. in their 2012 paper4. Analysis of EEG recordings across the 15 min rest period in Brokaw et al.1 also found that improved memory in the waking rest condition was associated with an increased slow EEG oscillation rhythm (0.3–1 Hz) and decreased alpha rhythm (8–12 Hz). Improved memory in the distractor task condition also correlated with more time spent thinking about the past and the future, and less time thinking about the current task. Together, these results suggested that waking states characterized by EEG slowing and spontaneous, unstructured thought facilitate memory consolidation.To more accurately estimate the effect of waking rest on memory in the current study, we conducted a quasi-exact replication of our 2016 paper , all of which closely followed the procedures of Brokaw et al. (2016). We predicted that, as in Brokaw et al.1, resting after hearing a short story would lead to significantly better free recall of story details at the delayed test. We also predicted that improved memory in the distractor task condition would correlate with more time spent thinking about the past and the future, and less time thinking about the current task.Through the Open Science Framework (OSF), we preregistered the hypothesis, procedure, and analyses for this project in advance of data collection ; a 15-item scale designed to assess mindfulness, a concept which is inversely correlated with “mind wandering”)40 and daydreaming ; assessing trait daydreaming frequency)41, which allowed us to conduct exploratory analyses of the relationship between spontaneous thought and the memory benefits of rest. We predicted that greater daydream frequency and mindwandering propensity would positively correlate with memory for the short story.We also anticipated spontaneous thought as a potential mediator of the effect of waking rest on memory, as spontaneous thought may arise from the reactivation of newly encoded memorieshttps://osf.io/w7x4p/). Unplanned analyses are noted.All of the primary confirmatory analyses were preregistered . Two raters scored the responses for correct recall (interrater agreement 97%), and one of these raters and another new rater scored the responses for false recall (interrater agreement 73%). Final scores used for analysis were the mean of the two raters’ scores.Short story free recall responses were scored by two raters blind to condition, who counted the number of correctly and falsely recalled items. Each short story contained 25 discrete pieces of information. To score correct recall, one point was given for each correctly recalled item, with total possible scores ranging from 0 to 25 points, following the procedure in the Wechsler Memory Scale ManualThree recall scores were calculated for each test: the number of correctly recalled items, the number of falsely recalled items, and the total number of recalled items . Raw change in recall was calculated as (delayed test score - immediate test score), and percent change was calculated as ((delayed score - immediate score)/immediate score).t(30) = 0.37, p = 0.71, d = 0.09; total recall: t(30) = 0.07, p = 0.95, d = 0.02)  = 0.83, p = 0.41, d = 0.15; total recall: t(30) = 1.26, p = 0.22, d = 0.25).Rest did not benefit raw change in memory relative to the distractor task  in correct, false, and total recall across the retention interval. As predicted, the results mirrored those for raw change: % change in correct, false, and total recall did not differ significantly between the waking rest and distractor task conditions. There was also no difference between conditions in baseline false recall or raw change in false recall  = 0.47, p = 0.64, d = 0.11), but did differ at the delayed test (t(30) = 2.04, p = 0.05, d = 0.40), when participants in the waking rest condition reported being sleepier. This was substantiated by a non-preregistered 2 (immediate test vs. delayed test) x 2 (waking rest vs. distractor task) ANOVA revealing a Time x Condition interaction: F(30) = 5.87, p = 0.02, ƞp2 = 0.16. However, as expected, there was no significant correlation between sleepiness at test and raw change in correctly recalled items across the retention interval, r(31) = 0.10, p = 0.56.As predicted, participants in the waking rest and distractor task conditions did not differ in sleepiness (SSS) at the immediate test (r(31) = −0.07, p = 0.70.Contrary to our hypothesis, there was no correlation between the time spent thinking about Snood, as reported in the distractor condition exit questionnaire, and the raw change in correct recall in the distractor condition, negatively correlated with change in correct recall over waking rest (raw change: r(31) = −0.51, p = 0.004; % change: r(31) = −0.47, p = 0.008; Fig. r(31) = 0.41, p = 0.03. This was contrary to our hypothesis that daydreaming frequency would correlate positively with change in correct recall over waking rest (raw and %), and that it would not correlate with change in false recall. Daydream frequency was also negatively correlated with raw change in total recall in the distractor task condition, r(31) = −0.39, p = 0.03. To explore the association between trait daydream frequency and subjective experience during the retention interval, we additionally ran non-preregistered correlations between daydream frequency score and subjective experience as assessed via the exit questionnaire. Daydream frequency correlated negatively with time in a “mind blank” state, r(31) = −0.42, p = 0.02, but was not associated with any other category of experience (Supplementary Table Trait mindwandering and daydreaming propensity both predicted memory retention across rest. Daydreaming frequency was strongly r(31) = −0.39, p = 0.045  = −0.48, p = 0.006). Additional analyses are reported in Supplementary Table Trait mindwandering scores from the MAAS correlated negatively with % change in false recall over waking rest, r1 = −0.39t(30) = 2.27, p = 0.03, d = 0.33). There was, however, no difference between conditions in ratings of how refreshed participants felt (t(30) = 0.19, p = 0.85).In line with our prediction and the above-reported tendency for participants to be sleepier following the rest vs. distractor condition, participants also rated their ability to concentrate at the delayed test as lower after waking rest, relative to after the distractor (t(30) = 2.99, p = 0.006, d = 0.62) and imagining the story (t(30) = 2.89, p = 0.007, d = 0.59) more frequently during rest than during the distractor task  = 2.56, p = 0.02, d = 0.52) and the future (t(30) = 3.10, p = 0.004. d = 0.75) during waking rest than during the distractor task and, specifically, more about something that happened last year or several years ago (t(30) = 2.36, p = 0.03, d = 0.43), the remainder of the day (t(30) = 2.48, p = 0.02, d = 0.61), and tomorrow to next week (t(30) = 2.42, p = 0.02, d = 0.54). Participants also reported spending more time doing “focused meditation” (t(30) = 2.27, p = 0.03; no participants reported spending any time doing focused meditation during the distractor task) and counting the time (t(30) = 2.42, p = 0.02, d = 0.64) during waking rest. In contrast, participants reported spending more time thinking about their current activity during the distractor task than during waking rest (i.e. thinking about Snood/resting with eyes closed t(30) = 6.38, p < 0.001, d = 1.49). Additional analyses are reported in Supplementary Table As expected, participants reported spending more time thinking about the past (r(31) = −0.42, p = 0.02. However, this correlation is likely spurious, being driven solely by a single participant who reported spending 60% of the time sleeping. Non-preregistered analyses confirmed that the non-significant effect of waking rest on memory was unaltered when this participant was excluded from analysis  = 0.22, p = 0.82; total recall: t(29) = 0.22, p = 0.83). Additionally, thinking about the story during waking rest or during the distractor task, as reported on the rehearsal questionnaire, was unrelated to memory . This may provide a more accurate estimate of the true size of rest’s effect than any single previous study. Taken together, the aggregated evidence is consistent with the existence of a memory benefit of waking rest. Given that effect sizes are often overestimated in early published studies describing a new effect of interest43, it is not surprising for subsequent research to reduce the estimate. Knowledge that the true effect of post-training rest on verbal memory may be in the medium-to-small effect range can usefully inform the planning of future studies, enabling researchers to plan for sample sizes that will power studies sufficiently to detect this effect.The summary effect computed from all available published studies is statistically significant, but suggests that the size of the effect of rest on verbal memory may be smaller than previously thought , a state that was negatively associated with daydream frequency in the current study.7. Accordingly, it is possible that hippocampal resource engagement during daydreaming and mindwandering functions to interfere with memory consolidation. This hypothesis is in line with two recent studies showing that engaging the hippocampal memory system via an autobiographical memory recall task impairs memory for previously learned information, even in the absence of directed attention to any external task15. Thus, it may be that the particular state of resting wakefulness that most strongly benefits memory is one during which spontaneous cognition is at a minimum and the default network is not strongly engaged.The hippocampus is a crucial element of the default network, and its engagement has been hypothesized to inhibit offline consolidation from occurringIn summary, together with previous studies, our current observations help to more accurately estimate the effect of waking rest on memory. Although large effect sizes were reported in early studies with this same experimental design, subsequent work, including the present failed replication, moves us toward a more modestly sized estimate of rest’s impact on verbal memory. This summary effect (d = 0.38) can serve as a useful guide for planning sufficiently powered future studies.https://osf.io/w7x4p/.All study procedures and analysis methods described here were preregistered on Open Science Framework prior to beginning data collection. The preregistration is publicly available here: et al.1 (dz = 0.60).A target sample size of n = 32 participants was set by determining the number of participants needed to achieve power of 0.90 for detecting the within-subjects effect of post-training rest vs. distractor task on raw improvement in memory score reported in Brokaw 49 , speaking English as their native language (n = 2 enrolled participants excluded); and having not already participated in a study using the same story task (n = 2 enrolled participants excluded). We concluded enrollment when we had reached n = 35 usable participants according to these criteria.Participants (n = 48) were tested in small group sessions of up to 5. Enrollment continued until we reached the target number of participants meeting our preregistered inclusion criteria of: Correctly following study instructions (n = 4 enrolled participants excluded), obtaining an average ≥5 hours of sleep on the 3 nights preceding participation , scoring ≤5 on the baseline Stanford Sleepiness ScaleAn additional n = 4 participants had to be excluded for outlying raw improvement scores . Thus, we arrived at a final sample size of n = 31 participants , who slept an average of 8.21 ± 0.91 SD hours on the three nights preceding the experiment; see Table Participants were recruited by email, advertisement, or word-of-mouth, and were compensated by receiving either $10/hr or credit for an introductory psychology course. All participants signed written informed consent. The study was approved by the Furman University Institutional Review Board, and the methods were carried out in accordance with the relevant guidelines and regulations.50. Participants were tested in group sessions of up to 5 individuals in a computer-lab setting. All participants within a single session were assigned to the same condition and counterbalancing order . There were four possible counterbalancing orders – waking rest or distractor task first and Story A or Story B first – which were assigned to experimental sessions in a block randomized order. Counterbalancing order had no effect on immediate or delayed story recall performance in either condition.The experimental timeline is illustrated in Fig. et al.1 closely, differing only in that (1) the current study did not include EEG recording, (2) the instructions for the distractor task were given at the beginning of both sessions, (3) up to five participants could complete the study together, and (4) there was a minimum 24 hr delay between conditions.The procedure followed that of Brokaw 51, and a 3-day retrospective sleep log. The experimenter then explained the rules of the distractor task, the computer game “Snood” (www.snoodworld.com). Participants practiced the game for one minute, then completed the Stanford Sleepiness Scale (SSS), a measure of state sleepiness.Upon arrival at the laboratory, participants signed informed consent, then completed initial paperwork including a demographic survey, the Epworth Sleepiness Scale 42. Immediately afterward, participants completed an initial free recall test (immediate test) during which they attempted to recall as many of the details of the story as possible. Afterward, participants began the 15 min retention interval, during which they either rested with their eyes closed or completed the distractor task (Snood). In the rest condition, participants were instructed to sit upright in their chairs with their eyes closed and to remain awake. They were not given any instructions about what to do mentally during the interval. In the distractor condition, participants played Snood for 15 min, starting a new game every time they won or lost. Immediately following the experimental phase, participants were given a delayed recall test. The research assistant was present in the room with participants for the duration of the experiment, and monitored participants to ensure they were complying with instructions (e.g. remaining sitting upright with eyes closed in the rest condition and playing Snood for the entirety of the distractor task condition).The experimenter then played an audio file of a short story (approx. 30 sec) about a school cafeteria worker who was robbed (Story A) or a truck driver who skidded off the road (Story B)Following the delayed recall test, participants completed the post-test SSS and completed an exit questionnaire that assessed subjective experience during the retention interval . This questionnaire included multiple-choice questions about whether participants rehearsed the story and followed task instructions, about their “thoughts, feelings, or daydreams” during the interval, and the proportion of the interval they spent in one or more of 13 pre-defined mental categories: “thinking about the past” , “imagining the future” , “thinking about the short story”, “thinking about [playing Snood]/[resting with eyes closed]”, “counting the time”, “mind was blank”, “meditating”, “sleeping”, “thinking about something else”, and “other”.40 and the Daydreaming Subscale of the Imaginal Processes Inventory (DFS)41. They were then debriefed and compensated.The first session ended after participants completed the exit questionnaire. Following the second session, participants also completed the Mindfulness Attention Awareness Scale (MAAS)Detailed information about the short story task and Snood can be found in Supplementary Materials.To integrate our current findings with the prior literature, we additionally calculated a meta-analytic summary effect across all studies of the impact of waking rest on verbal memory that used the same fundamental experimental design. This analysis was not pre-registered. Based on a literature search , we identified n = 11 prior studies that also tested the effect of a short (5–20 min) period of post-learning eyes closed rest on verbal memory in healthy controls, as compared to a visuomotor task control condition.1) the measure described as the primary outcome, or if that was not clear, 2) the first measure reported. If memory was tested at multiple time points (e.g. both immediately after rest and a week later), we selected the time point most comparable to that used in the current study . If multiple age groups were tested, we selected the effect that encompassed all ages tested, or if that information was not available, we selected the effect for the age group most comparable to that of our current study (young adults). All effect sizes were converted to standardized mean differences (SMD: www.cebm.brown.edu/openmeta.Details of the included studies are described in Supplementary Table Supplementary Information