Outside the laboratory, people need to pay attention to relevant objects that are typically multisensory, but it remains poorly understood how the underlying neurocognitive mechanisms develop. We investigated when adult-like mechanisms controlling one’s attentional selection of visual and multisensory objects emerge across childhood. Five-, 7-, and 9-year-olds were compared with adults in their performance on a computer game-like multisensory spatial cueing task, while 129-channel EEG was simultaneously recorded. Markers of attentional control were behavioural spatial cueing effects and the N2pc ERP component . In behaviour, adult-like visual attentional control was present from age 7 onwards, whereas multisensory control was absent in all children groups. In EEG, multivariate analyses of the activity over the N2pc time-window revealed stable brain activity patterns in children. Adult-like visual-attentional control EEG patterns were present age 7 onwards, while multisensory control activity patterns were found in 9-year-olds . By combining rigorous yet naturalistic paradigms with multivariate signal analyses, we demonstrated that visual attentional control seems to reach an adult-like state at ∼7 years, before adult-like multisensory control, emerging at ∼9 years. These results enrich our understanding of how attention in naturalistic settings develops. Objects in such environments are also multisensory in nature. Here, we investigated whether adults and children pay attention to visual and multisensory stimuli in a similar way, and through similar brain mechanisms.1.1task-set contingent attentional capture hypothesis (multisensory enhancement of attention capture). These findings suggest that findings from purely unisensory attentional research may be limited in explaining how attention to objects in space is controlled in real-world, multisensory settings. While we know relatively little about how adults control their attention towards multisensory objects, we know even less about how children do so, and if adults and children attend to multisensory objects using similar mechanisms.The brain integrates information across the senses, and so processes multisensory stimuli differently than unisensory stimuli. Multisensory processes lead to faster and more accurate behavioural responses  and imprpothesis . Additio1.2Typically children have weaker visual attentional control skills than adults . This caand brain mechanisms that children and adults use when paying attention to objects in naturalistic, multi-stimulus contexts (where location of the target (unlike other target attributes), is typically unknown. Such investigations are necessary for several reasons. First, in that context, one can benefit from using rigorous paradigms isolating relevant attentional control processes, such as the One way to create more complete and accurate theories of development of attention is by comparing the cognitive 1.3and multisensory information? As indirect evidence, we have previously shown that audiovisual distractors interfere with visual search in adults and 11-year-olds, but not 6-year-olds, for both colour  analyses of the N2pc component are better at capturing developmental changes in attentional control over multisensory stimuli than the traditional N2pc analyses. Briefly, an EN approach encompasses a set of multivariate reference-independent analyses of the global features of the electric field measured at the scalp . The maiWe had the following predictions. Behaviourally, in adults, we expected to replicate task-set contingent attentional capture (“TAC”) and multisensory enhancement of attentional capture , 38 7-year-olds , and 28 5-year-olds , and 39 adults .A total of 39 young adults and 115 primary school children participated in the study. The children sample consisted of 28 children from the fifth grade, 46 children from the third grade, and 41 from the first grade of primary schools located in the canton of Vaud, Switzerland. In the local school system, children enter formal education at age 4, but only transition to sitting at desks instead of open seating and receiving less play-oriented instruction in third grade, when they are aged 6–7 years. To reduce confusion due to school system specificities, each child group is henceforth referred to by their members’ majority age, that is: ‘5-year-olds’ (first grade), ‘7-year-olds’ (third grade), and ‘9-year-olds’ (fifth grade). Children were recruited from local schools, nurseries, public events and entertainment facilities. Recruitment took place from March 2017 to May 2019. Of the total number of children recruited, first, 18 were excluded due to failure to initiate the testing session or failure to complete the task with above chance-level accuracy (50 %), thus leading to exclusion of one 9-year-old, six 7-year-olds, and eleven 5-year-olds. Additionally, five additional participants  were excluded because of unusable EEG signals due to excessive noise. Data for adult “controls” was taken from one task that was part of a larger study. The final sample consisted of 26 9-year-olds , neurological problems , neurodevelopmental disorders , or learning difficulties , as indicated by parental report for children, or by direct report for adults. No children had an FSIQ under 70 which would warrant exclusion, as confirmed by an overall cognitive functioning assessment (see below). All research procedures were approved by the Cantonal Commission for the Ethics of Human Research (CER-VD). Informed consent was obtained from parents/caregivers and verbal assent was obtained from children before participating in the study.2.2All participants were tested at the Lausanne University Hospital Centre (CHUV). For children, the EEG session lasted between 1 h and 1 h30mins, including briefing, obtaining consent, the testing protocol, and breaks. For adults, the session took approximately 3 h (part of a larger study), but the data used here were acquired within the first 1 h-1 h30, akin to the child protocol. Children’s baseline overall cognitive level was also assessed , during a separate session on a different day. To this end, we used the Wechsler scale of intelligence for school-age WISC-V,  or prescIn the EEG session, participants completed a computer-game-like task where they searched for an elongated target diamond of a predefined colour  across different age groups. This, in turn, provided insights into the age at which such visual attentional control mechanisms develop. In turn, manipulating the visual-only or audiovisual of the distractor allowed us to investigate the occurrence of multisensory enhancement of capture (MSE) in different age groups. This enabled us to investigate the development of attentional control processes engaged by task-irrelevant multisensory stimuli. The two above mentioned manipulations related to the cue resulted in two factors: Cue Colour (target colour-cue – TCC vs. nontarget- colour-cue – NCC) and Cue Modality , and, consequently, 4 experimental conditions: TCCV, NCCV, TCCAV, NCCAV.The target array contained 4 elements (‘diamonds’) where 1 was always the colour-defined target. The targets and their preceding cues could have either a blue  or red  colour, and the target colour was counterbalanced across participants. The targets were given their diamond-like appearance by adding triangle shapes on the short sides of the bars and increasing and decreasing the luminance of certain sides of the bars by 20 %.2), as determined by a luxmeter placed at a position adjacent to participants’ eyes, measuring the luminance of the screen filled with each respective element’s colour. The means of three measurement values were averaged across colours and transformed from lux to cd/m2 in order to facilitate comparison with the results of Experimental sessions were conducted in a dimly lit, sound-attenuated room, with participants seated at a distance of 90 cm from a 23” LCD monitor with a resolution of 1080 × 1024 . All elements were spread equidistally along the circumference of an imaginary circle against a black background, at an angular distance of 2.1° from a central fixation point. All visual elements were approximately equiluminant  map’ which informed participants of the number of blocks remaining until the end, and during which participants could take a break and parents/caregivers could enter the testing room. To maintain motivation in younger participants, stickers on diamond-shaped sheets were offered during breaks following each session.2.3A 129-channel HydroCel Geodesic Sensor Net connected to a NetStation amplifier  was used to record continuous EEG data sampled at 1000 Hz. Electrode impedances were kept below 50kΩ, and electrodes were referenced online to Cz. Offline filtering involved a 0.1 Hz high-pass and 40 Hz low-pass as well as 50 Hz notch and a second-order Butterworth filter . Next, the EEG was segmented into peri-stimulus epochs from 100 ms before cue onset to 500 ms after cue onset. Epochs were then screened for transient noise, eye movements, and muscle artefacts using a semi-automated artefact rejection procedure. It has been noted previously that due to physiological differences between children and adults’ skulls and brains, these two groups require different artefact rejection criteria to prevent discarding clean EEG signal . Therefo splines . Averagewww.fbmlab.com/cartool-software/; Cleaned epochs were averaged, baseline corrected to the 100 ms pre-cue time interval, and re-referenced offline to the average reference. Next, due to persistent environmental noise present in the majority of the child and adult datasets even after initial filtering, an additional 50 Hz notch filter was applied. All of the above steps were done separately for ERPs from the four cue conditions, separately for cues in the left and right hemifield. To analyse cue-elicited lateralised ERPs, single-trial data from all conditions with cues presented on the left were relabelled to have electrodes over the left hemiscalp represent activity over the right hemiscalp, and vice versa. After relabelling, the “mirror cue-on-the-right” single-trial data and the veridical “cue-on-the-right” data were averaged together, creating a single lateralised average ERP for each of the 4 cue conditions. As a result of this, we obtained 4 different ERPs, one for each of the 4 conditions . All preprocessing- and EEG analyses, unless otherwise stated, were conducted using CarTool software  and multisensory enhancement of attention capture (MSE) in adults , we used2.4.1SDs from individual participant’s mean RTs were also removed. Overall, 26 % of all trials were removed . Next, to verify if RT spatial cueing modulations were preserved after correcting for children’s general cognitive slowing, each individual’s RT’s per condition was divided by their average overall RT, and then converted to a percentage .Analyses were focused on reaction-time (RT) spatial cueing effects. This measure was derived by subtracting the mean RTs for trials where the cue and target were in the same location from the mean RTs for trials where the cue and target location differed, separately for each of the 4 cue conditions, following 2.4.2Given that the N2pc is a well-understood correlate of adult visual attentional control, we began our ERP analyses by conducting a canonical N2pc analysis, involving a comparison of the contralateral and ipsilateral average ERPs elicited across the 4 cue conditions. This way, we could compare the N2pc’s elicited by our visual and audiovisual distractors with the existing N2pc research in adults and children. Furthermore, those analyses helped us better bridge the previous and present canonical-analysis’ N2pc results with our electrical neuroimaging  analyses of the N2pc. Thus, we used data from the entire 129-channel electrode montage in our analyses of the contralateral versus ipsilateral ERP voltage gradients within an EN approach.Since the aim of the current study was to identify the emergence of adult-like attentional control mechanisms in childhood, all ERP analyses in developmental groups followed a ‘normative’ framework. That is, the parameters typically used for canonical analyses of the N2pc in adult visual attention research were applied to children’s data. Below, we detail how we conducted the N2pc and EN analyses across age groups.2.4.2.1We extracted the mean amplitude for each of the 4 cue conditions within a prescribed time-window, separately for the contralateral and the ipsilateral posterior electrodes. We used electrodes e65 and e90 – the EGI 129-channel equivalents of the canonical PO7/8 electrodes e.g., , and the2.4.2.2We have introduced EN analyses in the Introduction. Thus, here we provide more information on how EN has already been used to analyse mechanisms governing N2pc, how we designed our EN analyses of the N2pc in this study, as well as on the EN measures themselves.Attentional control mechanisms can modulate the contralateral-to-ipsilateral gradients of voltage potentials across different conditions see . These gTo analyse the global mechanisms underlying N2pc’s during visual control and multisensory control across development, we first computed a difference ERP where we subtracted the voltages over the ipsilateral hemiscalp from the voltages over the contralateral hemiscalp, separately for each of the 4 cue conditions. This resulted in a 59-channel lateralised ERP (without the uninformative midline electrodes from the 129-electrode montage). Next, this difference ERP was mirrored onto the other side of the scalp, creating a “fake” 129-electrode montage . It was on these mirrored “fake” 129-electrode lateralised difference ERPs that we performed the EN response strength and topography analyses, across the 4 cue conditions, for each of the 4 age groups.2.4.2.2.1We used Global Field Power (GFP) to investigate whether attentional modulations of cue-elicited lateralised ERPs resulted from differential response strength within statistically indistinguishable brain networks. GFP is a timepoint-to-timepoint standard deviation of voltage across the scalp, and can be plotted as a single waveform, just like any regular waveform. In an EN framework, differences in response strength in line with the gain-control account would be readily detected as GFP differences between experimental conditions over the N2pc time-window . To mirr2.4.2.2.2Next, we investigated whether differences across the lateralised ERPs were driven by changes in electric scalp field topography, and in turn, changes in activated configurations of brain generators. To analyse topographic differences across conditions, we applied clustering to the group-averaged mirrored difference ERPs over their whole post-cue time course. Clustering can reveal periods of tens to hundreds of milliseconds of stable topographic activity, i.e., topographic “maps” . Specifically, every time-point over the adult N2pc time-window in the cue-induced mirror difference ERPs of each tested child was labelled by the adult topographical map with which it best correlated spatially. The final output for each participant was the number of timeframes (in milliseconds) that each adult topographical map characterised the child’s ERP in the adult canonical N2pc time-window. These map durations were submitted to separate three-way 2 × 2  ×  4 repeated-measures ANOVAs in each age group, with factors: Cue Colour (TCC vs. NCC) and Cue Modality (V vs. AV), and Map (Map1 vs. Map2 vs. Map3 vs. Map4) followed by post-hoc t-tests. Maps with durations under 10 contiguous timeframes were not included in the analyses. Greenhouse-Geisser corrections were applied where necessary to correct for violations of sphericity. Unless otherwise stated, map durations were reliably present across the time-windows of interest . Throughout the results, Holm-Bonferroni corrections were used to correct for multiple comparisons between map durations. Comparisons passed the correction unless otherwise stated.As part of our “normative” EN analyses, first we applied the TAAHC to the group-averaged 33.13.1.1F = 94.7, p <  0.001, ηp² = 0.7. Here, 5-year-olds were reliably slower than 7-year-olds (t(33) = 4.4, p <  0.001), who were slower than 9-year-olds (t(32) = 5.7, p <  0.001), who were in turn, slower than adults (t(32.5) = 5.1, p <  0.001). However, Age did not interact with any other factors . Nonetheless, to adequately investigate differences between adults and children, and the developmental trajectory of attentional control processes, we analysed the raw RT data from each age group separately.Mean RTs sped up progressively from 5-year-olds (1309 ms) through 7-year-olds (1107 ms) and 9-year-olds (836 ms) to adults (594 ms), which was reflected in a significant main effect of Age, F = 36.9, p <  0.001, ηp² = 0.5, driven by faster responses on trials with target colour-cues  than on trials with nontarget colour-cues . Adults also showed generally faster responses on trials with sounds  than with no sounds , F = 76.1, p <  0.001, ηp² = 0.7. Overall behavioural capture effects in adults were reliable, i.e. responses were faster for trials where the cue and target location were the same (600 ms) versus when they were different (624 ms), F = 110.9, p <  0.001, ηp² = 0.8. Further, as in the original Matusz and Eimer’s (2011) study, the adults’ overall behavioural capture effects differed depending on the colour of the cue, as shown by a 2-way Cue-Target Location x Cue Colour interaction, F = 161.5, p < 0.001, ηp² = 0.8 (this is the TAC effect). This effect was driven by statistically significant behavioural capture effects for the TCC condition  = 16.7, p <  0.001), but not the NCC condition  = 0.2, p =  0.8; F = 4.9, p = 0.03, ηp² = 0.1 (this is the MSE effect). This effect was driven by larger behavioural capture effects elicited by AV  = 10.8, p <  0.001) than by V cues  = 8.9, p <  0.001; F < 1) was not significant, and neither was the Cue-Target Location x Cue Colour x Cue Modality interaction . These results demonstrated that adults showed both reliable TAC and MSE in behaviour, replicating Firstly, in adults, there was a significant main effect of Cue Colour, F = 28.4, p <  0.001, ηp² = 0.5. Their overall behavioural capture effects were also reliable, with faster RTs for trials where the cue and target location were the same (839 ms) versus when they were different (870 ms), F = 68.9, p <  0.001, ηp² = 0.7. Overall speeding up of responses on AV compared to V trials now showed the level of a nonsignificant trend  = 0.3, p =  0.08, ηp² = 0.1). However, the main question was whether behavioural capture effects in 9-year-old children would be modulated by the cues’ matching of the target colour, as well as the audiovisual nature of the cues. Notably, like in adults, 9-year-olds did indeed show TAC, as evidenced by a 2-way interaction between Cue-Target Location and Cue Colour, F = 19.5, p < 0.001, ηp² = 0.4. This interaction was driven by significant capture effects for the TCC distractors  = 8.3, p <  0.001), but not for the NCC distractors  = 0.9, p =  0.7; F = 1.4, p =  0.3. Other interactions failed to reach statistical significance . With this, we can conclude that 9-year-olds showed reliable TAC, but not MSE, in behaviour.Like adults, 9-year-olds responded faster on TCC trials (843 ms) than on NCC trials (865 ms), F = 18.7, p <  0.001, ηp² = 0.3, and were also faster for trials with AV cues (1111 ms) than V cues (620 ms), F = 8.6, p =  0.006, ηp² = 0.2. Further, overall capture effects were again reliable, with faster responses on cue-target location same (1109 ms) versus different (1140 ms) trials, F = 14, p <  0.001, ηp² = 0.4. Just as in the two older groups, 7-year-olds, did show TAC, as shown by a Cue-Target Location x Cue Colour interaction, F = 6.4, p = 0.02, ηp² = 0.2. This was driven by reliable cueing effects elicited by TCC distractors  = 4.8, p <  0.001), but not by NCC distractors  = 0.6, p =  1; F = 2.1, p =  0.2. Other interactions also did not reach statistical significance . It thus appeared that 7-year-olds, like 9-year-olds before them, showed reliable TAC, but not MSE.In 7-year-olds, like in adults, responses were faster for trials with TCC cues (1112 ms) than for NCC cues (1138 ms), F = 14, p <  0.001, ηp² = 0.4, driven by faster responses for cue-target location same (1312 ms) versus different (1343 ms) trials. However, there was no evidence for either of the two key interactions, specifically, the Cue-Target Location x Cue Colour interaction  = 1.4, p = 0.2), or the Cue-Target Location x Cue Modality interaction  = 0.4, p = 0.5). In further contrast with the older age groups, overall RTs were not affected by the colour of the cue, as shown by a nonsignificant main effect of Cue Colour, F = 2.6, p =  0.1. In one final contrast, faster responses on AV versus V trials showed only a nonsignificant trend, F = 3.5, p =  0.07, ηp² = 0.1. No other interactions reached statistical significance . The 5-year olds, therefore, did not show reliable TAC nor MSE in behaviour.In 5-year-olds, as in the other age groups, we observed reliable overall attentional capture effects 3.1.2Even after correcting for children’s overall cognitive slowing, all child groups showed the same patterns of results as in the raw RT analyses. That is, 9-year-olds and 7-year-olds showed TAC but not MSE, and 5-year-olds did not show either effect. The full description of the results on data corrected for slowing can be found in Supplementary materials.3.1.3H test to test for differences between groups, and 3-way Friedman tests (or Durbin tests where there were no errors for a given condition) to test for differences within each age-group. Overall, error rates were highest in 5-year-olds (57 %), and steadily reduced in 7-year-olds (23 %), followed by 9-year-olds (12 %), and adults (6%), χ2(3) = 81.4, p <  0.001. In adults, error rates were modulated by Cue-Target Location χ2(1) = 8.7, p =  0.003, such that fewer errors were made on trials where the cue and target location was the same (5.5 %) than when they were different (6.6 %). Error rates were not significantly modulated by Cue Colour or Cue Modality . In 9-year-olds, 7-year-olds, and 5-year-olds alike, error rates were not significantly modulated by Cue-Target Location, Cue Colour or Cue Modality .Since error data were not normally distributed, we conducted a 1-way Kruskal–Wallis 3.23.2.1F = 17.8, p <  0.001, ηp² = 0.3, where the mean contralateral amplitude (-0.4μV), was larger than the ipsilateral amplitude (0.1μV). Consequently, the contra-ipsilateral difference had a mean overall amplitude of −0.5μV. As expected, cue-elicited N2pcs’ differed in their magnitude depending on the cue colour, as supported by a Contralaterality x Cue Colour 2-way interaction, F = 17, p <  0.001, ηp² = 0.3. This interaction was driven by a reliable N2pc for target colour-cues .In adults, there was a reliable overall N2pc, as demonstrated by a statistically significant main effect of Contralaterality, F = 8, p =  0.007, ηp² = 0.2. We first analysed this interaction as a function of Cue Modality. First, for AV cues, mean N2pc amplitudes elicited by TCCAV were larger (−0.8μV) than mean amplitudes for NCCAV cues (−0.2μV), t(38) = 5, p <  0.001. In contrast, for V cues, there was no statistically significant difference in mean N2pc amplitudes elicited by NCCV cues (−-0.3μV) and TCCV cues (−0.6μV), t(38) = 1.8, p =  0.2. When we analysed the 3-way interaction as a function of Cue Colour, for both TCC and NCC distractors, differences in mean N2pc amplitude between AV and V were at the level of a nonsignificant trend (t(38) = 1.8, p =  0.06, and t(38) = 1.4, p =  0.07, respectively). Other effects did not reach statistical significance (All F’s < 1), except the main effects of Cue Colour, F = 8.4, p =  0.006, ηp² = 0.2  = 7.1, p =  0.011, ηp² = 0.2 . Thus, although MSE was not observed in N2pc’s, adult’s overall ERP data was jointly modulated by visual and multisensory attentional control. This effect seemed to be driven by reliable difference between TCC and NCC distractors on trials where distractors were AV but not V.Interestingly, the N2pc amplitudes elicited by TCC and NCC cues were modulated by sound presence, as shown by a 3-way interaction between Contralaterality, Cue Colour, and Cue Modality, F = 0.4, p = 0.6; 7-year-olds: F = 0.04, p = 0.8; 5-year-olds: F = 0.2, p = 0.6; F’s < 1), and thus no presence of an N2pc.For the child age groups, 2 × 2  ×  2 repeated-measures ANOVAs were conducted on mean amplitude values from adult electrodes over the adult time-window. In no child group was there a significant main effect of Contralaterality (9-year-olds: 3.2.2F’s < 1). Full results can be found in Supplementary Materials: Supplemental GFP results. For graphical representations of the GFP results, we direct the reader to Supplemental Fig. 1 in the Supplementary Materials.An ANOVA on the average GFP values per condition revealed no significant main effects or interactions in adults, 9-year-olds, 7-year-olds, or 5-year-olds  = 18.3, p <  0.001, ηp² = 0.3, where Map4 predominated  the N2pc time-window across conditions  = 12, p <  0.001, ηp² = 0.2. Following up this interaction by the factor of Cue Colour showed that Map4 predominated responses to TCC (67 ms) compared to NCC distractors (40 ms), t(38) = 5.2, p <  0.001, while Map2 predominated responses to NCC (34ms) compared to TCC distractors (13ms), t(38) = 3.9, p =  0.004. Other maps did not differ significantly between TCC and NCC cues . Hereafter, differences in map predominance that are not reported here were not statistically significant (p’s > 0.1). Following up the interaction by the factor of Map revealed that for TCC cues, Map4 (67 ms) overall predominated the N2pc time-window compared to all other maps – Map1 (15 ms), t(38) = 7.7, Map2 (13 ms), t(38) = 8, and Map3 (25 ms), t(38) = 6.3, all p’s < 0.001, while no map differed in their predominance of responses to NCC distractors . These results suggest that Map4 drove the processing of TCC distractors, while no particular map was more implicated than others in the processing of NCC distractors. Finally, the map modulations by Cue Colour demonstrated here support the presence of TAC in adult ERP topography. Thus, it appeared that the Map × Cue Colour interaction was driven by modulations of Map2 and Map4 presence for different cue colours, where Map4 is especially implicated in the processing of target-colour cues.There was a 2-way interaction between Map and Cue Colour, F = 3.2, p =  0.027, ηp² = 0.1. A follow-up by Cue Modality revealed that Map2 predominated responses when cues were purely visual  than audiovisual , at the level of a nonsignificant trend, t = 2.8, p =  0.08. However, a follow-up by Map revealed that Map4 predominated the N2pc time-window compared to other maps for both AV cues  = 4.3, Map2 [17 ms], t(38) = 5.6, Map3 [26 ms], t(38) = 4.2, all p’s < 0.001), and for V cues  = 5.7, Map2 [30 ms], t(38) = 3.7, Map3 [19 ms], t(38) = 5.5, all p’s < 0.001). Taken together, it appeared that Map2 may be implicated more strongly in topographic modulations of lateralised ERPs by Cue Modality, whereas Map4 was the main map driving the processing of both AV and V cues.In contrast to canonical N2pc analysis results, topographic map presence over the N2pc time-window interacted with Cue Modality, as evidenced by a 2-way interaction, F = 5.4, p =  0.002, ηp² = 0.1. When followed up as a function of Cue Colour, for NCC distractors, Map2 predominated responses to V cues (50 ms) over AV cues (18 ms), t(38) = 4.7, p <  0.001. Yet, for TCC distractors, all map durations were comparable between V and AV cues . Next, when following up as a function of Cue Modality, for AV cues, Map4 predominated responses to TCC distractors (67 ms) compared to NCC distractors (40 ms), t(38) = 3.8, p =  0.004. Likewise, for V cues, Map4 predominated TCC (67ms) than NCC (39ms) distractor responses, t(38) = 3.6, p =  0.003. However, Map2 also predominated responses to NCC (50ms) over TCC distractors (10ms), for V cues, t(38) = 5.4, p <  0.001. Thus, maps that are sensitive to TAC and MSE appear to interact, suggesting that top-down visual attentional control and bottom-up multisensory attentional control may share neural generators.Finally, the 3-way Map x Cue Colour x Cue Modality interaction was significant, To explore if and when the above adult topographical EEG patterns are present in children, we submitted each child age-groups’ data within the 180–300 ms time-window to a fitting procedure, where child topographical data were labelled according to the adult template maps with they which they best correlated spatially.F = 9.2, p <  0.001, ηp² = 0.3, and, like in adults, Map4 predominately characterised ERPs during the N2pc time-window  = 3.4, p =  0.04, ηp² = 0.1. A follow up by Cue Modality found that Map3 predominated the responses to AV (27 ms) compared to V cues (11 ms), t(25) = 2.6, p =  0.02, while Map4 predominated responses to V (55ms) compared to AV cues (39ms), t(25) = 2.5, p =  0.02. However, the map that was sensitive to the (audio)visual nature of the cues in adults, Map2, was comparable in how it predominated responses to V cues (31ms) and AV cues (27ms), t(25) = 0.7, p = 1. In a follow-up as a function of Map, there were no significant differences between map predominance for AV cues . For V cues, however, Map4 (55 ms) predominated overall, compared to all other maps  = 4, Map2 [32 ms], t(25) = 3.7, Map3 [11 ms], t(25) = 5.8, all p’s < 0.001). In a marked contrast to adults, 9-year-olds did not show the other 2-way interaction of interest, Map × Cue Colour  = 1.3, p =  0.3). Other interactions also failed to reach statistical significance . Taken together, 9-year-olds seemed to show adult-like MSE . Even though they did not show a modulation of the adult MSE-sensitive map, 9-year-olds’ overall topographic results were like those of adults, with a predominance of Map4 across conditions.For 9-year-olds, the ANOVA revealed a main effect of Map, F = 9.7, p <  0.001, ηp² = 0.2, with a predominance of Map4, akin to the two older age groups . This included the 2-way interactions of interest, Map x Cue Colour  = 0.7, p =  0.6) and Map x Cue Modality  = 1.3, p =  0.3). We can therefore conclude that 7-year-olds’ topography did not show adult-like TAC or MSE, although their overall topographic pattern could be considered adult-like.In 7-year-olds, there was also a main effect of Map, F = 6.3, p <  0.001, ηp² = 0.2, but here, there was no clear map predominance pattern (F’s < 1), including the two 2-way interactions of interest, Map x Cue Colour  = 0.8, p =  0.4) and Map x Cue Modality  = 0.7, p =  0.5). With this, 5-year-olds seemed not to show adult-like TAC, MSE, or overall pattern of map presence.Finally, 5-year-olds also showed a main effect of Map, 4In cluttered learning environments like classrooms, children must focus their attention on relevant information and ignore unimportant information. There is modest research on the differences in neuro-cognitive mechanisms governing visual  attentional control between adults and children. In contrast, little is known about the development of multisensory attentional control mechanisms. Our study aimed to clarify how adult-like visual and multisensory attentional control mechanisms develop side by side over the course of primary education. We tested this by combining traditional behavioural and ERP measures of attentional selection with multivariate electrical neuroimaging analyses.4.1Behaviourally, we replicated in adults both task-set contingent visual attention capture (TAC) and multisensory enhancement of attention capture (MSE). We did so in a larger sample and with small adjustments to the Behavioural findings were extended by EN findings. In adults, topographical ERP analyses revealed two stable patterns of brain activity (template maps) that were each modulated by TAC and by MSE. Interestingly, the adult TAC-sensitive template map dominated the N2pc time-window overall - in adults, 7-, and 9-year-olds. However, in the child groups, the predominance of the adult TAC-sensitive map was not modulated by target-colour-matching, as those groups did not show evidence for a Map x Cue Colour interaction. Nonetheless, the child groups showed adult-like visual attentional control in behaviour also as well as the recruitment of brain networks modulated by distractor colour in adults. This at least indirectly supports that already 7-year-olds can deploy their top-down attention in a way that could be considered adult-like.Our youngest group, 5-year-olds, did not show a reliable TAC effect. This result contrasts with the only other study on TAC in young children , which f4.2irrelevant objects. Notably, however, we did not study multisensory integration per se, but rather cross-modal audio-visual interactions, which should be present already at age 5  the previously elusive sensitivity of visual attentional selection to bottom-up multisensory processing, and 2) adult-like attentional control processes in children as young as 7. The second finding is certainly promising for the use of EN to study the development, of attentional control and beyond. Developmental studies compare adults and children in their attentional skills e.g., . In shor5Taken together, our study revealed the developmental trajectory of a frequently studied visual attentional control mechanism that is task-set contingent attentional capture (TAC). We showed, both behaviourally and using an EN analytical framework, that TAC develops early in childhood (after the age of 5 years), and reaches adult-like state at 7 years of age. Though MSE, present in adults, was undetected in children’s behaviour or traditionally analysed EEG signals, an EN framework revealed spatially-selective brain mechanisms sensitive to the multisensory nature of distractors, at 8–9 years. Our findings underline the utility of combining traditional behavioural and EEG/ERP markers of visual attentional control with multivariate EEG analytical techniques for investigating the development of attentional control, and for identifying developmental differences and similarities in attentional control between adults and children.pawel.matusz@gmail.com) for data sharingUnfortunately, the current data set was acquired before both researchers and the broader public began to understand the scientific importance of data sharing. As we had not consented participating parents and children with an explicit data sharing statement, we cannot upload the current data set to an open data repository. Researchers interested in analysing the current data set are very welcome to contact the Corresponding Author  grants to P.J.M., as well as by the Fondation Asile des Aveugles.This work was supported by the Nora Turoman: Investigation, Formal analysis, Data curation, Software, Visualization, Writing - original draft. Ruxandra I. Tivadar: Software, Writing - review & editing. Chrysa Retsa: Software, Writing - review & editing. Anne M. Maillard: Methodology, Writing - review & editing. Gaia Scerif: Methodology, Writing - review & editing. Pawel J. Matusz: Conceptualization, Funding acquisition, Methodology, Resources, Formal analysis, Data curation, Software, Supervision, Writing - review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Neural crest cells (NCCs) contribute to several tissues during embryonic development. NCC formation depends on activation of tightly regulated molecular programs at the neural plate border (NPB) region, which initiate NCC specification and epithelial-to-mesenchymal transition (EMT). Although several approaches to investigate NCCs have been devised, these early events of NCC formation remain largely unknown in humans, and currently available cellular models have not investigated EMT. Here, we report that the E6 neural induction protocol converts human induced pluripotent stem cells into NPB-like cells (NBCs), from which NCCs can be efficiently derived. NBC-to-NCC induction recapitulates gene expression dynamics associated with NCC specification and EMT, including downregulation of NPB factors and upregulation of NCC specifiers, coupled with other EMT-associated cell-state changes, such as cadherin modulation and activation of TWIST1 and other EMT inducers. This strategy will be useful in future basic or translational research focusing on these early steps of NCC formation. •Human neural plate border-like cells (NBCs) emerge during the E6 neural induction•NBC-to-NCC induction recapitulates sequential steps of NCC specification and EMT•This approach provides a human cell model of early NCC development in vitro platform for investigating human early NCC development and associated disorders.Kobayashi et al. show that neural conversion of hiPSCs produces NPB-like cells that can be efficiently converted into NCCs. This enables observation and recapitulation of sequential molecular and cellular events involved in NC specification and EMT, and provides an  Neural crest cells (NCCs) are a multipotent cell population that plays pivotal roles in vertebrate embryonic development. In late-blastula stages, intermediate BMP levels specify cells residing between the prospective neural and non-neural ectoderm into the neural plate border (NPB), a broad competence domain that contributes to formation of the neural tube, neural crest (NC), and the preplacodal and non-neural ectoderm . Definitin vitro approach describing human NC EMT.Disturbances in any of the above steps may impair NC development and result in several human health conditions. These include congenital disorders arising from defects in NC derivatives, such as craniofacial syndromes, pigmentation disorders, Hirschprung disease, and others, and also aggressive cancers of NC origin, such as neuroblastoma and melanoma . Due to Here, we report a simple hiPSC-based procedure to investigate early NC formation. This procedure recapitulates gene expression dynamics and other cellular hallmarks associated with embryonic NPB cells transitioning from a neuroepithelial to an NC mesenchymal state, allowing for time series assessment of key molecular events orchestrating NC specification and EMT. This approach could be used to model human NC-related disorders, particularly those associated with these early events of NC development.OCT3/4 and NANOG were downregulated, while neural markers SOX2 and PAX6 showed increased mRNA expression on day 4, persisting until day 6  minimal medium, without blocking the BMP pathway, in 6 days . Three hil day 6 B. On dayo hiPSCs A. After o hiPSCs , was loco hiPSCs B. Finallo hiPSCs   involved in NC development and/or expressed in the NPB region (CDH1 (E-cadherin) and CDH2 (N-cadherin) are maintained and upregulated on day 4, respectively. CDH6 and CDH7, which are expressed in the NPB of chick and rat embryos . We alsTogether, the above findings show that an NPB-like identity arises on day 4 of the E6 neural induction, which recapitulates embryonic NPB expression patterns. Thus, we renamed this differentiation time point to “NPB-like cells” (NBCs), which were selected to undergo NC induction.SOX9 and SOX10, in addition to NC markers NGFR (p75) and VIM (Vimentin) , an Activin pathway blocker (SB431542), and a WNT pathway activator (CHIR99021), for 15 days . During imentin) A. Flow cimentin) B and 2Bʹ0+ cells C and 2Cʹ+/Vimentin+ smooth muscle  via differentiation into NC derivatives. NBC-NCCs were first converted to ectomesenchymal stem cells D, which h muscle F, and adh muscle G. Day 15ecursors H, in addecursors I. FinallPRICKLE1  ; after upregulation on day 0 (NBCs), NPB factor DLX5 was also downregulated, remaining active until day 7, and was later reactivated on day 15, possibly reflecting later NCC functions in pharyngeal arch patterning (TFAP2A/MSX2/ZIC1) or further increased (PAX3) until day 15  or were further upregulated (CDH11) from day 0 to 15 . Nevertheless, nuclear expression of SNAI2 could be observed in all time points, being strongly expressed in nuclear foci at the end of induction  is lost at intercellular contact sites as cells acquire mesenchymal features. Initially, N-cadherin was observed at those sites from days 0 to 4 in all samples Gʹ and S3nd FOXD3 E.Despite the variations in cadherin dynamics between days 2 and 8, we observed that NBC-NCCs were, around day 7, invariably composed of several dense, epithelial-like cell clusters surrounded by individualized, mesenchymal-like cells A and 4A′− cells, albeit not reaching statistical significance compared with day 0  and day 15 (5.30%) D and 4D′th day 0 F and 4F′+ cells appear on this day, we targeted DLX5 and TWIST1 with double immunostaining on days 0, 7, and 15 to further characterize EMT-associated cell-state changes. On day 0, we observed some TWIST1 expression, which largely did not overlap with DLX5  that affect endogenous BMP signaling and result in variable NCC conversion rates , increment or maintenance in expression of factors involved both in NPB and NC specification , and upregulation of NC specifiers/EMT inducers . These changes were accompanied by downregulation of CDH1/2 in favor of mesenchymal cadherins CDH6/7/11, a hallmark of NC EMT  approved the Three hiPSC lines derived from healthy individuals  were used in all experiments, except for immunostaining analysis of day 7 NBC-NCCs  and S4, All experiments were performed with at least n = 3 biological replicates.Three hiPSC lines were reprogrammed from dermal fibroblasts  and one from erythroblasts (F8799-1c1) according to previous protocols . hiPSCs 5 cells/cm2) in E8 medium with Normocin and 5 μM Y-27632, and 1 day post-seeding, medium was changed to E6 medium (Thermo Fisher Scientific) with Normocin and replenished daily for 6 days.hiPSCs were conditioned to E8 medium for at least two passages before differentiation toward NBCs via the E6 method . In brie4 cells/cm2) in E6 with 5 μM Y-27632. In the following day, medium was changed to NCC medium . Medium was changed every 3 days.NCC-derived ectomesenchymal stem cells were induced with mesenchymal medium consisting of DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 2 mM GlutaMAX, 0.1 mM non-essential amino acids (Thermo Fisher Scientific), and Normocin. NCCs were seeded at 2 × 104 cells/μL in mesenchymal medium, and 5-μL droplets were deposited in 24-well plates and incubated at 37°C in 5% CO2. After 45 min, 400 μL of chondrogenesis medium was carefully added to each well. Alcian blue staining was performed according to the manufacturer's instructions. Undifferentiated ectomesenchymal stem cells were used as negative controls.To produce smooth muscle, cells were treated with DMEM/F12 supplemented with 5% FBS and 10 ng/mL transforming growth factor β1 (PeproTech) for 3 weeks  with med4 cells/cm2 on Matrigel-coated dishes and cultured for 40 days in NC medium supplemented with 20 ng/mL heregulin-β1 (PeproTech) , 10 ng/mL brain-derived neurotrophic factor, 10 ng/mL glial cell line-derived neurotrophic factor, 10 ng/mL NT-3 (PeproTech), 10 ng/mL NGF (BioLegend), 0.5 mM db-cAMP, and 200 μM ascorbic acid , and 1 μM SJ000291942 (Sigma). Medium was changed every 3 days.For Schwann precursors, NBC-NCCs were seeded at 5 × 10proTech) ; medium bic acid . MelanocACTB, TBP, HMBS, GAPDH, and HPRT1). Relative expression was calculated as elsewhere (One microgram of RNA was obtained with a NucleoSpin RNA II kit (Macherey-Nagel) and converted into cDNA with Superscript IV (Thermo Fisher Scientific) according to the manufacturer's recommendations. Reactions were performed with 2× Fast SYBR Green PCR Master Mix (Thermo Fisher Scientific) and 50–400 nM of each primer . Fluoreslsewhere .Cells were fixed in 4% paraformaldehyde for 20 min at RT and permeabilized with PBS 0.2% Triton X-100 for 30 min followed by blocking with PBS 5% BSA for 1 h at RT. Cells were incubated with primary antibodies overnight at 4°C under gentle agitation, washed 3× with PBS, and incubated with secondary antibodies at 4°C for 1 h. Cells were counterstaining with DAPI for 2 min at RT. Antibodies used in this study are detailed in To assess expression of NPB and EMT markers, cells were dissociated with Accutase, washed once with PBS, and fixation, permeabilization, and antibody incubation were performed with FIX & PERM (Thermo Fisher Scientific). Cells were incubated for 1 h with primary antibodies at 4°C, and 1 h with secondary antibodies at 4°C . To accoDay 15 NBC-NCCs were double stained for p75/HNK-1 with conjugated antibodies. Ectomesenchymal stem cells were stained with antibodies for CD29, CD31, CD45, CD90, CD73, and CD166. Expression was quantified in relation to appropriate immunoglobulin-matching isotype controls to calibrate for non-specific staining .At least 5,000 events were acquired in a FACSAria II equipment and analyzed on FlowJo.Gene expression values were log-transformed before statistical analyses. One-way ANOVA was conducted to compare the effect of differentiation (days) on gene expression across samples. When means were significantly different (p < 0.05), Dunnett's multiple comparison test was performed, and significance was indicated in comparison with day 0 of differentiation, unless otherwise stated. Analyses were carried out on GraphPad Prism.G.S.K. and C.M.M. conceptualized the work and designed the experiments. G.S.K., C.M.M., D.d.P.M., G.P.-G., G.S.P.H., L.C.C.-J., E.G., and M.R.P.-B. contributed with data acquisition, analysis and/or interpretation. G.S.K., C.M.M., and M.R.P.-B. wrote the manuscript.


Kuphus polythalamius . Based on its 16S rRNA sequence, the endosymbiont falls within a clade that includes the as-yet-uncultivated thioautotrophic symbionts of a marine ciliate and hydrothermal vent gastropods, uncultivated marine sediment bacteria, and a free-living sulfur-oxidizing bacterium ODIII6, all of which belong to the Gammaproteobacteria. The endosymbiont is Gram-negative, rod-shaped and has a single polar flagellum when grown in culture. This bacterium can be grown chemolithoautotrophically on a chemically defined medium supplemented with either hydrogen sulfide, thiosulfate, tetrathionate or elemental sulfur. The closed-circular genome has a DNA G+C content of 60.1 mol% and is 4.79 Mbp in size with a large nitrogenase cluster spanning nearly 40 kbp. The diazotrophic capability was confirmed by growing the strain on chemolithoautotrophic thiosulfate-based medium without a combined source of fixed nitrogen. The bacterium is also capable of heterotrophic growth on organic acids such as acetate and propionate. The pH, temperature and salinity optima for chemolithoautotrophic growth on thiosulfate were found to be 8.5, 34 °C and 0.2 M NaCl, respectively. To our knowledge, this is the first report of pure culture of a thioautotrophic animal symbiont. The type strain of Thiosocius teredinicola is PMS-2141T.STBD.0c.01aT (=DSM 108030T).A chemolithoautotrophic sulfur-oxidizing, diazotrophic, facultatively heterotrophic, endosymbiotic bacterium, designated as strain 2141T, was isolated from the gills of the giant shipworm  Kuphus polythalamius, the world’s longest bivalve , no growth of strain 2141T was observed. Various other C1-compounds  that may potentially be present in the environment of the host Many nitrogen-fixing bacteria grow best under microaerophilic conditions, at least in part because nitrogenases are irreversibly inactivated by oxygen. We tested the aerotolerance of strain 2141T on soft-agar STBA medium (0.2 % w/v Bacto agar) with or without combined nitrogen source (ammonium chloride). In the presence of combined nitrogen, growth was observed from the surface to the bottom of the agar stab, while in the absence of combined nitrogen, growth was limited to subsurface regions of the agar, signifying a preference for microaerobic conditions during diazotrophic growth. The capacity of these symbionts to utilize nitrate and nitrite  were tested during growth under thioautotrophic conditions by replacing ammonium chloride in STB50 medium. Under aerobic conditions, large colonies formed on nitrate plates, while small colonies developed on nitrite plates, suggesting that strain 2141T can assimilate nitrate and to a lesser extent, nitrite. Chemolithoautotrophic growth was also seen on both nitrate and nitrite plates under anaerobic conditions (GasPak Anaerobic Pouch). Yeast extract (0.1 % w/v) also supported growth under oxic growth conditions.+2 and Mg+2 ions similar to those found in seawater. No growth was seen on STBA medium modified to contain reduced Ca+2 and Mg+2 concentrations (0.5 mM CaCl2·2H2O and 0.05 mM MgCl2·6H2O); while growth was observed with 10 mM CaCl2·2H2O and 50 mM MgCl2·6H2O.The effects of temperature, salt concentration and pH on cultures grown on STBA were determined under thioautotrophic conditions. Growth of strain 2141T was observed between 30 and 37 °C, with the optimum around 34 °C, while no growth was observed at 25 °C and 40 °C. Salt tolerance was tested by varying the concentration of NaCl from 0.0 M to 1.0 M, at 0.1 M increments in the liquid culture. The optimum salinity was 0.2 M NaCl, but growth was observed in NaCl concentrations ranging from 0.0 M to 0.7 M. Growth of strain 2141T was observed in media with an initial pH of 5.5 to 8.5, with pH 8.5 being the optimum. The pH buffer systems used were as follows: pH 5.5, 6.0 and 6.5 with 20 mM MES; pH 7.0, 7.5 and 8.0 with 20 mM HEPES; pH 8.5 with 20 mM TAPS; and pH 9.0 and 9.5 with 20 mM CHES. Three days after growth was initiated at pH 8.5, a dramatic decline in pH (>3 pH units) was observed, presumably due to the build-up of sulfate, the end-product of sulfur metabolism in thioautotrophic bacteria. Typical of marine bacteria, growth of strain 2141T requires concentrations of CaThiosocius .Thiosocius is proposed to accommodate six thioautotrophic endosymbiotic bacterial strains isolated from the gill tissues of the giant shipworm Kuphus polythalamius, collected in two locations in the Philippines. All strains share greater than 99.8 % nucleotide identity within a 1265 bp region of the small subunit (16S) ribosomal RNA gene.The monospecific genus Thiosocius teredinicola.Gram-negative straight rods, that are on an average 1.7 µm long and 0.5 µm wide, have a single polar flagellum. Cells display carboxysomes and sulfur storage globules within the cytoplasm. Capable of chemolithoautotrophic growth utilizing thiosulfate as an energy source, and bicarbonate as a sole carbon source, diazotrophic, mesophilic, requires media containing elevated Ca and Mg concentrations reflecting those characteristic of seawater, capable of heterotrophic growth utilizing organic acids or alcohols, aerobic, microaerobic or facultative anaerobe via nitrate or nitrite respiration. The type species is Thiosocius teredinicola .d-fructose, glutamine and glutamate. The FAME profile was largely composed of C16 : 1 ω7c and/or C16 : 1 ω6c, palmitic acid (C16 : 0) and (C18 : 1 ω7c). The pH, temperature and salinity optima for growth were found to be 8.5, 34 °C and 0.2 M NaCl, respectively. The closed-circular genome has been sequenced and has a G+C content of 60.1 % and is 4.79 Mbp in size. The type strain PMS-2141T.STBD.0c.01aT (=DSM 108030T) was isolated from a sediment-dwelling Kuphus polythalamius from specimen ID PMS-2133X collected in a marine bay in the Philippines.Gram-negative rods, that are on an average 1.7 µm long and 0.5 µm wide, have a single polar flagellum, bearing carboxysomes and sulfur storage globules. Capable of chemolithoautotrophic growth on sulfide, thiosulfate, tetrathionate, or elemental sulfur on a completely chemically defined marine salt medium. Diazotrophic when grown in the absence of combined nitrogen. Assimilates nitrate and nitrite during aerobic thioautotrophic growth on thiosulfate. Can respire nitrate and nitrite anaerobically during thioautotrophic growth on thiosulfate. Grows heterotrophically on acetate, propionate, pyruvate, malate, succinate, fumarate, glycerol, ethanol, Click here for additional data file.


Escherichia coli-produced monophosphoryl lipid A (MPL), named EcML, in enhancing the immunogenicity and efficacy of an influenza vaccine. Similar to MPL, EcML activated dendritic cells and enhanced the antigen processing of cells in vitro. Using ovalbumin (OVA) as a model antigen, EcML increased OVA-specific antibody production, cytotoxic T lymphocyte activity. The safety of EcML was demonstrated as being similar to that of MPL by showing not significant in vitro cell cytotoxicity but transient systemic inflammatory responses within 24 h in OVA immunized mice. Importantly, mice vaccinated with pandemic H1N1 (pH1N1) vaccine antigen, combined with EcML, were fully protected from pH1N1 virus infection by enhanced influenza-specific antibody titers, hemagglutination inhibition titers, and IFN-γ- secreting cells. Taken together, our results strongly suggest that EcML might be a promising vaccine adjuvant for preventing influenza virus infection.Emerging influenza viruses pose an extreme global risk to human health, resulting in an urgent need for effective vaccination against influenza infection. Adjuvants are vital components that can improve vaccine efficacy, yet only a few adjuvants have been licensed in human vaccines. Here, we investigate the adjuvant effects of  The influenza virus poses a serious global threat to human health and can cause pandemics associated with high mortality worldwide . While iSalmonella minnesota R595. MPL has been demonstrated to stimulate innate immunity via TLR4 [Escherichia coli-produced MPL (named EcML) has been previously produced by direct extraction from genetically engineered E. coli [Adjuvants targeting pattern-recognition receptors, such as Toll-like receptors (TLRs), are part of a broad approach to stimulate innate immune responses, thereby enhancing antigen-specific immunity and subsequently improving vaccine efficacy . Recentlvia TLR4  and promvia TLR4 . Even thvia TLR4 , the com E. coli .In this study, we first investigated the action mechanisms of EcML to increase innate immunity and tested its adjuvanticity using the model antigen ovalbumin (OVA). EcML triggered the activation of bone marrow-derived dendritic cells (BMDCs) via TLR4 and increased the antigen processing of cells. Compared to OVA alone, OVA plus EcML-immunized mice showed an enhancement in OVA-specific humoral and cellular immune responses. Finally, we explored the adjuvanticity of EcML in enhancing the pandemic H1N1  influenza vaccine efficacy. EcML improved the protective efficacy of the pH1N1 vaccine antigen in mice with 100% survival after viral challenge by increasing influenza-specific antibody (Ab) titers, hemagglutination inhibition (HI) titers, and cytotoxic T lymphocyte (CTL) activity. Collectively, our results strongly suggest that EcML might be a promising adjuvant candidate for influenza vaccines.Six-week-old female C57BL/6 mice were purchased from Orient Bio . Six-week-old female wild-type C3H/HeN and congenic TLR4-defective C3H/HeJ mice were purchased from Central Lab. Animal Inc. . All animals were housed in a specific pathogen-free (SPF) facility at the Korea Research Institute of Bioscience and Biotechnology (KRIBB). All experiments employing mice were reviewed and approved by the Institutional Animal Care and Use Committee of the KRIBB. Immature BMDCs were generated using Roswell Park Memorial Institute 1640 media (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 U/mL penicillin, 100 mg/mL streptomycin (Gibco), 20 ng/mL murine granulocyte-macrophage colony-stimulating factor , and 10 ng/mL murine IL-4 (Peprotech) for 7 days followed by changing with fresh media every 3 days, as previously described ,22.E. coli KHSC0055 and formulated as aqueous formulations using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and chloroform, and was then evaporated to remove chloroform. The dried EcML was rehydrated in ultrapure water at 0.45 mg/mL, and suspended by sonication at 60 °C. The MPL from S. minnesota R595 was purchased from InvivoGen  and aqueously formulated using the same processes as for EcML. The resulting EcML and MPL were stored at 4 °C for further use. Alum (Alhydrogel adjuvant 2%) was obtained from InvivoGen.EcML was produced by Eubiologics. Co., Ltd.  as described previously  with slig for 10 min at 4 °C, filtrated through 0.45 μm pore size membrane filters , and then stored at −80 °C for further use. All viral experiments were implemented under conditions of biosafety level 2.The pH1N1 influenza virus was grown in 9- to 10-day-old SPF embryonated chicken eggs  for 48 h at 37 °C. The viruses were harvested from the allantoic fluids of the eggs by centrifugation at 3500× TM automated cell counter .Immature BMDCs were stimulated with PBS as a negative control, 0.625, 1.25, 2.5, and 5 μg/mL EcML, 1 μg/mL LPS or 0.5% Triton X-100 (as a positive control) for 24 h at 37 °C. Cell viability was measured using Trypan blue stain 0.4% with the Countess To investigate the BMDC activation, the cells were stimulated with PBS as a negative control, 0.625, 1.25, 2.5, and 5 μg/mL EcML, or 5 μg/mL MPL for 24 h at 37 °C. The stimulated cells were stained with PE-conjugated monoclonal antibodies (mAbs) against mouse CD40, CD80, CD86, major histocompatibility complex (MHC) class II, and isotype-matched control mAbs . To examine the antigen processing of BMDCs, we incubated the cells with 5 µg/mL DQ™-OVA, which is OVA conjugated with BODIPY FL dye (a self-quenched dye that emits fluorescence upon proteolytic degradation) (Thermo Fisher Scientific) alone or mixed with either 2.5 μg/mL EcML or MPL for 5 h at 37 °C. The cells were acquired on FACSCalibur flow cytometers (BD Biosciences), and the data were collected and analyzed using FlowJo software version 10 .g for 15 min at 4 °C, and concentrations of total proteins were determined using a bicinchoninic acid protein assay (Thermo Fisher Scientific). The cell lysates were separated by 12% SDS-PAGE and transferred to PVDF membranes (Merck KGaA). The blots were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 and probed with anti-phospho-IκBα rabbit mAb  for 16 h at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) Ab . The blots were then visualized by chemiluminescence using an enhanced chemiluminescent substrate kit . The membrane was then subsequently stripped and reprobed with anti-β-actin mAb  as a loading control.Immature BMDCs generated from C3H/HeN and C3H/HeJ mice were serum-deprived for 3 h and then stimulated with EcML at 0.625, 1.25, and 2.5 μg/mL or 2.5 µg/mL MPL for 30 min at 37 °C. The cells were lysed with a lysis buffer containing protease inhibitors  plus a phosphatase inhibitor cocktail (Sigma-Aldrich). The cell lysates were collected by centrifugation at 13,000× 50) of the pH1N1 influenza virus. The body weight and survival of the challenged mice were monitored for 14 days after infection. Mice that lost significantly more than 25% of their body weight were considered to have reached the experimental endpoint and were sacrificed.C57BL/6 mice were intramuscularly (i.m.) immunized with 10 µg OVA protein (Sigma-Aldrich) alone or in combination with 0.625, 1.25, 2.5, or 5 µg EcML on days 0, 14, and 28. Spleens and sera were collected 2 weeks after the last immunization. In a separate experiment, the mice were i.m. immunized with 10 µg OVA protein alone or mixed with either 2.5 µg EcML or 2.5 μg MPL. The body temperature of the immunized mice was measured at indicated time points using Thermalert TH-5 . In addition, sera were collected from the mice. In a separate experiment, the mice were i.m. immunized with 10 µg OVA protein alone or mixed with either 25 µg alum, 2.5 µg EcML, or 2.5 µg MPL on days 0, 14, and 28. Spleens and sera were collected on day 14 after the last immunization. In the influenza experiment, the mice were i.m. immunized with pH1N1 split vaccine antigen , which contained 0.05 μg hemagglutinin (HA), alone or mixed with 25 μg alum, 2.5 μg EcML, or 2.5 μg MPL on days 0 and 14. Fourteen days after the last immunization, spleens and sera were collected from the vaccinated mice. On day 14 after the final vaccination, the mice were intranasally (i.n.) challenged with a lethal dose .Levels of cytokines in the cell culture supernatants were measured using OptEIA kits (BD Biosciences) according to the manufacturer’s instructions. The production of antigen-specific IgG, IgG1, and IgG2b Abs in the sera of the immunized mice were determined by indirect ELISA. Briefly, MaxiSorp 96-well plates (Thermo Fisher Scientific) were coated overnight at 4 °C with 100 µL of 1 µg/mL OVA protein or 0.5 µg/mL pH1N1 split vaccine antigen in carbonate solution, pH 9.5. The plates were blocked with 5% skim milk in PBS for 2 h at 37 °C and were washed with 0.05% Tween-20 in PBS. The plates were then incubated with 100 µL of diluted sera from the vaccinated mice for 2 h at 37 °C, followed by incubation with 100 µL of HRP-conjugated anti-mouse IgG , and anti-mouse IgG1 or IgG2b Abs  for 1 h at 37 °C. After washing, the reactions were developed with the chromogenic tetramethylbenzidine substrate (BD Biosciences) and then terminated with 2N H5 cells/well onto purified anti-IFN-γ-coated ELISPOT plates. The cells were treated with 0.5 µg/well of OVA257–264 peptides  or the 500 median tissue culture infectious dose (TCID50)/well of UV-inactivated pH1N1 influenza virus for 3 days at 37 °C. The spot-forming units (SFUs) of antigen-specific IFN-γ-secreting cells were calculated using an ELISPOT plate reader .The frequencies of antigen-specific IFN-γ-secreting cells were evaluated using a mouse ELISPOT kit (BD Biosciences), as previously described ,23. BrieTo measure the systemic inflammatory responses, sera from the immunized mice were collected. Levels of inflammatory cytokines in the pooled sera (n = 5 per group) were measured using the Lengendplex mouse inflammation panel  according to the manufacturer’s instructions.t-test and one-way ANOVA, followed by Bonferroni’s correction (ANOVA/Bonferroni), respectively. The p values less than 0.05 (p < 0.05) were considered to be statistically significant. All the analyses were implemented using GraphPad Prism software .All of the data were presented as the means ± standard deviations (SDs) and represented three independent experiments. Statistically significant differences between the two and multiple groups were assessed using the two-tailed Student’s To examine whether EcML can cause cell cytotoxicity in vitro, we first treated immature BMDCs with various concentrations of EcML , 1 µg/mL LPS, or 0.5% Triton X-100 for 24 h at 37 °C. The cell cytotoxicity was investigated using a Trypan blue assay. As shown in p < 0.01). In particular, BMDCs treated with EcML produced much higher levels of TNF-α cytokine than those treated with MPL at the same concentration (5 µg/mL)   B. We als5 µg/mL) C. The tr < 0.01) D. Collecp < 0.001)  B.As NF-κB activation is a major signaling pathway through which TLR4 induces innate immune responses , we examp < 0.01) (p < 0.05) (p < 0.01)  on days 0, 14, and 28. Two weeks after the last immunization, the levels of OVA-specific IgG, IgG1, and IgG2b were measured in the sera of the immunized mice using ELISA. Compared to the OVA alone group, OVA-specific IgG levels significantly increased in the groups immunized with OVA plus 1.25, 2.5, or 5 μg EcML when using serum dilution factors of 300, 900, and 2700 ( < 0.01) A. There  < 0.05) B. OVA-sp < 0.01) C. While 257–264 peptides, which are restricted peptides presented to MHC class I molecules. We observed that the numbers of OVA257–264 peptide-specific IFN-γ-producing cells increased significantly in mice vaccinated with OVA plus EcML as compared to mice vaccinated with OVA alone (p < 0.001). Nevertheless, no statistically significant differences were observed among the OVA plus EcML groups  (p < 0.001) (257–264 peptide-specific IFN-γ-secreting cells was significantly higher in the EcML–OVA group (69 ± 27 SFUs) than those in the other groups . We consistently observed that the level of released IFN-γ cytokine was significantly increased in the EcML–OVA group (627 ± 50 pg/mL) compared to those of the other groups , when using splenocyte culture supernatants upon ex vivo stimulation with OVA protein , MPL (MPL–OVA), or EcML (EcML–OVA) on days 0, 14, and 28. Mice immunized with either OVA protein alone or PBS were used as the negative controls. Two weeks after the final immunization, the levels of OVA-specific Abs in the sera of immunized mice were measured using ELISA. Our results showed that the levels of OVA-specific IgG were significantly higher in the EcML–OVA group than in the OVA alone group when using serum dilution factors of 300, 900, 2700, 8100, and 72,900 ( < 0.05) A. The in< 0.001) B. There  protein D. CollecTo investigate whether EcML induces systemic inflammatory responses, we immunized C57BL/6 mice with 10 µg OVA protein alone or in combination with 2.5 µg EcML (EcML–OVA). MPL was used for comparison, and PBS was used as a negative control. Body temperature was measured at 6, 24, 48, 72, and 96 h post-injection. The production of systemic inflammatory cytokines, including IL-1α, IFN-γ, TNF-α, MCP-1, IL-12p70, IL-1β, IL-6, IL-17A, and GM-CSF, was determined in the sera of the immunized mice. As shown in 50) of the pH1N1 influenza virus. As shown in To evaluate whether EcML improves the influenza vaccine efficacy, we examined the effects of EcML on the protection against viral infection resulting from the pH1N1 vaccine. Mice were vaccinated with the pH1N1 split vaccine antigen mixed with alum , MPL (MPL–vaccine), or EcML (EcML–vaccine) on days 0 and 14. Vaccine antigen alone or PBS were used as controls. Fourteen days after the last immunization, the immunized mice were i.n. challenged with a lethal dose (50 LDp < 0.01), vaccine alone , and alum–vaccine  groups. There was no significant difference in the HI titers of the EcML–vaccine and MPL–vaccine groups  (p < 0.001), vaccine alone , and alum–vaccine  groups. No significant difference in the cell number was observed between the EcML–vaccine and the MPL–vaccine  groups (p < 0.001). There was no significant difference in the cytokine levels of the EcML–vaccine and MPL–vaccine  groups  C. Moreov) groups D. Additi) groups E. CollecE. coli-produced MPL (named EcML) has recently been produced by direct extraction from an engineered E. coli [E. coli strain is a sustainable source and can be easily grown at a very low cost and EcML is directly extracted from the bioengineered E. coli without hydrolysis, indicating that the manufacture of EcML is more cost-effective than that of MPL [Despite the availability of annual vaccinations, influenza remains a significant cause of human infectious disease and, thus, there is a push to improve vaccine effectiveness. An adjuvant capable of enhancing vaccine immunogenicity without compromising safety would be beneficial to improving influenza vaccines. However, the main influenza vaccine adjuvants, including alum, MF59, and AS03, have limitations such as their weak induction of cellular immune responses and safety issues ,5,6,7,8  E. coli . The E. t of MPL . Here, wDCs play a major role in innate immunity and serve as a significant link between innate and adaptive immunity . Thus, tThe safety of a vaccine adjuvant is paramount as vaccines are given prophylactically to healthy individuals. A previous study reported that Lipid A, a toxic domain of LPS, was not detected after the purification of EcML, suggesting that EcML is not contaminated with LPS . EcML ha257–264 peptide-specific IFN-γ-producing T cells and the levels of IFN-γ release, indicating that EcML robustly enhances antigen-specific CTL activity. Additionally, we observed that EcML was better than MPL in improving OVA-specific humoral and cellular immune responses. We speculated that the adjuvanticity of EcML could be with the reason behind the enhanced levels of inflammatory responses observed for EcML compared to MPL. Currently, alum and MF59 are mainly used in influenza vaccines [A novel vaccine adjuvant could have the ability to improve both antigen-specific humoral and cellular immunity . Consistvaccines  and may MPL has been reported to enhance the protective efficacy of the influenza vaccine . We obseE. coli [In an attempt to seek a potent vaccine adjuvant capable of enhancing both cellular and humoral immune responses, we assessed EcML—an MPL extracted from the engineered E. coli . EcML wa


Innate immunity is the first line of defense against bacterial infection and is initiated by macrophages. Sorting nexin 25 (SNX25) is an SNX family member and is reported to negatively regulate TGF-β signaling by enhancing TGF receptor degradation. However, few studies have focused on the relationship between SNX25 and the immune system. We knocked down SNX25 expression in macrophages and examined inflammatory cytokine expression, a hallmark of innate immunity, after lipopolysaccharide stimulation. SNX25 knockdown increased proinflammatory cytokine expression in RAW 264.7 cells. In addition, SNX25 knockdown activated the NF-κB signal by promoting ubiquitination of IκBα. These results suggest that SNX25 inhibits the NF-κB signal and thereby regulates proinflammatory cytokine expression in macrophages. Among ts (DUBs) . Althougand DUBs –8, the dSNXs have been identified from mammals through to yeasts . They plThe macrophage cell line RAW 264.7 was cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were grown in a humidified chamber containing an atmosphere of 95% air/5% CO2 at 37°C.GAUAUUAUGACCAAUCCUU, antisense; AAGGAUUGGUCAUAAUAUC), SNX25 siRNA-2  was transfected into RAW 264.7 using Lipofectamine RNAiMAX Transfection Reagent  according to the manufacturer’s instructions. Cells were used for experiments 24h after siRNA transfection.SNX25 siRNA-1  and sodium vanadate. The lysates were then sonicated. The homogenate was centrifuged at 20,600 g for 5 min, and the protein concentration in the supernatant was measured using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein per lane were electrophoresed on SDS-polyacrylamide gels, and then transferred to a polyvinylidene difluoride membrane. After blocking with 5% non-fat milk in phosphate-buffered saline with 0.05% Tween 20 for 1.5 h, proteins of interest were allowed to bind, during an overnight incubation at 4°C, with anti-SNX25 , anti-IL-1β , anti-IL-6 , anti-TNF-α , anti-IκBα , anti-phospho-IκBα , anti-p65 , anti-phospho-p65 , anti-JNK , anti-phospho-JNK , anti-p38 , anti-phospho-p38 , anti-ERK , anti-phospho-ERK , anti-phospho-IKKβ , anti-α-Tubulin , anti-LaminB1  or anti-GAPDH  antibody. Anti-rabbit or -mouse horseradish peroxidase-conjugated antibody was used as a secondary antibody, followed by enhanced chemiluminescence Western blotting detection reagents . Without LPS treatment, the expression level of cytokine was almost none, and in some cases, it was below the detection sensitivity. Therefore, we examined the time-course of target protein expression and compared at the point with the highest expression level of target protein. For quantification of expression level of target protein, the band intensities of scramble siRNA (control) and SNX25 siRNA-treated sample were measured and calculated the intensity ratio of SNX25 siRNA-sample to control. SNX25 siRNA-treated sample data were expressed as fold changes to control (scrambled siRNA) data and the same analysis was repeated for every three to seven independent experiments.AGCCATGTACGTAGCCATCC; antisense: CTCTCAGCTGTGGTGGTGAA), IL-1β primer , TNF-α primer (AATTCACTGGAGCCTCGAATG), IL-6 primer  and SNX25 primer .Total RNA was extracted using a NucleoSpin RNA kit  and reverse-transcribed using a QuantiTect Reverse Transcription kit  with random primers. Real-time quantitative PCR was conducted in a Thermal Cycler Dice , using THUNDERBIRD SYBR qPCR Mix . PCR primers used in this study were β-actin primer  according to the manufacturer’s instructions.P < 0.05, ** P < 0.01, *** P < 0.001. All n and P values are indicated in figure legends.Quantitative analysis was performed from at least three independent experimental groups. All data are shown as mean ± SD. Statistical analysis was performed using Welch’s t test for two groups or Tukey–Kramer method for multiple groups. Statistical significance is indicated as asterisks. * P = 0.0014; TNF-α, SNX25 siRNA, 1.8 ± 0.68-fold control P = 0.041; IL-6, SNX25 siRNA, 2.2 ± 1.2-fold control P = 0.063)  . SNX25 siRNA-2 decreased the SNX25 mRNA level more effectively  . SNX25 s= 0.063) . We also= 0.063) . SNX25 k= 0.048) . These rP = 0.032)  and NF-κB signal are important factors in proinflammatory cytokine expression. We therefore examined whether SNX25 knockdown affects MAPK phosphorylation at 20 min after LPS stimulation because all MAPK phosphorylation reached a peak at 20 min after LPS stimulation . ERK and= 0.032) . This re= 0.032)  and 2; iP = 0.012). In addition, SNX25 knockdown increased nuclear p65 though the difference was not statistically significant  , which is a hallmark of NF-κB activation . p65 pho = 0.15) . These rP = 0.009) (P = 0.046) (P = 0.00080) though there was no obvious difference in the expression of p-IKKβ at the peak time, 10min after LPS stimulation  . IκBα is= 0.046) . The expmulation . As an amulation , Ub-IκBαSorting nexins play key roles in membrane trafficking, membrane remodeling, and organelle motility  and someWe focused on innate immunity using the macrophage cell line RAW 264.7. To examine whether SNX25 affects innate immunity, we examined proinflammatory cytokine expression in SNX25 knockdown RAW 264.7 cells treated with LPS. We found that SNX25 knockdown increased the expression of IL-1β, TNF-α, and IL-6 Figs  and 2. Wβ-TrCP, a specific E3 ubiquitin ligase of IκBα, and activation of deubiquitinating enzymes (DUBs) against IκBα [The MAPK pathway has an important role in cytokine expression ; howevernst IκBα . Anothernst IκBα . Their cBesides SNX17, other SNXs are also reported to be associated with the ubiquitin-proteasome pathway. For example, SNX16 activates c-Myc signaling by inhibiting ubiquitin-mediated proteasomal degradation of eukaryotic translation elongation factor 1A2 in colorectal cancer development . This stWe also found that SNX25 knockdown decreases phosphorylation of IKKβ. This indicates that SNX25 knockdown suppresses a molecule(s) upstream of IKKβ. In addition, we found SNX25 knockdown did not affect the expression of TLR4 (data not shown). SNX25 is reported to downregulate TGFβ signaling. Therefore, these results suggest that TGFβ signaling is activated by SNX25 knockdown, resulting in downregulation of TLR4 signaling because TGFβ signaling inhibits TLR4 signaling . On the In conclusion, we showed several functional roles of SNX25 in TLR4 signaling such as ubiquitination of IκBα and the expression of inflammatory cytokines . FurtherIn the case of SNX25-knockdowned cell, ubiquitination and proteasome degradation of IκBα are enhanced. p65 and p50, released from the complex with IκBα, translocate into nucleus and activate cytokine transcription and expression.S1 Fig(A) SNX25 siRNA-1 and siRNA-2 were transfected into RAW 264.7 cells. RT-qPCR analyzed the relative mRNA expression of SNX25. β-actin was used as endogenous control. Data are expressed as means ± SD. n = 3 ** P<0.01, Tukey–Kramer method. (B) SNX25 siRNA-2 was transfected into RAW 264.7 cells. RAW 264.7 cells were incubated for 24h or 48h. The protein level of SNX25 was analyzed by Western blotting. GAPDH was used as a loading control.(TIF)Click here for additional data file.S2 Fig(A-D) RAW 264.7 cells were stimulated with LPS (1μg/ml) for 10min, 20min, 30min or 1h. The expression of p-ERK, p-p38, p-JNK, IκBα, p65, p-IκBα, p-p65, p-IKKβ was analyzed by Western blotting. ERK, p-38, JNK, GAPDH, LaminB1 and α-Tubulin was used as a loading control.(TIF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


Scientific Reports 10.1038/s41598-017-18917-3, published online 11 January 2018Correction to: This Article contains errors in Figure 1a.The red square used to denote > 0.5 GPa UTS increase at 30 J and the grey circle used to denote referenced data points were omitted from the key. Furthermore, the 1h reference numbers were incorrectly given as “11, 26-29”.The correct version of Figure


Farnesol is a sesquiterpene from propolis and citrus fruit that shows promising anti-bacterial activity for caries treatment and prevention, but its hydrophobicity limits the clinical application. We aimed to develop the novel polymeric micelles (PMs) containing a kind of derivative of farnesol and a ligand of pyrophosphate (PPi) that mediated PMs to adhere tightly with the tooth enamel.Streptococcus mutans while being fed with a high-sucrose diet. The anti-caries efficacy of free Far can be improved significantly by PPi-Far-PMs through the effective binding of it with tooth enamel via PPi.Farnesal (Far) was derived from farnesol and successfully linked to PEG via an acid-labile hydrazone bond to form PEG-hyd-Far, which was then conjugated to PPi and loaded into PMs to form the aimed novel drug delivery system, PPi-Far-PMs. The in vitro test about the binding of PPi-Far-PMs to hydroxyapatite showed that PPi-Far-PMs could bind rapidly to hydroxyapatite and quickly release Far under the acidic conditions. Results from the mechanical testing and the micro-computed tomography indicated that PPi-Far-PMs could restore the microarchitecture of teeth with caries. Moreover, PPi-Far-PMs diminished the incidence and severity of smooth and sulcal surface caries in rats that were infected with This novel drug-delivery system may be useful for the treatment and prevention of dental caries as well as the targeting therapy of anti-bacterial drugs in the oral disease. Streptococcus mutans and other organisms that colonize the surface of teeth   , 23, in )6(OH)2] , 25. Fin1H and 13C NMR confirmed the synthesis of PPi-PEG-hyd-Far as described below.To develop a pH-sensitive dentotropic polymeric conjugate for the formulations of polymeric micelles, we synthesized the PPi-PEG-hyd-Far according to the synthetic scheme in Fig. 1  showing δ (ppm) 7.01 , 6.67 , 4.27 , 3.64 , 1.45 . The 13C NMR  showed δ (ppm) 70.71, 28.40. Compound 2  showing δ (ppm) 4.30–4.19 , 3.87 , 3.62 , 2.45 , 1.44 . The 13C NMR spectrum  showed δ (ppm) 155.82, 70.67, 69.36, 68.88, 65.47, 64.93, 28.37. The 31P NMR spectrum  showed δ (ppm) -9.49 . Compound 3  showing δ (ppm) 6.27 , 4.27–4.21 , 3.65 , 1.87 . The 13C NMR spectrum  showed δ (ppm) 70.72, 69.60, 64.71. Compound 4  showing δ (ppm) 8.63 , 7.80 , 6.08 , 5.07 , 4.33 , 3.63 , 2.21 , 2.12 , 2.03 , 1.96 , 1.85–1.82 , 1.66 , 1.57 . The 13C NMR spectrum  showed δ (ppm) 136.05, 131.67, 124.40, 124.29, 123.32, 123.13, 72.80, 70.70, 70.40, 69.48, 40.20, 39.84, 26.84, 26.21, 25.90, 17.89, 17.47, 16.20.Compound 2000-PLA2000 polymeric conjugate were self-assembled into PPi-Far-PMs using a film hydration procedure. Dynamic light scattering showed the resulting blank PMs to have an average diameter of 15.95 ± 0.10 nm with a polydispersity index of 0.086 ± 0.008 . Intra- and inter-day relative standard deviation (RSD) was less than 10%, and Far extraction recoveries were 99.09 ± 0.48%  at low concentrations, 99.84 ± 0.10%  at medium concentrations, and 101.63 ± 0.04%  at high concentrations. These results suggest that our HPLC method was robust and reliable. Using this method, we measured drug loading and encapsulation efficiencies to be 9.10 ± 0.70% and 76.4 ± 2.10% for Far-PMs, and 9.51 ± 0.40% and 78.30 ± 1.40% for PPi-Far-PMs.We developed an HPLC method to calculate drug loading and encapsulation efficiencies of the PMs. The retention time of Far in PMs was consistent with that of the Far standard, and no interference signals were observed near the characteristic peak. The Far standard curve was linear over the range of 10–400 μg/mL  and MBC  significantly. These results indicate that PPi-Far-PMs can inhibit the growth of S. mutans in vivo. All animals remained in good health during the experiment and the body-weight increase was similar to that of the normal rats rats , PPi-Far-PMs (P < 0.001), or CHX (P < 0.0001)  Table b. FarnesP < 0.0001), or CHX  . Meanwhile, we found that BMD and BV/TV of molars were significantly higher with the low-dose PPi-Far-PMs  than with distilled water . This reminds us that the drug-delivery system of PPi-Far-PMs can reduce the dosage of the Far and prevent effectively the dental caries. We attribute these results to the ability of PPi-Far-PMs killing bacteria in the acidic microenvironment of dental plaques and binding efficiently to hydroxyapatite, and thereby inhibited demineralization and promoted re-mineralization.We assayed the anti-caries activity of PPi-Far-PMs in a rat model of caries induced by the combination of nic diet , 36. PPiS. mutans UA159. We further showed that incorporating Far into PPi-Far-PMs made it much more effective than free farnesol at treating dental caries in a rat model. This suggests that PMs can improve the water solubility and prolong the retention time in the oral cavity. This novel pH-sensitive drug delivery system shows potential for targeted anti-bacterial treatment against dental caries, and it may be useful for delivering other agents to treat disease in the oral cavity.In this study, we developed the drug-delivery system of PPi-Far-PMs that binds efficiently and rapidly to hydroxyapatite, and that releases Far selectively in the acidic microenvironment of dental plaque. We showed in vitro that Far on its own has similar anti-bacterial activity as its parent farnesol against cariogenic 2000-NHNH2 was obtained from Xi’an Ruixi Biological Technology , and mPEG2000-PLA2000 was purchased from Jinan Daigang Biomaterials . Mitis Salivarius agar was purchased from BD Biosciences . Brain heart infusion broth and agar were purchased from Hopebio . S. mutans UA159 (ATCC 700610) was purchased from the China Center of Industrial Culture Collection .Far, farnesol, and TBAP were supplied by Sigma-Aldrich . Chlorhexidine digluconate (CHX) was obtained from Macklin . HO-PEG2000-NHNH2 was protected with a t-butyloxy carbonyl (Boc) group, and then subjected to esterification and bromination to give PPi-PEG-NHNH-Boc. The Boc group was removed and subsequent hydrazide reaction generated PPi-PEG-hyd-Far, whose identity was confirmed using 1H and 13C NMR. The specific reaction conditions for each compound are detailed below.The pH-sensitive dentotropic polymeric conjugate PPi-PEG-hyd-Far was synthesized as shown in Fig. 2000-NHNH2  in methanol. The mixture was cooled to 0 °C and di-tert-butyl dicarbonate  was added slowly to the reaction solution. The reaction mixture was stirred at 0 °C for 1 h, then warmed to room temperature and stirred for 24 h. The solution was concentrated under vacuum, and the product was precipitated in anhydrous ether three times, dried overnight under vacuum and stored at − 20 °C for an overall yield of 72.4%.Triethylamine  was added to a stirred solution of HO-PEG1  in anhydrous dichloromethane (DCM). The solution was cooled to 0 °C, then 4-dimethylaminopyridine  and N,N′-dicyclohexylcarbodiimide  were added slowly, and the mixture was stirred at room temperature for 24 h. The reaction mixture was filtered and concentrated under vacuum, and the product was precipitated in anhydrous ether. The precipitate was filtered and dialyzed against water for 24 h. TBAP , previously dissolved in anhydrous acetonitrile (CH3CN), was added slowly to the dialyzed product, and the mixture was stirred at room temperature for 12 h. The solution was concentrated under vacuum, and the product was precipitated into anhydrous ether. The product was filtered, dialyzed against NaCl solution and then dialyzed against water at 0 °C for 10 h. The purified product was freeze–dried and stored at − 20 °C for overall yield of 80.7%.Bromoacetic acid  was added to a stirred solution of compound 2  was de-protected using zinc bromide  in dichloromethane (DCM) for 4 h. The solution was filtered and concentrated under vacuum, and the product was precipitated in anhydrous ether three times and against water at 0 °C for 10 h. Then the purified product was dried overnight under vacuum and stored at − 20 °C with overall yield of 80.20%.Compound 3  and Far  were dissolved in anhydrous methanol, and acetic acid  was added to the reaction solution. After stirring at room temperature for 48 h, the solution was concentrated under vacuum, and the product was precipitated in anhydrous ether three times. Then the purified product was dried overnight under vacuum and stored at − 20 °C with overall yield of 79.80%.Compound 2000-PLA2000 (A), PEG-hyd-Far (B), PPi-PEG-hyd-Far (C), and Far (D). The weight ratios of components A/B/C/D were optimized using the central composite design [The membrane hydration method  was usede design , 39 to bThe same method and the additional reagent FITC-PEG (E) were used to prepare FITC-labeled PMs in the following weight ratios: Far-PMs-FITC, A/B/D/E: 20/20/4/1; and PPi-Far-PMs-FITC, A/C/D/E: 20/20/4/1.The size and zeta potential of polymeric micelles were measured using dynamic light scattering . Morphology of blank PMs and PPi-Far-PMs were observed using transmission electron microscopy . Critical micelle concentration (CMC) was determined using fluorescence spectroscopy and pyrene as the hydrophobic fluorescent probe.Encapsulation and drug-loading efficiencies were calculated using the ultrafiltration method and an ultrafiltration column with a molecular weight cut-off of 3 kDa . Far was quantified on an Agilent ZORBAXSB-C18 column  at 25 °C attached to an Agilent 1260 HPLC system . The mobile phase was acetonitrile and ultrapure water  and the flow rate was 1 mL/min. The detection wavelength was 216 nm. Encapsulation efficiency was calculated using the equation:Drug-loading efficiency was calculated using the equation:Release of Far from PPi-Far-PMs was investigated in vitro using the dialysis method. Briefly, 1 mL of PPi-Far-PMs or free Far were placed into separate dialysis bags with a molecular weight cut-off of 1 kDa  and dialyzed at 37 °C against phosphate-buffered saline (PBS) at pH 4.5 and 7.4 with gentle stirring. At different time points , 0.5 mL of release medium was removed and replaced with fresh medium. Far was quantified by HPLC as described above, and experiments were performed three times.As we all know, the most important component of the tooth enamel is hydroxyapatite . To mimi2HPO4 . This tube was inverted and soaked into the beaker filled with a solution of Ca(CH3COO)2 . This reaction system, which mimics bone mineralization conditions, was incubated at 37 °C for 10 days. Care was taken to ensure that the top and bottom surfaces of the submucosa membrane remained in contact with liquid [Small intestinal submucosa was prepared as described . The reah liquid . MorpholSolutions of FITC-labeled PPi-Far-PMs and Far-PMs were mixed with biotechnological or commercial hydroxyapatite in round-bottom flasks (at pH 7.4). After incubation with gentle stirring for 30 min at room temperature, the mixture was filtered, washed with PBS three times, and freeze-dried. Finally, the hydroxyapatite was observed under a fluorescence microscope .g, 5 min) and the supernatant was collected. The amount of Far in the micelle supernatant after binding (Wleft) was analyzed by HPLC as described above. The binding rate (%) at each time point was calculated as follows: Binding rate (%) = /Wtotal × 100%.For analysis of binding kinetics, PPi-Far-PMs or Far-PMs (1 mL) were mixed with either kind of hydroxyapatite particles (50 mg) and incubated at room temperature. At certain time points , hydroxyapatite was removed by centrifugation  glycerol. These stocks were streaked onto Mitis Salivarius agar and incubated anaerobically for 48 h at 37 °C under an atmosphere of 80% N2, 10% H2 and 10% CO2. Then a single colony of bacteria was inoculated into brain heart infusion broth, and the culture was incubated anaerobically for 12 h at 37 °C. Bacterial growth was assayed by measurement of absorbance at 600 nm. Optical densities were converted to CFU/mL using the conversion  was assayed as a solvent control. MBC was defined as the lowest concentration that showed no surviving bacteria on the agar after incubation for 48 h at 37 °C. For MBC determination, medium in micropores without bacterial growth was picked up with a sterile inoculating loop and plated onto brain heart infusion blood agar supplemented with 5% defibrillated sheep blood. These experiments were performed three times.Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Far were determined against dilution . The iniSprague–Dawley rats were obtained from the Laboratory Animal Center of Southwest Medical University (Luzhou China). All animal experiments were approved by the Animal Ethics Committee of this university (permit 2017060012) and carried out in accordance with the Luzhou municipal government guidelines on animal care and use.S. mutans prior to inoculation were removed from the study on day 21. Then each rat was inoculated with 1 mL S. mutans (7 × 108 CFU/mL) every day for 7 days. After being checked for infection, 29-day-old rats were randomly divided into nine groups (n = 7), and their teeth were topically treated twice daily for 5 weeks with a camel hair brush coated with the following treatments: (a) distilled water (negative control), (b) 15% ethanol , (c) farnesol (1.10 mg/mL), (d) farnesal , (e) chlorhexidine gluconate , (f) blank PMs, (g) Far-PMs (1.10 mg/mL Far) and (h) PPi-Far-PMs (1.10 mg/mL Far), ensuring that the drug kept working with the teeth for 1 min. All animals were weighed weekly, and physical appearance was recorded daily. At the end of the 5 weeks, saliva was collected and inoculated onto Mitis Salivarius agar with bacitracin (Sigma) to estimate the S. mutans population, and on brain heart infusion agar with 5% sheep blood to determine the total colony count. Finally, animals were sacrificed and the teeth were collected for further assessment.To establish a rat model of dental caries, animals were fed a cariogenic diet with 56% sucrose  and given 5% sucrose water to drink ad libitum , 43. AftTo evaluate the anti-caries activity of PPi-Far-PMs, teeth were stained with 0.4% murexide solution, and caries were scored according to Keyes’ system . A stereRat teeth were collected and assessed in terms of mechanical characteristics and microarchitecture. Compressive strength of teeth was measured using a universal testing machine  at 37 ± 0.5 °C. A stress–strain curve was recorded as vertical tooth occlusal pressure was applied at 1 mm/min. After the samples were broken, the compression modulus (slope of stress–strain curve), compressive strength, compressive yield stress, and maximum stress (the highest point of stress–strain curve) were calculated base on the stress–strain curve. Microarchitecture of molars was evaluated using high-resolution micro-computed tomography . The following parameters were used: voltage, 80 kV; current, 80 μA; exposure time, 2.96 s; scan resolution, 14 μm/slice; and total rotation angle, 360° increasing in 0.5° increments. Bone mineral density (BMD) and bone volume per tissue volume (BV/TV) were estimated from the three-dimensional reconstructions.t test or Tamhane’s T2 test. P < 0.05 was regarded as statistically significant.All data were expressed as mean ± standard deviation (SD) and analyzed using GraphPad Prism 6.0 . Differences between treatment groups were analyzed for significance using the Student’s Additional file 1: Figure S1.1H NMR and 13C NMR spectra of synthetic compound 1: tert-butyl 2-(2-(2-hydroxyethoxy)acetyl)hydrazine-1-carboxylate (HO-PEG-NHNH-Boc). Figure S2.1H NMR, 13C NMR and 31P NMR spectra of synthetic compound 2: sodium 2,2-dimethyl-4,7,13-trioxo-3,9,12-trioxa-5,6-diazatetradecan-14-yl hydrogen diphosphate (PPi-PEG-NHNH-Boc). Figure S3.1H NMR and 13C NMR spectra of synthetic compound 3: sodium 2-(2-(2-hydrazinyl-2-oxoethoxy)ethoxy)-2-oxoethyl hydrogen diphosphate (PPi-PEG-NHNH2). Figure S4.1H NMR and 13C NMR spectra of synthetic compound 4: sodium 2-oxo-2--3,7,11-trimethyldodeca-2,6,10-trien-1-ylidene)hydrazinyl)ethoxy)ethoxy)ethyl hydrogen diphosphate (PPi-PEG-hyd-Far)) is available in the online version of this article.