Sleep is vital for human health and wellbeing, and sleep disturbances are comorbid to many mental and physiological disorders. Music consistently improves subjective sleep quality, whereas results for objective sleep parameters diverge. These inconsistencies might be due to inter-individual differences. Here, 27 female subjects listened to either music or a control text before a 90 minutes nap in a within-subjects design. We show that music improved subjective sleep quality as compared to the text condition. In all participants, music resulted in a reduced amount of sleep stage N1 during the nap. In addition, music significantly increased the amount of slow-wave sleep (SWS) and increased the low/high frequency power ratio. However, these effects occurred only in participants with a low suggestibility index. We conclude that listening to music before a nap can improve subjective and objective sleep parameters in some participants. However, sleep disturbances are highly common in our society8, with increased prevalence in ageing9 as well as among people at risk of or suffering from a psychiatric disorder10. The use of sleep enhancing medicine is problematic, as its effectiveness decreases across time and may lead to addiction. Consequently, researchers need to empirically validate the effectiveness of non-pharmacological and easy to implement tools to support healthy sleep.Sleep plays an important role for maintaining physical and mental health11. In a survey in over 500 patients with sleep disorders, over 50% reported to use music as sleep aid12. In a meta-analysis based on six included studies in a total of 314 patients, Jespersen and colleagues reported that music helped to improve subjective sleep quality in insomnia patients13. Similarly, sedative music while resting could effectively improve subjectively rated sleep in patients with sleep complaints14. In 20 included trials, another meta-analysis showed positive effects of music based interventions before bedtime on PSQI scores, sleep onset latency and sleep efficiency in patients suffering from primary insomnia15. Positive effects of listening to music on subjectively evaluated sleep were found in toddlers when listening throughout the naptime, preschool children and young adults listening 45 minutes at naptime and/or bedtime18. Also in older women, two further studies reported shorter sleep onset time, less nocturnal awakenings and better sleep quality and satisfaction as measured with a questionnaire and sleep logs when listening to music when going to bed20. In sum, the positive effects of listening to music on subjective ratings of sleep quality are well established across different age groups including both healthy participants and patients.Listening to music is a widely used tool to improve sleep. In an online survey in a general population 62% (out of n = 651 respondents) stated to have at least once used music to help them sleep21 did not find differences in polysomnographic measures when subjects in a within design either listened to music, tones or neither tones nor music after lights off until continuous sleep was observed. Similarly, Chang et al.22 did not observe any positive effects of music playing when lying in bed on objective measures of sleep onset, total sleep time, sleep interruption and sleep efficiency, in spite of positive effects of music on subjective sleep quality. Only Chen et al.23 reported effects of music on objective sleep parameters: One hour of listening to music after subjects went to bed significantly decreased the amount of stage N2 sleep, and increased deep SWS only in a subgroup of participants with long sleep latencies. However, further, more fine grained analyses of sleep-related oscillations are missing in this study. This paper points towards the possibilities that the effects of music on objective sleep parameters might depend on certain individual differences.In contrast to subjective sleep quality, empirical findings on the effects of music on objectively measured sleep are scarce and inconsistent. For example, Lazic and Ogilvie24. Suggestibility accounts for strong individual differences in the effects of hypnotic interventions, but also outside of hypnosis, like for instance “placebo” effects25 (but see26). Also, a suggestion to not attribute meaning to Stroop words reduced the Stroop effect in highly suggestible subjects irrespective of whether a hypnotic state was established before or not27. It was even shown that suggestions impact on memories, beliefs and behaviors28. In a series of studies29, we have shown that hypnotic suggestion to sleep deeper increases deep SWS and slow wave activity (SWA) in high suggestible healthy participants. No enhancement of SWS was observed for low suggestible females. A similar pattern of results occurred in a conceptual replication of this study in elderly women30. As we here applied the same study design as in those previous experiments, we were interested if the effect of music on sleep also depends on expectations and self-suggestions. We expect that the effect of music on objective sleep parameters and sleep related oscillations in the electroencephalogram (EEG) is stronger in high vs. low suggestible participants.One possible factor being involved in individual differences in the effects of non-pharmacological interventions on objective sleep may be suggestibility. Suggestibility describes the ability of a person to respond to suggestions in terms of perceptual, cognitive, neural and bodily processes31. It was pre-selected in a pilot study due to its best effects on subjective ratings of sleep quality. As it is supposed to induce deep sleep through its frequencies of 0.01–2 Hz we also analyzed brain activity during listening to the sound. To foster the role of subjective expectations and beliefs, subjects were explicitly informed that the music composition used was specifically designed to deepen sleep. Consequently we also assumed to find increased low and reduced high frequency EEG activity during the nap following the music condition. In order to test possible consequences of altered sleep on cognitive performance, we additionally introduced memory tests before and after the nap.In the current study we tested if listening to music as compared to a spoken text before a midday nap improves sleep quality in high vs. low suggestible females. We measured sleep objectively using polysomnography. The experimental design was identical to our previous studies on the effects of hypnotic suggestions on sleep, except that a musical piece instead of the standardized hypnotic suggestion was used. The musical piece was composed by Dr. Lee Bartel for promoting sleep, called “Drifting into Delta”trackF = 4.36, p = 0.048, eta2 = 0.15). The interaction with suggestibility did not reach significance (p = 0.15).As expected from previous studies, subjects reported better subjective sleep after music  as compared to the control text (3.28 ± 0.18) as indicated by a significant main effect of sound on the dependent variable subjective sleep quality  = 5.39, p = 0.029, eta2 = 0.18, see Fig. p ≥ 0.15 see Table p = 0.035), whereas a non-significant reduction of SWS  occurred in high suggestible participants  = 4.37, p = 0.047, eta2 = 0.15), See Fig. p = 0.027). In contrast, no difference in time spent in SWS occurred for high suggestible participants . Both results were not affected when considering that during listening to music, 9 low and 11 high suggestible subjects fell asleep while 4 low and 3 high suggestibles did not .A chi-square test comparing the number of subjects falling asleep and suggestibility was non-significant (Chi2(1) = 0.31, p = 0.58). During listening to the text, 11 low and 11 high suggestibles fell asleep, while 2 low and 3 high suggestibles did not (Chi2(1) = 0.69, p = 0.16).For objective measures of sleep architecture, only one parameter revealed a significant effect: Listening to music before the 90 min nap decreased the time spent in N1 sleep (6.13 ± 0.73 min) as compared to the control condition  = 6.39, p = 0.018, eta2 = 0.20), as demonstrated in Table p > 0.15), we refrained from further analyses of this finding due to the very low sample size in this analysis.Additionally, the interaction between sound and suggestibility for REM sleep was significant . Therefore, the poweranalyses are based on the absolute muV values in the single frequency bands.For a more fine grained analysis of the effects of music on sleep, we compared power of sleep-related brain oscillations during NREM sleep stages N2 and SWS between the music and the control condition. The analyses were conducted using repeated measure ANOVAs with within-subject factors FCP , hemisphere (right vs. left) and type of sound (music vs. text) and between-subjects factor suggestibility (high vs. low). Total power  did not differ significantly between the two experimental sessions (32 and33). Higher values thus mean a higher proportion of low than high frequency power in the signal during NREM sleep and might be associated with more restorative sleep. Music significantly increased the low/high frequency ratio during sleep as compared to the control text only in low suggestible subjects  = 5.72, p = 0.025, eta2 = 0.19). The post-hoc t-test showed a significantly higher ratio after music (154.07 ± 32.69) than after text (89.67 ± 15.46), t(12) = −2.45, p = 0.031), while this difference was non-significant in high suggestible subjects (p = 0.65). The effects were wide-spread across the whole scalp, see Fig. F = 3.90, p = 0.027, eta2 = 0.14). Following up on this revealed that in high suggestibles, FCP * sound was non-significant (p = 0.64), while the interaction was significant in low suggestible subjects  = 3.40, p = 0.05, eta2 = 0.22. Here, all post-hoc tests were significant and showed a higher ratio in frontal (t(12) = 2.25, p = 0.044), central (t(12) = 2.57, p = 0.024) and parietal electrodes (t(12) = 2.71), p = 0.019). All other effects with sound or suggestibility in this ANOVA were non-significant (p > 0.06).We focused on the ratio between low and high frequencies during NREM sleep, as this marker has been discussed as indicator for restorative sleep  = 3.37, p = 0.08, eta2 = 0.12). No other main effects or interaction with those two factors were significant . When only investigating SWA in SWS this pattern remained. Please note however, that in this analysis 2 low suggestible subjects had to be excluded as they did not show SWS in one of the sessions. Examining beta power alone revealed a significant three-way interaction between sound, suggestibility and FCP  = 6.55, p = 0.003, eta2 = 0.21). While in high suggestibles, beta power did not differ depending on sound (p > 0.80) or FCP* sound (p > 0.10), a main effect of sound  = 5.46, p = 0.038, eta2 = 0.31) and its interaction with FCP  = 4.56, p = 0.021, eta2 = 0.28) were found in low suggestibles. After listening to music, beta power was lower in low suggestibles (0.59 ± 0.09) than after text (0.67 ± 0.08). This difference was most pronounced in frontal electrodes (t(12) = −2.55, p = 0.026)) and central electrodes (t(12) = −2.26, p = 0.043) and not significant in parietal area (t(12) = −1.99, p = 0.070)).Analyzing SWA alone revealed a trend in the interaction between suggestibility and sound (p > 0.20).We did neither find effects of sound in theta nor in alpha power  = 6.06, p = 0.02, eta2 = 0.20. The interaction between sound and suggestibility was a trend  = 3.90, p = 0.06, eta2 = 0.14) and further specified by the three-way interaction between sound, FCP and suggestibility  = 6.20, p = 0.004, eta2 = 0.20). In low suggestibles, sigma band power was higher in frontal and central electrodes after text compared to music (t(12) = −2.69, p = 0.02 and t(12) = −2.31, p = 0.04, respectively), while in high suggestibles, all direct comparisons were non-significant , see Fig. p > 0.20). Due to significant effects in the power range of spindle activity, we analyzed number and density of spindles during NREM sleep. All effects were however non-significant . Also no significant main effects occurred . All effects of density and number of parietal fast spindles were p > 0.50.Interestingly, power in the sigma band was reduced after music (1.19 ± 0.09) compared to the text condition 1.26 ± 0.10), 6 ± 0.10,p > 0.80). Also, neither the main effect of sound nor suggestibility reached significance (both p > 0.60). Similarly, during subsequent sleep, heart rate did neither differ depending on sound (p = 0.44), suggestibility (p > 0.70) nor sound * suggestibility (p > 0.60).As low suggestible participants profited more from relaxing music, it might have been possible that music induced stronger responses on the autonomic and central nervous system already during the listening period. In the ANOVA with sound and suggestibility on heart rate during listening, the interaction was not significant  = 4.79, p = 0.038, eta2 = 0.16). In low suggestibles, the main effect of sound and all its interactions were p > 0.08 with non-significantly higher values during music than sound. In high suggestibles all follow-up tests were p > 0.20. The same pattern was true for the four-way interaction with FCP  = 4.21, p = 0.020, eta2 = 0.14) and its follow-up tests . All main effects and interactions with sound or suggestibility were p ≥ 0.09 in the alpha and p > 0.10 in the theta and sigma band .In this analysis, we observed a significant three-way interaction between sound, suggestibility and hemisphere for the SWA/beta ratio (p > 0.09). Thus, attentional processes after sleep did not differ dependent on condition.To investigate effects of altered sleep pattern on post-nap cognitive performance, we included vigilance task after sleep. Here, performance was equal in both groups and after both conditions concerning reaction times, number of reactions and error rate  = 4.45, p = 0.05, eta2 = 0.16) demonstrated that maintenance of memory performance across sleep was higher after listening to the text (99.89% ± 1.24) than across sleep after listening to music (94.95% ± 1.61). The other effects were p > 0.10. PAL performance across sleep with presleep memory set to 100% were 96.00 ± 1.87% after music and 101.25 ± 2.09% after text in low suggestibles and 94.04 ± 2.56% after music and 98.72 ± 1.46% after text in high suggestibles. Both differences were p > 0.10. The difference between PAL performance across nap after listening to music vs. text did not correlate with the difference in any sleep stage  nor subjective sleep quality (p > 0.20), nor the difference in any power band .Also, we measured memory consolidation across sleep by presenting paired associate learning task (PAL) before and after the nap. For this parameter, a main effect of sound  was also reduced during NREM sleep in the nap after listening to music versus listening to a text. Thus, overall, it can be concluded that listening to relaxing music before a nap can reduce time spent in N1 and the power of high frequency bands.Here we tested if listening to relaxing music before a nap helps improving subjective and objective sleep quality of a nap in young healthy females. Our results showed a generally improving effect of listening to music on subjectively rated sleep quality. Subjects reported better sleep quality after having listened to music compared to listening to a control text. This is in line with previous studies showing beneficial effects of music on sleep ratingsAs we have seen in previous studies on the effect of hypnotic suggestions on a nap in healthy young and older females, it was important to consider subjects’ suggestibility. This measure indicates how sensitive subjects react to suggestions given externally. Concerning hypnosis, only those who were responsive for suggestions benefitted from the verbal, hypnotic intervention. To investigate whether suggestibility is also a relevant factor when testing the effect of music on sleep, we included this additional factor. We found that low suggestible subjects benefited from an increase in the amount of SWS of about 46% compared to their sleep after listening to the text. Whether this increase was to the detriment of the amount of REM sleep cannot be answered in this study due to a very limited amount of subjects reaching REM sleep at all during this nap. Additionally, as this was only a nap study we cannot make substantiated statements concerning REM sleep which usually takes place late in a sleep epoch. To confirm a possible trade-off between SWS and REM sleep the study should follow a nighttime design.34, we expected reduced high and increased low frequency power after listening to the relaxing music before sleep. A ratio quantifying the proportion of high and low frequency power in the NREM signal was calculated by dividing SWA power by beta power. Thus, higher values indicate a higher proportion of low compared to high frequencies in the signal. Low values had been referred to as an indication of worse sleep quality34 and less restorative sleep35. Here we found higher SWA/beta ratios during sleep after listening to music than after listening to the text in low suggestible subjects. No changes were observed in high suggestible participants. These results using objective sleep data suggest that low suggestibles might have experienced a more restorative sleep after listening to music.Also in a more fine grained analysis of oscillatory power bands during sleep, the level suggestibility significantly altered the effects of music. As studies have shown that insomniacs often experience low sleep quality when a high amount of high frequency penetrates their sleep36), while here we report the opposite case. A possible explanation is that music reduces more long-lasting sigma power in low suggestible which might be unrelated to discrete sleep spindles .In spite of more restorative sleep, sigma power in frontal and central brain regions during sleep was significantly reduced in low suggestibles after listening to music. However, we did not observe a reduction in sleep spindles in this condition, in spite of the reduction in sigma power. Previous studies have reported changes in spindle density without changes in sigma power  discussed that familiar, self-chosen in contrast to given music might be less analgesic and anxiolytic11. These authors showed that particularly subjects with musical engagement use music to improve sleep. In our data, the distribution of subjects playing an instrument did not differ between high and low suggestible subjects. However, it might have possibly required a more precise measure of familiarity with music to test this assumption. Besides, effectiveness of a sleep-related intervention depends on whether sleep destabilization is triggered by psychological  or physical  factors. Being explicitly and verbally guided by hypnotic suggestions could thus be more expedient when rumination must be stopped while reducing physical tension could be better achieved by a more indirect and open form of relaxation intervention. Unfortunately, we did not measure those aspects in our sample to test group differences in this respect. Cardiac responses during the listening period and brain activity during wakeful listening remained unaffected. Probably, a combination of both, suggestions shaded with relaxing music could be beneficial for both needs and thus, all subjects could improve their sleep.Interestingly, memory consolidation across the sleep interval was significantly reduced after listening to music as compared to the control text in the entire sample. The reduction in memory is particularly puzzling, because listening to non-verbal music after learning should induce less interference than the verbal control text. As SWS has been implicated in processes of memory consolidation during sleepThe importance of focusing on subjective and objective measures when investigating effects of any intervention on sleep was demonstrated by the fact that in neither of the groups the ratio or the amount of SWS was correlated with subjective sleep quality rating. Possibly separate mechanisms affect subjective and objective measures leading to converging results.In sum, here we show that subjective sleep quality in young healthy females’ naps can be improved by relaxing music to some extent. Music reduced time spent in N1 and arousing high frequency power during following NREM sleep while leading to improved subjective sleep quality. This method seemed to be particularly beneficial for subjects low in suggestibility, although this conclusion should be tested critically in further studies and possible other confounding factors should be considered. Taken together, our results support the conclusion that music is a non-pharmacological, low-risk and low– cost tool to improve sleep on a subjective and objective level. To what extent this is applicable to sleep disturbances cannot yet be answered.p = 0.29), four indicated to practice any kind of relaxation exercise. The subjects were German natives or had advanced German skills. They did neither regularly take naps, nor suffer from a diagnosed sleep disorder nor consume drugs. Hormonal contraceptives were allowed. None did shift work or intercontinental flights within 6 weeks before participation. For the experimental days they were asked to refrain from caffeine and alcohol, get up between 7 and 8 a.m. and to not do any sports until the session. Subjects received oral and written study information and gave their written informed consent before participation. They were paid 140 CHF for full participation. The Ethic Committee of the University of Zurich approved the study and the experiment was performed in accordance with the existing regulations.Thirty-two healthy, right-handed women  participated. To avoid gender effects we excluded males. Three subjects had to be excluded due to lacking sleep in one of the sessions, reporting naps in the week before the experiment or heavy cough during the session. In two subjects suggestibility could not be measured, one subject did not correctly understand the declarative memory test and was excluded in that analysis. The final sample consisted of 27 subjects (aged 23.22 ± 3.85 years). Ten of them indicated to play an instrument . Subjects were awakened 90 minutes after switching the lights off and asked to fill out the sleep quality questionnaire and perform again on the memory tasks. After each session, the sleep diary for the upcoming week was handed out.31. It is supposed to induce deep sleep through its frequencies of 0.01–2 Hz, based on treatises on the influence of auditory pulsing of 0.25 to 2 Hz frequencies on oscillatory coherence40. Please find the spectrogram displaying the contained frequencies in the Supplementary Material. The series “Music to Promote Sleep” out of which we took this piece was tested before in patients suffering from fibromyalgia and showed positive effects on subjective sleep quality41.Music: Before the experimental naps, subjects either listened to a music tape or a spoken text. The selection of the musical piece was performed in a pilot study: Five 15 minutes-pieces of music, composed to induce sleep, were selected according to internet evaluations . Fifteen subjects listened to this selection before falling asleep and rated the tapes the next morning. According to subjectively reported enhancement of sleep quality, shortened sleep latency and better recovery, the best rated tape was chosen for the study. It was a piece composed by Dr. Lee Bartel for promoting sleep, called “Drifting into Delta”tracket al.29 and was a documentary about mineral deposits. A text was chosen as control to exclude a simple effect of listening. It was spoken by a male voice in normal speed and intonation.Text: The text in the control condition was taken from Cordi Both tapes played for about 14 minutes. They were presented at a volume of 45–50 dB through loudspeakers before falling asleep. The order was determined randomly across subjects. Before playing the sound we instructed the subjects to relax and to get themselves into the tape, trying to let pass any thoughts that might come up and to not stuck on them. Subjects were allowed to fall asleep whenever possible.42. As a measure for subjective sleep quality we analyzed the mean of an item asking to rate the previous sleep on 7 adjectives. We excluded the adjective “extensive” as the nap was limited in time by protocol. Higher values indicate better ratings. Data of one subject is missing.Standardized Sleep Inventory for the Assessment of Sleep – Revised Version, SFA – R: It assesses subjectively reported sleep behavior within the last sleep period (adapted to a nap here) on several dimensions43. The global score ranges from 0 to 21, while 5 is the cutoff value for sleep difficulties. Overall, the sample scored 4.41 ± 2.26 ranging from 1 to 10 (5 subjects scoring >5). High and low suggestible subjects did not differ on this score (p = 0.83).Pittsburg Sleep Quality Index, PSQI: The PSQI measures general subjective sleep quality within the last monthSleep diary: To control sleep behavior one week before each of the experimental sessions, a sleep diary was used. We used the information about the wake up time to verify that subjects got up between 7 and 8 a.m. on experimental days.44. After listening to a tape with a recorded hypnosis, subjects are confronted with a questionnaire on their experiences during listening. According to their rating about how strongly they had reacted to the given suggestions, they were grouped as high  or low suggestible . The groups differed significantly on those means, t(25) = −7.78, p < 0.001, but not on age (p > 0.50).Harvard Group Scale of Hypnotic Susceptibility, Form A, HGSHS: A: This is a widely used, standardized tool to determine hypnotic suggestibility45. The words are presented consecutively in EPrime in black font on a white computer screen. After a 500 ms fixation cross, the first word of the pair was presented for 1000 ms, followed by a blank interval of 200 ms separating it from the according second word of the pair. Another blank interval of 500 ms was displayed before the next fixation cross separating the previous from the following word pair. During cued recall, the first word of the pair was displayed until the subject remembered the according second word or indicated that it was forgotten. No feedback was given. The order during recall was different from the order during learning, but stable across subjects. Recall was tested directly before the nap and afterwards in the same order. Memory performance was defined as the number of correctly recalled words after sleep relative to the presleep performance which was set to 100%.Word pair associate learning task, PAL: In this episodic memory task, subjects are asked to remember as many of the presented word pairs as possible for a later cued recallThe procedural memory task and its results are reported in the Supplementary Material.Psychomotor vigilance task (PVT): To overcome sleep inertia and to control for possible attentional differences before the memory tests after the nap, a vigilance task was applied. Subjects were confronted with a black screen on which a timer started to count upwards at unforeseen times. As soon as this was recognized, subjects were asked to press the space bar. Error rate and reaction time were analyzed.Sleep was measured with the Geodesic EEG System 400 series  including high density electroencephalogram (EEG) with 128 electrodes, electromyogram (EMG), electrooculogram (EOG) and electrocardiogram (ECG). Data was collected with a sampling rate of 500 Hz, impedances were kept below 50 kΩ. Data was filtered and scored according to the criteria of the American Association of Sleep Medicine (AASM) . Three coworkers blind to condition scored the data at the electrodes F4, C4, P4, O2, HEOG, VEOG and EMG. In case of disagreement, a forth blind scorer was consulted.32 and Maes et al.33 we focused on the ratio between low and high frequencies during NREM sleep as an indicator for restorative sleep. SWA power was divided by beta power and hence higher values would indicate slowing of the EEG activity.Power analyses were based on Fast Fourier Transformations (FFT). During data preprocessing we excluded the electrodes on the outer edge of the EEG cap , filtered the data between 0.1 and 50 Hz and applied a 50 Hz Notch Filter. We re-referenced data to the average of both mastoids. The signal that was recorded while the music and the control file were presented was segmented into epochs of 4096 data points (~8 seconds) with an overlap of 409 points to account for the later applied Hamming-window of 10% and semi-automatically corrected for artefacts within the wake periods in the 14 minutes in which subjects listened to the tape. The same was done for segments scored as NREM sleep. Afterwards, the FFT was calculated with a Hamming-window of 10% and a resolution of 0.2 Hz. From this analysis, we exported the power in the respective areas (muV*Hz) for slow-wave activity, SWA (0.5–4.5 Hz), theta (4.5–8 Hz), alpha (8–11 Hz), sigma (11–15 Hz), beta (15–30 Hz) and the total power (0.5–50 Hz). Similarly to KrystalFor the analyses on spindle density, we selected the derivations Fz, Cz and Pz for the analysis of the spindles across all sleep stages. On those, we performed a frequency extraction for frequency power of 11–15 Hz and added Spindle on and off markers to identify its on- and offset. Before automatic artifact correction detecting a maximal allowed difference of 200 muV within 200 ms in the EMG channel, we segmented the data into 1024 data point wide epochs (~2 seconds). Spindle events were counted for which the power signal exceeded a fixed threshold (±10 muV) for an interval lasting 0.5–3 sec. We extracted the amount of detected spindles (=spindle count) in those epochs separately for each sleep stage and separately for fast (13–15 Hz) and slow spindles (11–13 Hz). We then calculated spindle density per 1 minute within each sleep stage, also within NREM sleep (N2 + N3).Heart rate: We analyzed electrocardial data with Kubios HRV Version 3.1 2. We first ran an automatic artifact correction on the unfiltered data that eliminated ectopic beats and artifacts based in dRR series. Afterwards, we excluded further visually detectable artifacts resulting from e.g. movements. Mean heart rate (HR) was measured for the time in which the subjects listened to the tapes and the following sleep episode separately. We could include all except 2 subjects for who the signal was too bad in one of the two sessions .p = 0.05.The study followed a crossover design with a within-subjects comparison of the two naps. Data was analyzed using SPSS 23. The repeated measures analysis of variance (ANOVA) included the within subjects factor “sound” (music versus text) and the between subjects factor “suggestibility” (high versus low). When the Mauchly-Test was significant, we adapted values with Greenhouse-Geisser. We also adapted values when the Levene Test indicated uneven variances. Only the significant main effects and interactions were further investigated using paired samples t-tests according to Fisher’s protected LSD test. The level of significance was set to Supplementary Material


The availability of take home naloxone (THN) was increased for Canadians in 2016, including access to kits via pharmacies. Unlike typical over-the-counter (OTC) and prescription drugs, THN kits may be stored in non-standard conditions, including in vehicles, backpacks, and out of doors. To evaluate whether these non-standard storage conditions affect stability, we investigated the impact of heat and freeze-thaw cycling on naloxone hydrochloride stability.To assess the effect of heat, naloxone hydrochloride ampoules were exposed to 80 °C in a temperature-controlled oven for 8 h followed by 16 h at room temperature. To assess the effect of freeze-thaw cycles, naloxone hydrochloride ampoules were exposed to − 20 °C for 16 h followed by 8 h at 4 °C. The impact of these conditions on naloxone hydrochloride stability was evaluated each day for 1 week and after 2 and 4 weeks. The concentration of remaining naloxone hydrochloride was quantified using high-performance liquid chromatography (HPLC). Naloxone hydrochloride ampoules stored at room temperature served as the experimental control.Naloxone hydrochloride ampoules exhibit no changes in drug concentration following exposure to heat or freeze-thaw cycles for up to 28 days compared to ampoules maintained at room temperature (as indicated in the product monograph).Naloxone hydrochloride remains chemically stable following exposure to heat or freeze-thaw cycles after 28 days. If THN kits are stored in non-standard conditions (for up to 28 days) the active naloxone is likely to remain stable. Despite this, pharmacists should continue to emphasize the importance of appropriate storage of THN kits to ensure optimal efficacy should naloxone administration be required in an emergency situation.The online version of this article (10.1186/s12954-019-0288-4) contains supplementary material, which is available to authorized users. In North America, opioid-related overdose is the leading cause of death in individuals between the ages of 18 and 35 , 5. SincThe availability of THN outside of healthcare settings raises concerns regarding naloxone stability when exposed to conditions outside of the 15–30 °C range as indicated in the product monograph . For exaPrevious studies have investigated the consequence of naloxone storage under unconventional conditions. However, they are limited by the fact that they do not reflect conditions that may be encountered if THN kits were left, either intentionally or unintentionally, within a vehicle. For example, although temperatures as high as 70 °C have been evaluated, internal vehicle temperatures can reach up to 80 °C , 13. SevNaloxone hydrochloride ampoules (1 mL) were obtained from Sandoz . Naloxone hydrochloride reference standard was purchased from Cayman Chemical . HPLC grade 100% methanol (Product # 34860) was purchased from Sigma-Aldrich . Both 0.1% trifluoroacetic acid (TFA) in acetonitrile (Product #LS121-4) and 0.1% TFA in water (Product #LS119-4) were purchased from Fisher Chemical .Heat: Ampoules were placed in an 80 °C temperature-controlled oven for 8 h and transferred to room temperature for 16 h. Freeze-thaw: Ampoules were placed in a − 20 °C freezer for 16 h and transferred to a 4 °C refrigerator for 8 h. The effects of these conditions on naloxone hydrochloride stability were evaluated on the following days: 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28. To reduce the possibility of secondary reactions, exposure to thermal cycling proceeded with the day 28 ampoule and ended with the day 1 sample. Therefore, all ampoules were exposed for the same duration of time until the HPLC analysis. Ampoules stored at room temperature served as the experimental control.Naloxone hydrochloride ampoules were thermally stressed to either heat or freeze-thaw conditions on a daily basis for up to 4 weeks. Naloxone hydrochloride was detected and quantified using the Waters Alliance e2695 Separations Module  at a wavelength of 282 nm. The system was equipped with a Waters 2489 UV/Visible Detector (Waters Corporation) and a SunFire C18 column , which was used to achieve chemical separation at 25 °C. The mobile phase consisted of a 70:30 ratio of 0.1% TFA in water to 0.1% TFA in acetonitrile, pumped at a flow rate of 1.0 mL/min. Each sample was evaluated with three independent 50-μL volume injections into the column. Data was collected and analyzed using Empower Software (Waters Corporation). Naloxone hydrochloride identity was confirmed using retention time matching with a reference standard  vs the corresponding concentration (x-axis). The calibration plot generated the linear function y = 7,602,326.35x − 8640.01 with R2 = 0.9997. The concentration of naloxone hydrochloride in thermally stressed samples was determined using the measured AUC (y) and solving for concentration (x).A calibration plot was generated using the naloxone hydrochloride reference compound. A 500 μg/mL stock of naloxone hydrochloride dissolved in HPLC grade 100% methanol was filtered using a 0.45-um filter and diluted to the following concentrations: 200, 100, 50, 25, 10, 5, 3, and 1 μg/mL. Each sample was quantified by HPLC using the conditions specified above. The calibration plot was obtained by plotting the area under the curve (AUC) of each peak  or freeze-thaw cycles (− 20 °C for 16 h followed by 4 °C for 8 h) on naloxone hydrochloride stability. Regardless of duration and exposure to either condition, no detectable reduction in naloxone hydrochloride concentration was detected by HPLC, demonstrating drug stability under these conditions.The stability of naloxone hydrochloride exposed to thermal stresses has been investigated by several studies. However, findings have been variable due to inconsistencies in experimental design, duration of exposure, and methodology. For example, older studies have found that naloxone remains stable under thermal stress whereas newer studies demonstrate heat-dependent degradation , 14–16. In contrast with these findings, newer studies have found that naloxone hydrochloride exhibits temperature-dependent degradation. Gammon et al. exposed naloxone hydrochloride to fluctuating temperatures of − 6 °C and + 54 °C every 12 h, and concentration was measured weekly for up to 4 weeks using HPLC . By the sustained temperatures of − 20 °C, + 45 °C, or alternating weekly between the two) for 1 month. The most degradation occurred in the presence of heat with only 39% of the original concentration remaining after 4 weeks. Several factors may explain the degree of degradation observed. First, although ampoules were subject to moderate heat, the exposure was sustained for the duration of the study. Second, only one vial was used per thermal condition, which was sampled weekly. Therefore, the humidity and oxidation may have contributed to the extent of degradation. It is also important to note that an experimental control was not included in the study. Since HPLC was used to identify and quantify naloxone in this study, further characterization of degradation products by liquid chromatography-mass spectrometry (LC-MS) would have been useful in their overall analysis.Armenian et al. also observed naloxone hydrochloride exhibits temperature-dependent degradation . NaloxonThe limitations described above, in addition to other factors, were considered for our experimental design to reduce the impact of confounding factors. For example, no ampoules were tampered with until the end of the study where samples were run simultaneously via HPLC. This eliminated any impact that humidity or oxidation may have had on naloxone stability. Since all samples were run on HPLC at the same time, other external factors  could also be controlled. Each sample was also measured in triplicate readings to ensure homogeneity of ampoule contents. Importantly, our study also included control ampoules, which were stored according to the manufacturer’s storage conditions.However, this study is not without its limitations. First, we attempted to emulate conditions that would be generally encountered upon vehicle storage in winter or summer months. However, these conditions vary considerably. Ideally, assembled THN kits should have been stored in a car parked outside in the winter or summer months. Depending on the location within Canada, internal vehicle temperatures may differ. Temperature extremes may also be dampened by the THN kit housing and the location of placement within the vehicle. Second, although ampoules retain naloxone concentrations following thermal stress, we did not make any functional assessment of the naloxone samples  in vitro or in vivo. Third, although ampoules were not tampered with until the end of the study, we still cannot eliminate secondary reactions or degradation that may occur from time of last exposure until the HPLC run. However, because ampoules were stored according to the product monograph during this time and all ampoules were exposed for the same duration of time, any effect would be negligible. Last, it would be useful to extend the duration of exposure beyond 1 month to assess how long naloxone hydrochloride can tolerate these conditions before the compound begins to break down.This study is the first in Canada to assess the practical impact of heat and freeze-thaw cycles on naloxone hydrochloride stability. Our findings suggest that exposure of naloxone hydrochloride ampoules to temperatures up to 80 °C or daily freeze thaw cycles for 1 month appear to have no impact on its stability. Therefore, naloxone hydrochloride ampoules should remain efficacious should THN kits be temporarily stored outside of the recommended storage conditions. Despite naloxone’s stability, and because the clinical implications were not evaluated, pharmacists should continue to counsel patients on the importance of appropriate naloxone storage to ensure optimal efficacy. Furthermore, if any signs of precipitate become visible in the ampoule, the ampoules should be replaced. However, the naloxone itself remains stable in solution, and injection of such a product is warranted to reverse a life-threatening opioid overdose.Additional file 1:A sample naloxone chromatogram. (TIF 1072 kb)


Endothelial cells form the interior layer of blood vessels and, as such, are constantly exposed to shear stress and mechanical strain. While the impact of shear stress on angiogenesis is widely studied, the role of mechanical strain is less understood. To this end, endothelial cells and fibroblasts are cocultured under oscillatory strain to create a vessel network. The two cell types show distinctly different sensitivities to the mechanical stimulation. The fibroblasts, sense the stress directly, and respond by increased alignment, proliferation, differentiation, and migration, facilitated by YAP translocation into the nucleus. In contrast, the endothelial cells form aligned vessels by tracking fibroblast alignment. YAP inhibition in constructs under mechanical strain results in vessel destruction whereas less damage is observed in the YAP‐inhibited static control. Moreover, the mechanical stimulation enhances vessel development and stabilization. Additionally, vessel orientation is preserved upon implantation into a mouse dorsal window chamber and promotes the invading host vessels to orient in the same manner. This study sheds light on the mechanisms by which mechanical strain affects the development of blood vessels within engineered tissues. This can be further utilized to engineer a more organized and stable vasculature suitable for transplantation of engineered grafts. To do so, ECs and fibroblasts were cocultured in gelfoam, a collagen stretchable scaffold, and subjected to oscillatory stretch for 21 d. Our results demonstrated that fibroblasts were more responsive to the stress stimulus as compared to ECs. The mechanical strain induced YAP translocation to fibroblast nuclei, which, in turn, increased their proliferation and differentiation. Moreover, distinct alignment and migration of the fibroblasts cells, alongside vessel development, stabilization, and alignment and a subsequent increase in cell secretion of pro‐angiogenesis cytokines, were observed. Since oscillatory strain was previously shown to affect YAP expression,22.1FigureTo determine which cell type senses the stretch stimulus, cell alignment of monocultured ECs and fibroblasts on a gelfoam scaffold exposed to oscillatory strain was examined. On day 14, fibroblasts displayed a distinct orientation and alignment, whereas ECs were randomly arranged Figure1A,B. HWhen culturing fibroblast‐seeded gelfoam constructs for 12 d under oscillatory stretch or static conditions and then adding ECs for an additional 5 d without applying stretch, the endothelial vessels followed the fibroblast alignment, and were significantly more oriented when compared to the unstretched control group  were cut to 1 cm × 0.5 cm. Mechanical stimulation was applied on the constructs 1 d postseeding, using an EBERs TC‐3 bioreactor, with uniaxial oscillatory stretching or compression  of 20% strain and 1 Hz frequency, was applied for 21 d.Cell Culture and Inhibitors: Human adipose microvascular endothelial cells  lentivirally transduced with ZsGreen fluorescent protein, were grown in endothelial cell medium (ScienceCell) supplemented with 5% fetal bovine serum (FBS) (ScienceCell) and endothelial cell growth supplement (ScienceCell), and were used for 5–9 passages. Neonatal normal human dermal fibroblasts expressing red fluorescent protein (HNDFs‐RFP) (Angio‐Proteomie) were grown in Dulbecco's modified Eagle medium (DMEM) (Gibco), supplemented with 10% FBS (HyClone), 1% nonessential amino acids (NEAAs), 0.2% β‐mercaptoethanol (Sigma‐Aldrich), and 1% penicillin‐streptomycin solution (PEN STREP) . 3D vascularized constructs were obtained by coseeding endothelial cells  and support cells  on the gelfoam scaffolds, by mixing the cells, seeding them in a small volume of medium (20 µL), and incubating them for 15 min before adding medium. Verteporfin  was added to the culture medium 3 d postseeding, and then constructs were cultured for 7 d. Alternatively, blebbistatin  was added to the culture medium 3 d postseeding, constructs were cultured for 14 d.Whole‐Mount and Cryosection Immunofluorescence Staining: Constructs were fixated in paraformaldehyde (4%) for 20 min, and then permeabilized with 0.3% Triton X‐100 (Bio Lab Ltd.) for 10 min. Constructs were then washed with PBS and immersed in BSA solution  overnight. Samples were then incubated with the following primary antibodies overnight at 4 °C: mouse anti‐human Ki‐67 , mouse anti‐human αSMA , goat anti‐human PDGFRβ , mouse anti‐human NG2 , mouse anti‐human collagen IV , goat anti‐human VE‐cadherin , mouse anti‐human YAP , rabbit anti‐human β‐catenin , or mouse anti‐human AMOT . Constructs were then treated with Cy3‐labeled , Cy5‐labeled , or Alexa‐488‐labeled  secondary antibodies and DAPI (Sigma‐Aldrich), for 2 h, at room temperature.Scanning Electron Microscopy (SEM) Imaging: Scaffold morphology was examined using a SEM (Zeiss Ultra‐Plus FEG‐SEM). Scaffolds without cells were carbon‐coated using a Polaron carbon coater (Quorum Technologies). Cell‐embedded scaffolds were fixed in 2.5% (vol/vol) glutaraldehyde in 0.1 m cacodylate buffer (Sigma‐Aldrich) for 5 min, followed by dehydration in a gradient of 70, 85, 95, and 100% ethanol, with a 5 min incubation in each solution. Scaffolds were then immersed in hexamethyldisilazane (Sigma‐Aldrich) for 5 min and air‐dried at room temperature, before being coated with a gold‐palladium mixture using a Polaron gold coater.Cytokine Array and ELISA: Medium was collected on days 7, 14, and 21 postseeding. Cytokine quantification was measured using the Human Angiogenesis Array GS1 (RayBiotech). Ang1 was quantified using the Human ANGPT1 ELISA kit (RayBiotech).Construct Imaging and Image Analysis: Whole vascularized constructs were imaged with a confocal microscope , using 2.5×, 5×, 20×, and 63× oil immersion lenses. Imaris software (BITPLANE) was used to detect YAP, PDGFRβ, and NG2 localization in the 3D image. All other image analyses were quantified using self‐written algorithms in MATLAB: all images were transformed into a binary image and then analyzed using different algorithms.Cells, nuclei, vessels, and collagen IV orientation were determined using the orientation module in the regionprops function in MATLAB software. Cell migration pattern, VE‐cadherin morphogenesis, and vessel quality under blebbistatin treatment were measured by the eccentricity parameter using regionprops algorithm in the MATLAB software. Ki67 pixel density was calculated by dividing Ki67 staining‐positive pixels by DAPI staining‐positive pixels. AMOT vessel expression was calculated by determining the number of pixels with both a positive vessel (ZsGreen) signal and AMOT‐positive signal.Animal Experiments: Animal studies and surgical procedures were conducted according to protocols approved by the Technion animal ethics committee. Dorsal window chamber assembly followed the protocol by Palmer et al.−1)‐xylazine (10 mg kg−1), delivered via a 30gauge needle. On the following 2 d, mice were subcutaneously injected with buprenorphine (0.05 mg kg−1) every 12 h. To create an aseptic working area, the dorsal region of the mouse torso and the entire tail were cleaned with chlorhexidine solution and iodine. To assemble the window, mouse skin was stretched and sutured to a stabilizing device using 4‐0 silk sutures, after which, two window frames were sutured to each other using 4‐0 silk sutures. The skin was then cut and removed along the circumference of the window and then the area was washed with warm saline and covered with a 13 mm cover slip glass , and fixed with a snap ring. The mice were monitored daily to assess general health.Scaffold Implantation: 2 or 3 d after implantation of the dorsal skinfold window chamber, mice were anesthetized via intraperitoneal injection of a mixture of ketamine (100 mg kg−1)‐xylazine (10 mg kg−1), delivered via a 30 gauge needle. The snap ring and the cover slip glass were removed, followed by a saline wash of the tissue. The scaffold was then placed in the middle of the window groove and then covered with a new cover slip. The window was then closed with the snap ring. Mice were monitored daily and imaged using intravital microscopy.Intravital Imaging: Intravital microscopy  was performed on days 11 and 18 postimplantation. To track the host vasculature in the mice within the graft area, Alexa flour647 anti‐mouse CD31 (Biolegend) was intravenously injected via the tail vein. The antibody was allowed to circulate for 30 min before mice were anesthetized via intraperitoneal injection of ketamine (100 mg kg−1)‐xylazine (10 mg kg−1), delivered using a 30 gauge needle. Tetramethylrhodamineisothiocyanate‐dextran,  was then intravenously injected through the tail vein. Mouse body temperature was maintained during the entire imaging session with a heating chamber.Statistical Analysis: Presented data include the mean ± standard deviation. Two‐way analysis of variance (ANOVA) was performed to examine the influence of two independent categorical variables, followed by Bonferroni's multiple comparison tests. Results were considered significant for p < 0.05. Statistical analysis was performed using GraphPad Software, a computerized statistical program. All analyses were performed in at least biological triplicates.The authors declare no conflict of interest.SupplementaryClick here for additional data file.


N‐carbamoyl‐l‐glutamate (NCG), the N‐acetyl‐l‐glutamate analogue used to treat N‐acetylglutamate synthase deficiency, has been proposed as potential therapy of carbamoyl phosphate synthetase 1 deficiency (CPS1D). Previous findings in five CPS1D patients suggest that NCG‐responsiveness could be mutation‐specific. We report on a patient with CPS1D, homozygous for the novel p.(Pro1211Arg) CPS1 mutation, who presented at 9 days of life with hyperammonemic coma which was successfully treated with emergency measures. He remained metabolically stable on merely oral NCG, arginine, and modest protein restriction. Ammonia scavengers were only added after poor dietary compliance following solid food intake at age 1 year. The patient received a liver transplantation at 3.9 years of age, having normal cognitive, motor, and quality of life scores despite repeated but successfully treated episodes of hyperammonemia. Studies using recombinantly produced mutant CPS1 confirmed the partial nature of the CPS1D triggered by the p.(Pro1211Arg) mutation. This mutation decreased the solubility and yield of CPS1 as expected for increased tendency to misfold, and reduced the thermal stability, maximum specific activity , and apparent affinity (~5‐fold reduction) for ATP of the purified enzyme. By increasing the saturation of the NAG site in vivo, NCG could stabilize CPS1 and minimize the decrease in the effective affinity of the enzyme for ATP. These observations, together with prior experience, support the ascertainment of clinical responsiveness to NCG in CPS1 deficient patients, particularly when decreased stability or lowered affinity for NAG of the mutant enzyme are suspected or proven. N‐carbamoylglutamate, a drug that should be clinically tested for responsiveness in CPS1 deficient patients.The novel p.(Pro1211Arg) CPS1 mutation causes partial but life‐threatening CPS1 deficiency that may be alleviated by administration of 1CPS1 gene mutations.Carbamoyl phosphate synthetase 1 deficiency (CPS1D), a rare autosomal recessive inborn error of the urea cycle, is due to loss‐of‐function N‐carbamoyl‐l‐glutamate (NCG) use. This deacylase‐resistant analogue of N‐acetyl‐l‐glutamate (NAG), the essential activator of CPS1,Km for glutamateCarbamoyl phosphate synthetase 1 deficiency prognosis remains poor,Although advocated for many years,22.1All the interventions of this retrospective, observational review of medical records were intended for diagnosis and treatment and thus did not require ethical committee review. Written informed consent was obtained from the patient's mother for clinical and laboratory procedures and for data publication.CPS1 gene, using blood DNA as template and appropriate oligonucleotide pairs (sequences will be provided upon request). Reference genomic and mRNA sequences are those with respective GenBank entries NG_008285.1 and NM_001875.4 (https://www.ncbi.nlm.nih.gov/nucleotide/). Nucleotide numbering of the mRNA starts at the A of the ATG translation initiation codon (codon 1). The translated protein sequence of human CPS1 corresponds to entry P31327 of UniProtKB (https://www.uniprot.org/uniprot/).Carbamoyl phosphate synthetase 1 deficiency was suggested by the clinical and laboratory data see . It was 2.2GCACACAAACC3′ and 5′ GTTTGTGTGCGCAGCATCAGAGTG3′ (the changed nucleotide is underlined), corroborating the correctness of the change and the lack of unwanted changes by automated Sanger sequencing. Wild type and mutant CPS1 were purified from baculovirus‐infected insect cell extracts by Ni2+‐affinity chromatography.Human mature CPS1 was produced recombinantly in a baculovirus/insect cell system.2.34Cl, 50 mM KHCO3, 5 mM adenosine‐5‐triphosphate (ATP), 25 mM MgSO4, 10 mM NAG, 5 mM L‐ornithine HCl, and 4 U/mL of pure Enterococcus faecalis ornithine transcarbamylase (produced recombinantly in our laboratory). To study substrate kinetics and NAG activation, the concentration of each substrate or of NAG was varied individually while other assay components were kept fixed at the concentrations used in the standard reaction assay, with MgSO4 being always in 20 mM excess over ATP. Kinetic data were fitted to hyperbolae .Carbamoyl phosphate synthetase 1 activity was determined colorimetrically as citrulline productionFor monitoring the thermal stability of CPS1, a solution of 0.5 mg/mL of this enzyme (wild type or mutant) in 50 mM glycylglycine pH 7.4, 20 mM imidazole, 0.5 M NaCl, 2 mM dithiotreitol (DTT), and 10% glycerol was heated 15 minutes at the indicated temperature, then chilled to 0°C, followed by immediate CPS1 activity determination at 37°C as described above.2.4https://www.ebi.ac.uk/Tools/msa/clustalo)http://genetics.bwh.harvard.edu/pph2; HumVar‐trained dataset),http://mutpred2.mutdb.org),http://www.mutationtaster.org)probably damaging, possibly damaging, and benign, with the highest probability score being 1. The score given by MutPred2 is the probability that a given amino acid change is deleterious/disease associated. Pymol  was used for visual structural analysis and for depicting the CPS1 structure.Amino acid conservation was determined by sequence alignment with Clustal . This base change was not found in the EXAC (http://exac.broadinstitute.org/transcript/ENST00000233072), 1000 Genomes (https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/), dbSNP (https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?geneId=1373), or specific CPS1D LOVD databases (https://databases.lovd.nl/shared/genes/CPS1) and therefore it is a novel mutation. The proline that is mutated is invariant among all types of CPS. Three pathogenicity prediction servers  when compared with that of wild‐type CPS1  mutation by introducing it into recombinantly produced human CPS1. The mutant protein was expressed similarly to wild‐type CPS1 in the insect cell system used  mutation is shown here to disfavor proper CPS1 folding, also causing protein destabilization and negatively affecting intrinsic catalysis parameters, lowering the l Figure A.5 The dl Figure B. Thus, P Figure B so dispKmATP of CPS1,KmATP. A subsaturation effect due to decreased affinity for NAG could explain the NCG sensitivity of the p.(Arg850Cys) mutation found in subject 5 of the previous 3‐day trial of NCG in CPS1D,KaNAG.The apparent positive effect of NCG in our patient could stem from the selection for a well‐folded stable CPS1 conformation by the simultaneous binding of NCG and MgATP to the enzyme. This previously reported chaperoning effecthe p.Arg80Cys mutaThis is an observational retrospective patient study that did not involve any human experimentation or research‐based patient intervention, as all interventions and decisions made were intended to diagnose and treat the patient. In any case, no aspect of the present report is in contradiction with the Helsinki Declaration of 1975, as revised in 2000.Sufin Yap has received honoraria, travel and accommodation support from Orphan Europe‐Recordati Rare Disease for participation in industry‐sponsored symposia, advisory board meetings, and master classes and attending scientific meetings. Nadine Gougeard, Antony R. Hart, and Belén Barcelona declare that they have no conflict of interest. Vicente Rubio has received honoraria, travel and accommodation from Orphan Europe‐Recordati Rare Disease for participation in company‐sponsored symposia, and advisory board meetings.Written informed consent for the publication of these data was obtained from the patient's mother.This article does not contain any studies with animal subjects performed by any of the authors.6S.Y. diagnosed and treated the patient, conceived the study, obtained consent, provided the clinical data and Figure


Euglena agilis Carter was exposed to the polycyclic aromatic hydrocarbon (PAH) anthracene for 96 h under optimal photosynthetically active radiation (PAR), and responses of growth, photosynthetic pigment production, and photosynthetic efficiency were assessed. Anthracene reduced the growth rate (μ) and levels of chlorophyll a (Chl a), chlorophyll b (Chl b), and total carotenoids. The growth rate was more sensitive than photosynthetic parameters, with a median effective concentration (EC50) of 4.28 mg L−1. Between 5 and 15 mg L−1, anthracene inhibited the maximum quantum yield (Fv/Fm) of photosystem II (PSII) and the maximum photosynthetic electron transport rate through PSII (rETRmax) with EC50 values of 14.88 and 11.8 mg L−1, respectively. At all anthracene concentrations, intracellular reactive oxygen species (ROS) were elevated, indicating increased oxidative stress. Anthracene presumably reduced the PSII efficiency of photochemical energy regulation and altered the photochemistry through intracellular ROS formation. Acute exposure to PAHs may induce severe physiological changes in phytoplankton cells, which may influence vital ecological processes within the aquatic environments. Additionally, growth and Chl a content may serve as sensitive risk assessment parameters of anthracene toxicity in water management since EC50 values for both overlap with anthracene levels (8.3 mg L−1) permitted by the US Environmental Protection Agency (USEPA).The freshwater flagellate alga  PAHs are hydrophobic in nature and their persistence in the environment is a consequence of their low water solubility2. Due to their toxic, carcinogenic, mutagenic and photosensitising effects, especially their ability to generate singlet oxygen and other ROS, PAHs are considered one of the most prevalent groups of aquatic contaminants of high global concern3.Polycyclic aromatic hydrocarbons (PAHs) comprise a diverse family of hydrocarbons, each composed of two or more fused benzene rings. They are ubiquitous in terrestrial and aquatic ecosystems and are introduced by natural and man-made processes such as volcanic eruptions, crude oil spills, fossil fuel combustion, oil refining, automobile exhausts and industrial effluents4. Anthracene adversely affects the growth and photosynthesis of natural phytoplankton communities, with a toxicity threshold value less than its aqueous solubility1. Moreover, it is one of the most rapidly modified hydrocarbons and is assumed to be a strong photosensitizer5 that induces intracellular oxidative stress and blockage of the photosynthetic electron transport chain6 through the formation of ROS. Given the wide occurrence and high toxicity to aquatic organisms, anthracene is now on the list of priority pollutants, with a recommended water quality criterion of 8.3 mg L−1 and interim water quality guidelines of 0.012 μg L−1 for the protection of freshwater life set by the Canadian Council of Ministers of the Environment8.Anthracene is a three-ring, low molecular weight PAH with relatively high water solubility than other toxic PAHs9. Various studies have reported the toxic effects of PAHs to freshwater algae in terms of growth, photosynthesis, and respiration, with special attention given to interactions with solar and ultraviolet (UV) radiation12. However, Brack et al.13 suggested that anthracene toxicity is largely independent of irradiation and the intact compound itself contributes to toxicity. EC50 values reported for anthracene in freshwater microalgae range between 0.024 and 5 mg L−1 . Phytoplankton is the main biomass producers in aquatic ecosystems, contributing ca. 50% of the atmospheric carbon dioxide fixation25. Any negative effects of anthracene on the growth and photosynthesis of phytoplankton would be detrimental to entire aquatic ecosystems and food chains.The ultimate goal of bioassay tests is to provide representative and incorporative criteria regarding exposure conditions, thereby improving risk assessment and management of water quality. In this respect, multiple, rather than single, endpoint assays may be more reliable for comprehensive risk assessment of toxicants. Such an approach may facilitate insight into the mechanisms of toxicity and provide information on the relative sensitivity of selected parameters to toxicant concentration and/or exposure duration, thereby establishing methods for detecting changes caused by particular phytotoxicantsEuglena agilis Carter was cultured in mineral medium (pH 5)26 in 1 L Erlenmeyer flasks at 25 °C under white fluorescent irradiance  of 30 µmol photons m–2 s−1  on a 16:8 h light:dark (LD) cycle. All experiments were performed using cells at the exponential growth phase.2; SPL Life Sciences, Gyeonggi-Do, Korea) with a test volume of 2 mL per well. Equal volumes of cell suspension and anthracene stock solutions were mixed to obtain final concentrations of 0.625, 1.25, 2.5, 5, 10 and 15 mg L−1, along with untreated controls. The concentration of the carrier solvent did not exceed 0.2% v/v of the test culture volume. The initial cell density was 10 ± 0.5 × 104 cells mL−1 of suspension. An additional solvent toxicity test (96 h) was conducted with a maximum DMSO concentration of 0.2% v/v. Organisms were exposed to nominal concentrations of anthracene and all treatments were performed in triplicate. The well plates were covered with parafilm to avoid evaporation and mixing of the volatile toxicant.Anthracene  was purchased from Sigma Aldrich  and test solutions at the desired concentrations were prepared by serial dilution from stocks in high-performance liquid chromatography (HPLC)-grade dimethyl sulphoxide . Microplate toxicity tests of 96 h in duration were conducted in 24-well cell culture plates  and t2 , respectively.Growth rates were determined by measuring the number of cells in each well on the first and final days using a hemocytometer . The specific growth rate (μ) was calculated using the following formula:27. Briefly, 1 mL cell suspension was collected from each replicate culture and centrifuged before extraction, and 1 mL of 90% v/v acetone was added followed by vigorous vortexing and centrifugation at 10,000 × g for 5 min at 4 °C. Supernatants were withdrawn and their optical density was measured spectrophotometrically at 470, 664 and 647 nm using an S-3100 UV/Vis spectrophotometer . Pigment concentrations are expressed as μg mL−1 of suspension.Photosynthetic pigment content was estimated using standard protocolsa fluorescence was measured using a pulse amplitude modulation (PAM) imaging instrument  as a proxy for photosynthetic performance. For measurement of maximum quantum yield (Fv/Fm) and electron transport rate (ETR), samples were kept in the dark for 10–15 min and then subjected to pulsed light emitted by a diode at ~0.15 µmol photons m−2 s−1 to obtain the initial fluorescence yield (Fo), which denotes the fluorescence yield when all PSII reaction centres are open with fully oxidized plastoquinone A(QA). A saturation pulse of ~5000 µmol photons m−2 s−1 emitted by a built-in halogen lamp was then applied to produce the maximum fluorescence yield (Fm), which is induced by a short saturating pulse of actinic light that reduces all QA. The maximum PSII quantum yield (Fv/Fm) was then derived from the equation (Fm − Fo)/Fm.Chl −2 s−1. The relative electron transport rate (rETR) was calculated by multiplying the effective quantum yield  by photon flux density (PFD) and plotting against PFD. The ETR is relative because the absorbance of light by cells was not measured. Maximum electron transport rate (ETRmax) was derived from the hyperbolic tangent formula rETR = ETRmax ∗ tanh (α/I/ETRmax), adapted from Jassby and Platt28, where α indicates the electron transport rate under light-limited conditions. Alterations in Chl a fluorescence due to changes in non-photochemical quenching (NPQ) and photochemical quenching (qP) were calculated from (Fm − F′m)/F′m and (F′m − Ft)/(F′m − F′o), respectively29.Rapid light curves were produced using 10 s pulses of actinic light increased stepwise from 0 to 335  μmol photons mE. agilis treated with anthracene. DCFH-DA  solubilized in ethanol was added to the cell suspension and incubated on a shaker at room temperature in the dark for 1 h30. The fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a Spectra MAX Gemini EM microplate fluorescence reader . The relative production of ROS is represented as relative fluorescence units (RFU).The oxidant-sensing fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate  was used to detect intracellular ROS generation in p < 0.05) using the JMP software . Multiple comparison tests based on the least significant difference (LSD) were then carried out to find significant differences (p < 0.05) from controls and between treatments. The effective concentration at which 50% inhibition occurs (EC50) was estimated by the linear interpolation method using ToxCalc 5.0 . The coefficient of variation (CV), the standard deviation expressed as a percentage of the mean, was calculated to estimate the precision of test values.Data are presented as means ± 95% confidence intervals (CI). All parameters were compared across treatments with one-way analysis of variance  or photosynthetic efficiency  of E. agilis, even at the maximum concentration of 0.2% (v/v) in the growth medium  had no significant inhibitory effects on cell growth . The EC50 value for growth was 4.28 mg L−1  had a pronounced effect on photosynthetic pigments content in E. agilis. The most abundant pigment in E. agilis was Chl a (7.14 μg mL−1) followed by carotenoids (1.72 μg mL−1) and Chl b (1.25 μg mL−1). At the lowest test concentration (0.625 mg L−1), there were significant reductions in Chl a, Chl b and total carotenoids of up to 20%, 16%, and 17%, respectively, while at the highest concentration, reductions of 58%, 64%, and 49% were observed , Chl b  and total carotenoids , respectively , there were significant reductions in dark fluorescence , which reflects emission by excited Chl a molecules in the antennae structure of PSII, and in maximal fluorescence , which can be attributed to severe loss of pigments and/or inactivation of PSII reaction centres. It is evident that at higher anthracene doses, reduction in the number of cells and pigment levels resulted in an overall decline in the light-harvesting by E. agilis. Moreover, no significant variation  in F0 was observed between 0–2.5 mg L–1 anthracene, despite significant reductions in the concentration of Chl a  was recorded in our control E. agilis population, comparable to the value reported previously for Euglena gracilis21. Fv/Fm did not significantly vary up to 1.25 mg L−1 anthracene  by 17% (5 mg L−1), 36% (10 mg L−1), and 55% (15 mg L−1) with an EC50 of 13.74 mg L−1 . This decline in Chl a fluorescence quenching can be attributed to impairment of electron transport downstream from PSII and an elevated reduction of the PQ pool45. NPQ is produced through the generation of an H+ electrochemical gradient across the thylakoid membranes46 and is an indicator of absorbed energy that is dissipated through heat loss and other non-photochemical mechanisms. We assume that a severe reduction in the photosynthetic process at high anthracene levels likely reduces the magnitude of the pH gradient, thereby affecting NPQ values, consistent with the view of González–Moreno et al.47 who reported similar observations on fluorescence quenching in E. gracilis under salt stress. Severe reduction in Fv/Fm of E. gracilis exposed to NaCl had resulted in a diminution of the pH gradient across the thylakoid membranes and subsequently, up to 95% reduction in NPQ47.We noted that the reduction in NPQ Fig. . Althougmax was not significantly affected up to 1.25 mg L−1 anthracene, whereas 5, 10 and 15 mg L−1 doses resulted in 18%, 46%, and 56% reductions, respectively, with an EC50 value of 11.8 mg L−1  and the absorbed energy cannot be used in photochemistry6. A probable consequence of inhibition of the electron transport chain at PSII is the transfer of energy from triplet chlorophylls to oxygen, forming singlet oxygen species, which induces oxidative damage of cells48. The generation of free radicals and subsequent intracellular oxidative stress is a prominent mechanism of anthracene toxicity in freshwater phytoplankton38.In the present study, anthracene reduced the ETR across PSII at higher concentrations Fig. . rETR isE. agilis cells was measured, a significant increase was observed at all dosages , indicating a rise in intracellular ROS levels  can be attributed to reduced cell growth and diminished enzyme activities, although values were still significantly higher (p < 0.5) than in controls. The major site of ROS production in photosynthetic organisms is the disrupted electron transport chain across PSII49. We report here, for the first time in freshwater microalga taxa, the significant elevation of intracellular ROS levels under anthracene stress. In Euglena spp., ROS play a significant role in metal toxicity45, UV damage and defense mechanisms50. However, ROS, generated by chemical stressors, trigger adverse effects through multifaceted actions inside the cell. They attack thylakoid lipids and initiate peroxyl radical chain reactions, eventually destroying membranes and pigment-protein complexes45. Moreover, in chloroplasts, ROS cause lipid peroxidation, which results in the disruption of photosynthetic pigments, and the inactivation and degradation of RuBisCo and other components of the Calvin cycle51. Our results correspond to Babu et al.52, who found that 1,2-dihydroanthraquinone, a photoproduct of anthracene, inhibited photosynthetic electron transport, leading to the overproduction of O2− and subsequent oxidation of proteins, membranes, and pigments in Lemna gibba.When DCFH-DA fluorescence emission in anthracene exposed 53. The superoxide anion radicals (O2−) produced in the electron transport chain is a precursor for many other ROS species. They are rapidly converted to hydrogen peroxide (H2O2) and subsequently to hydroxy radicals (OH•) by enzymatic reactions54. DCFH-DA does not directly react with O2− but can be oxidized to highly fluorescent DCF by H2O2 and OH• radicals55. Thus, DCFH-DA probing of ROS revealed the overall cellular redox status of E. agilis under anthracene stress. Higher ROS also reflect the inefficiency of both photochemical pathways and protective regulatory mechanisms to process the excitation energy at PSII. Our data suggest that ROS generation and consequential oxidative stress play a pivotal role in acute anthracene toxicity in the model organism, E. agilis. We detected significant ROS levels under optimal PAR irradiation, where photo-modification of the parent compound is less likely. Under high oxidative damage, Euglena relies on the activation of antioxidant enzymes such as ascorbate peroxidase (APX) and glutathione peroxidase (GPX)56, and biosynthesis of antioxidant metabolites such as reduced glutathione (GSH) and its derivatives57. Furthermore, some canonical metabolites act as indicators of oxidative damage, such as malondialdehyde (MDA)58. Thus, antioxidant/oxidant responses upon anthracene exposure may represent a promising area for further investigation.DCFH-DA is more suitable to estimate total ROS production rather than as a probe for a particular type of ROS59. As stated before, anthracene reduced the Fv/Fm in a dose-dependent manner, and the photon energy requirements for a complete reduction of QA decrease drastically. This explains the gradual reduction in Y(II) with increasing anthracene doses and a corresponding increase in Y(NO) ) is a significant stress response, suggesting potential damage to the photosynthetic apparatus exerted by anthracene. Moreover, the declining Y(NPQ) is an indicator of the failure in regulated non-photochemical quenching mechanisms to process the excess energy at PSII. These PSII quantum yield parameters collectively indicates the reduced efficiency of photochemical energy regulation imposed by anthracene exposure.We further analyzed the three photochemical quantum yields of PSII measured by imaging PAM to describe the response of PSII photochemistry to anthracene Fig. . Y(II) rNO) Fig. . Y(NO) dOW) of 4.5460 and, therefore, multi-well plate assay is likely to underestimate the toxicity potential, because a loss of exposure concentration due to lipophilicity is expected for hydrophobic compounds with KOW > 461. We controlled the evaporative loss of the toxicant by sealing the well plates, however, loss in nominal concentration due to physicochemical properties of the tested PAH may have adversely affected the toxicity thresholds reported here. Nevertheless, our data confirmed the mode of anthracene toxicity in E. agilis through Chl a fluorescence technique and the role of ROS in the overall toxicity response.On the downside of our methodology, anthracene has a higher n-octanol/water partition coefficient , rETRE. agilis cells, indicating a rise in the intracellular ROS levels. A probable source of generation of ROS would be an inhibition of the electron transport chain at PSII which would have transferred energy from triplet chlorophylls to oxygen, forming singlet oxygen species. A corresponding increase in Y(NO) with increasing anthracene also confirmed that the fraction of absorbed energy might have been used for the generation of free radicals (ROS) via an apparent catalytic transfer of electrons that occurred from the reduced PQ pool to O2.There was a significant increase in DCFH-DA fluorescence emission in anthracene exposed a content of E. agilis may serve as sensitive risk assessment parameters of anthracene toxicity in water management since EC50 values for both overlap with anthracene levels (8.3 mg L−1) permitted by the US Environmental Protection Agency (USEPA).Growth and Chl Five principal conclusions derived from this study are:


Accumulating evidence suggests an impact of gestational weight gain (GWG) on pregnancy outcomes; however, data on cardiometabolic risk factors later in life have not been comprehensively studied. This study aimed to evaluate the relationship between GWG and cardiometabolic risk in offspring aged 7 years.We included a total of 905 mother–child pairs who enrolled in the follow-up visit of the multicentre Hyperglycemia and Adverse Pregnancy Outcome study, at the Hong Kong Centre. Women were classified as having gained weight below, within or exceeding the 2009 Institute of Medicine (IOM) guidelines. A standardised GWG according to pre-pregnancy BMI categories was calculated to explore for any quadratic relationship.p values of all the traits: 4.6 × 10−9 < p < 0.0390) than women who were within the recommended range of weight gain during pregnancy. Meanwhile, women who gained less weight than outlined in the recommendations had offspring with increased risks of hypertension and insulin resistance, compared with those who gained weight within the recommended range (7.9 × 10−3 < p < 0.0477). Quadratic relationships for diastolic blood pressure, AUC for insulin, pancreatic beta cell function and insulin sensitivity index were confirmed in the analysis of standardised GWG (1.4 × 10−3 < pquadratic < 0.0282). Further adjustment for current BMI noticeably attenuated the observed associations.Independent of pre-pregnancy BMI, gestational hyperglycaemia and other confounders, women who gained more weight than the IOM recommendations had offspring with a larger body size and increased odds of adiposity, hypertension and insulin resistance  contains peer-reviewed but unedited supplementary material, which is available to authorised users. Type 2 diabetes has been recognised as a familial disease transmitted across generations through genetic, epigenetic and environmental circumstances including in utero exposures. To illustrate the role of early-life exposures in programming of disease, previous studies have reported evidence of an excess maternal inheritance of type 2 diabetes and cardiometabolic risk in offspring compared with the pattern of paternal inheritance , 2.Barker’s hypothesis, focusing on the association of prenatal undernutrition with the risk of cardiovascular and metabolic disease in adult life, has been corroborated by the study of individuals exposed to the Dutch famine . More reWith the recent upward shift of maternal weight gain during pregnancy, increasing evidence suggests a separate role for maternal gestational weight gain (GWG) on offspring cardiometabolic risk . The majority of studies addressing GWG in metabolic disorders have found a positive association between maternal GWG and adiposity in offspring , 8, althTo help clinicians monitor an appropriate GWG, the Institute of Medicine (IOM) reviewed the impact of weight gain during pregnancy on both maternal and fetal outcomes, and in 2009 established new guidelines for optimal GWG according to pre-pregnant BMI . The IOMBy using follow-up data on mother–child pairs from the multicentre Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study, procured at the Hong Kong study centre , the curThe study methods have been described previously . At the This study was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong. Research staff explained the study objective and procedures to both the mother and the child. The children’s parents or legal guardians, as appropriate, provided written informed consent.2). We defined total GWG as the difference between the mother’s weight at delivery and her pre-pregnancy weight. Women were classified as gaining weight ‘below’, ‘within’, or ‘exceeding’ recommendations, according to their pre-pregnancy BMI category, on the basis of the 2009 IOM guidelines for healthy pregnancy weight gain [Maternal weight at delivery was abstracted by research staff from the medical records. Pre-pregnancy BMI was calculated as self-reported pre-pregnancy weight (kg) divided by the square of the measured height ; meani was the mid-point of the range of GWG recommended by IOM in the ith pre-pregnancy BMI category; SDi was (upperi − loweri)/2; and upperi and loweri was the upper and lower ranges of GWG recommended by IOM in the ith pre-pregnancy BMI category, respectively. For example, given that the recommended range of GWG in the ‘underweight’ group is from 12.5 to 18 kg, meani = (18 + 12.5)/2 = 15.25 kg, and SDi = (18–12.5)/2 = 2.75 kg.Within each BMI category, the standardised GWG was computed as 2. The sum of skinfold thickness was calculated as the sum of the biceps, triceps, subscapular and iliac skinfold thicknesses. Systolic and diastolic blood pressure (SBP and DBP) were measured in triplicate, and the mean readings were used. SBP and DBP for age-, sex-, and height-specific percentiles were computed based on data released from the National High Blood Pressure Education Program Working Group on High Blood Pressure in Children and Adolescents [Anthropometric indices including body weight, body height, waist and hip circumference, and biceps, triceps, subscapular and iliac skinfold thicknesses were measured. BMI was calculated as weight/heightlescents .glu) and insulin (AUCins) during the OGTT at 0–120 min were calculated using the trapezoid rule.Participants were examined in the morning after an overnight fast. All individuals underwent an OGTT with a glucose load of 1.75 g/kg body weight or a full 75 g glucose load if they weighed ≥42.8 kg , with blood taken at 0, 15, 30, 60 and 120 min to measure plasma glucose and insulin levels. Plasma glucose was measured by the hexokinase method, using an automated analyser . Plasma insulin was analysed using an immunoassay analyser . Fasting blood samples were also collected to measure lipid profile. AUCs for glucose  ÷ 22.5 . Beta ce÷ AUCglu .www.r-project.org/). p < 0.05 was considered statistically significant. Data are presented as percentage (n), mean ± SD, or median (Q1–Q3). Comparison between IOM categories of GWG was performed by χ2 test, one-way ANOVA or Kruskal-Wallis test, as appropriate.All analyses were performed using either SPSS for Windows v22  or R 2.15.1  and postnatal periods (history of breastfeeding and childhood exercise level); model 4—model 3 + birthweight; and model 5—model 4 + childhood BMI. Individuals with missing data points for any variables included in the linear regression model were removed from the analysis. A mean of 3.9% of individuals were excluded in each regression analysis. The resulting residuals were transformed to a z score for normally distributed traits, and transformed to approximate normality using an inverse standard normal function for the skewed traits.Each normally distributed trait was winsorised by replacing any data value above the mean plus 4 SD of the sample data by the mean plus 4 SD, and any value below the mean minus 4 SD by the mean minus 4 SD. A total of 0.5% of the data were replaced. All traits were then adjusted for covariates using linear regression as follows: model 1—child’s sex, age and/or height as appropriate; model 2—model 1 + family history ; model 3—model 2 + environmental factors during prenatal . In general, women who gained excessive weight during pregnancy were younger and had longer gestational periods, a higher primary Caesarean delivery rate and a greater BMI before pregnancy, at delivery and at 7 years after delivery. Offspring born to mothers with excessive GWG were heavier and longer, showed greater adiposity and had higher umbilical cord blood C-peptide concentrations at birth.The characteristics of the women and newborn offspring included in this analysis are listed in ESM Table ins, were more insulin resistant (higher HOMA-IR and lower Matsuda ISI) and showed a greater insulin response  than the offspring of mothers who achieved the weight gain target during pregnancy  (model 1 in Table ins), insulin sensitivity (Matsuda ISI) and beta cell function  (pexceeding < 0.05), although the association of GWG with HOMA-IR and SBP percentile was no longer significant in models 2–4 (pexceeding > 0.05) (Table pbelow < 0.0407) and pancreatic beta cell function (0.0184 < pbelow < 0.0538) was marginally significant, while its association with AUCins  and Matsuda ISI  were slightly mitigated in models 2–4 , greater BMI, waist circumference and hip circumference, and higher blood pressure , FPI, 2 h insulin and AUCins, increased risks of insulin resistance (Matsuda ISI) and an enhanced insulin response (pancreatic beta cell function) in the children of women with either more or less weight gain than recommended during pregnancy (1.4 × 10−3 < pquadratic < 0.0282) , DBP percentile , AUCins , pancreatic beta cell function  and Matsuda ISI . In addition, we observed a positive linear relationship for weight (7.5 × 10−4 < plinear < 0.0224 and pquadratic > 0.05) and hip circumference (5.8 × 10−3 < plinear < 0.0573 and pquadratic > 0.05) in models 2–4  and AUCAccumulating evidence suggests that adiposity and related cardiometabolic risk factors in the offspring are influenced by maternal weight gain during pregnancy. Using the follow-up data of 905 mother–child pairs from the HAPO study, we extended observations from earlier studies by (1) examining both linear and non-linear associations between maternal GWG and cardiometabolic risk factors in offspring aged 7 years, and (2) including data from OGTTs. To achieve a better understanding of the causal pathways, we have also systematically considered the potential confounding effects of the clinical risk factors in these relationships.pexceeding = 0.0706) ; for example, offspring may inherit their mother’s genetic potential to gain weight. Moreover, mothers and children may share obesogenic dietary patterns (e.g. a preference for fatty food) and lifestyle habits , particularly in early childhood , which mThere is increasing recognition of the potential metabolic impact of maternal adiposity, suggesting that high maternal plasma concentrations of glucose, NEFA and amino acids result in a sustained modulation of appetite control, neuroendocrine functioning or energy metabolism in the developing fetus. The fetus may thus become more vulnerable to an obesogenic environment, leading to adiposity and related cardiometabolic diseases in later life. Human studies of individuals undergoing bariatric surgery highlighted the impact of intrauterine environment on health outcomes in the offspring. A study that included 49 mothers and their 111 children aged 2.5–26 years demonstrated that offspring born after maternal biliopancreatic diversion bariatric surgery had a markedly lower prevalence of macrosomia, a reduced risk of severe obesity, greater insulin sensitivity and improved lipid profiles in adolescence, compared with their siblings born prior to the surgery . In addiThe current study has unique strengths. Our mother–child cohort was prospectively designed, with comprehensive clinical assessments of childhood cardiometabolic risk factors including glucose and insulin levels during OGTTs. The availability of detailed measures collected in early life and childhood permitted meaningful and stepwise adjustment for a large number of potential confounders. Owing to the design of the HAPO study, the mothers and their doctors were blinded to the OGTT results and hence did not receive any dietary or medical treatment for hyperglycaemia during pregnancy, which would have confounded similar analyses in other studies.r2 = 0.899), supporting the validity of the self-reported data. Second, the IOM recommendations for GWG were developed in a cohort consisting largely of healthy white women, and used the standard BMI thresholds to define the categories of GWG. As adults in Asia have different BMI classification thresholds for overweight and obesity, further studies with sufficiently large sample sizes are required to determine the optimal GWG for Asian populations. Third, 41.8% (n = 697) of participants were non-eligible, declined the follow-up visit or were not contactable. Compared with children who attended the follow-up visit, we found no evidence of discrepancy in the distributions of GWG between two groups  and risk of stunted growth in the offspring if maternal GWG is inadequate. Finally, long-term follow-up of these children is necessary to evaluate the effect of maternal GWG on cardiometabolic risk in adolescence and adulthood.ESM(PDF 1.82 mb)