This involved much turn-taking and predicting what others would do. A matched control group, the Synchrony group, did similar activities but in synchrony, with less prediction and no turn-taking. Task complexity increased over the six sessions. Psychometric testing before and after the series showed that the Turn-taking group increased in empathy on self-report (Toronto Empathy Questionnaire) and behavioral (‘Reading the Mind in the Eyes’) measures, and in the General subtest of the Culture-Free Self-Esteem Inventory. While more work is needed to confirm the conclusions for relevant demographic groups, the current results point to the social value of musical and linguistic activities that mimic entrained, tightly coordinated parameters of everyday conversational interaction, in which, at any one time, individuals act as equal participants who have different roles.This study describes a preliminary test of the hypothesis that, when people engage in musical and linguistic activities designed to enhance the interactive, turn-taking properties of typical conversation, they benefit in ways that enhance empathy and self-esteem, relative to people who experience activities that are similar except that synchronous action is emphasized, with no interactional turn-taking. Twenty-two 12–14 year olds identified as socially vulnerable  received six enjoyable 1-h sessions of musical improvisation, language games that developed sensitivity to linguistic rhythm and melody, and cross-over activities like rap. The Turn-taking group ( In a seper se . Physiolper se ,c. BroadJoint rhythmic actions require temporal entrainment, manifest in group music-making as a shared pulse. Neuroscientific research confirms that a shared musical pulse induces temporal entrainment between ensemble players . LikewisOne recent line of phonetic research indicates that shared pulse may be very important in conversation in that it seems to indicate positive social alignment at turn transitions . The terIn related work, In musical performance, there are many parallels with conversational turns and turn transitions. For example, call and response songs, but in these, each turn may last much longer than the average conversational turn, and the exchange of turns is formalized so that prediction is easy. A closer parallel with conversation is when a solo line is improvised together with an improvised accompaniment that provides additional, brief, musical interest between phrases. Such structures are inherent in musical genres like gospel singing and jazz ensembles. They were discussed for example by The music therapy community also typically accepts that turn-taking in musical interaction leads to enhanced social behaviors since it mirrors early mother-infant interaction . No systPulling these literatures and observations together, we reasoned as follows. Joint music-making is typically rewarding and enhances group affiliation, presumably at least partly through shared rhythmicity—shared pulse. One could expect this experience to generalize to shared rhythmicity in language, especially since people typically report finding pleasant conversation rewarding, but the problem with this is that there is no evidence for a sustained steady pulse in ordinary conversation. However, since there is evidence for rhythmicity being established at the end of one talker’s turn, and maintained across the turn by the next talker, albeit briefly, this might provide not just sufficient reward for the sense of affiliation to develop, but a more effective type of affiliative action (or reward) than simply maintaining joint rhythmic action without a need for significant interaction. Support for this speculation comes from the well-established behaviorist observation that partial (intermittent and random) positive reinforcement facilitates longer-lasting learning than regular reinforcement. Venturing beyond behaviorist terms, we could conceptualize this as engendering hope. Moreover, entrainment at turn transitions ensures that each interactant attends closely to the other’s behavior and tries to predict when their own turn will start. Such close attention to others helps choral, synchronous rhythmic action, but is by no means essential to it as long as the external pulse itself is predictable. Furthermore, such turn-taking interaction, with quasi-independent yet tightly coordinated roles, could potentially offer richer sources of information about one’s interactant(s) than does playing or singing in synchrony with a shared pulse. We therefore asked whether shared rhythmicity is most beneficial to social interaction when joint activities focus attention onto rhythm across interactive turns. Specifically, we predicted that positive changes in empathy and self-esteem will be greater when activities are targeted to enhance joint interpersonal attention to shared rhythm at turn transitions, rather than to enhance general synchronicity of rhythm across longer stretches of behavior without need for interaction.Since temporal entrainment is a general process experienced by the vast majority of people, then if the hypothesis is right, everyone should benefit from attention to shared rhythmicity at turn transitions. We chose to test this hypothesis with moderately socially-vulnerable people, for three reasons. First, as a service: this type of population could benefit from enhanced empathy and self-esteem as well as better communication skills. Second, to avoid ceiling and floor effects: most people in socially-vulnerable populations are likely to have moderately low empathy and self-esteem. Assuming an ogival function in the rate at which change can be achieved, to start from moderate scores should maximize the chances of inducing measurable change from short-term intervention, compared with people who score at either extreme on these measures. Thirdly, for this proof-of-concept study we wanted to avoid disabilities that would make execution of the tasks difficult: people who have physical or learning challenges might take longer, or need more individualized methods, to benefit.The final program was carried out with young teenagers who were identified as having emotional problems that affected them both socially and academically. We chose this demographic group since they fulfilled the three criteria above and their schools’ inclusion units could accommodate the program.We measured empathy rather than prosocial behavior because empathy can be assumed to lead to prosocial behavior, and can be measured reliably via validated questionnaire, whereas it proved impractical to objectively or subjectively measure prosocial behavior of young teenagers in their schools. Self-esteem was likewise assessed by questionnaire.In sum, this study developed and delivered a program of enjoyable musical and verbal interactive activities (games) that stress shared rhythm, and are intended to enhance empathy, self-esteem and prosocial behavior, including more effective interactive communication. We delivered the program in two contrasting ways to two groups of participants. A Turn-taking group spent significant time learning to ‘pass turns’ fluently between them, modeling properties of conversational interaction, translated as needed into music. A parallel Synchrony group did most of the same musical and language tasks, but always in synchrony, typical of much Western group music-making. We predicted that positive changes in empathy and self-esteem would be greater for the Turn-taking group, whose games were targeted to enhance joint attention to shared rhythm across interactive turns , rather than to enhance attention to general synchronicity of rhythm. The hypothesis is novel, and the training requires several weeks’ commitment from participants, so this first study is necessarily exploratory. Its promising results are reported in full to allow further investigation.Twenty-four children were recruited by the Special Educational Needs Coordinators (SENCOs) at two Cambridgeshire (United Kingdom) state schools, of whom 22 completed the course. The children who dropped out, one boy and one girl, were from the same school. The boy was asked to leave because his behavior prevented the group from doing any activity, and the girl’s parents preferred her to attend another class scheduled at the same time. All children attended the ‘inclusion’ unit of their school. They were selected by SENCOs as suffering from low self-esteem and/or empathy, and related problems such as anxiety, and as likely to enjoy the sessions offered in that they liked music. Because of the emphasis on interpersonal interaction, no child had a diagnosis of autism or Asperger syndrome, or presented primarily with autistic traits. Mean ages at time of first testing (see next Sections) were as follows. Girls: 12.89 years, range 12.14–14.13; boys 13.08, range 12.24–14.18. Mean age of the Turn-taking group  was 12.84 years, range 12.26–14.18, and of the Synchrony group 13.12 years, range 12.14–14.13.Fully informed parental consent was obtained, and each child also signed consent forms before the first session, and at the end of the last session. Information provided, and the forms themselves, were approved by the Ethics Committee of the School of Humanities and Social Sciences, University of Cambridge. Each child who completed the course received a £25 gift card in the last session and received a certificate from the University of Cambridge Music Faculty.We used a fully counterbalanced 2 × 2 between-groups design with Treatment Group (Turn-taking or Synchrony) and Sex  as independent variables. Dependent variables were scores on psychometric tests, administered once before sessions began, and once afterward.Since the boy and girl who dropped out were from the same school, there were still equal total numbers (11) in each Treatment group: five girls and six boys in Turn-taking, and six girls and five boys in Synchrony. While group sizes of 11 risk low statistical power, access to near-clinical populations such as these is difficult, and working with them is challenging. For a first test of concept, accepting Ns of this size was a practical decision dictated by budget and access.1 , djembe drums, and tambourines. These were chosen from experience in related work  and piloSix sessions of 50–55 min were held weekly, with either five or six children in each group see  above. CEach child was tested individually in a quiet room at their school within 3 days before the first music and language session, and 3–11 days after the last session. Tests were administered by the first author, who holds a degree in psychology and is trained and experienced in psychological testing. After establishing rapport, testing took place in the order TEQ, RMIE, CFSEI-3. Several children struggled with reading some questions, so, for uniformity, each was read aloud unless the child found this annoying. If the child said they did not understand a word or item, it was explained. A neutral presentation and rate of speech were used at all times, each sentence or phrase receiving the same intonation across instances. This was possible because the first author is also a phonetician. Each child sat directly in front of the test paper, with the tester to their left, and wrote their responses independently, without intervention from the tester.Testing sessions took between 25 and 45 min, with the vast majority lasting about 30 min. Rests were allowed during and between tests, during which the child chatted informally with the tester.Testing after the last session followed the same procedure. For School1, testing took place 10–11 days after the course , and for School2 it was 3–5 days afterward except for one child. This Turn-taking boy was tested at home 7 days after the sessions finished, because he had moved to live in another town.Results were scored by the first author after all second tests were concluded. All were triple-checked.For both Turn-taking and Synchrony groups, activities comprised a mix of instrumental and vocal music-making, with or without body percussion, and language games including conversations and related activities, poetry and rapping. A typical session would start with djembe drumming, move on to balaphons with a particular musical or linguistic goal, then alternate between language and musical activities, or activities that involve both. Sessions always finished with free improvisation in which each participant chose which instrument(s) to use. By Session 6, many participants were playing on multiple instruments with impressive invention. Many activities took place with the whole group participating, others with the group divided into  sections, or in pairs who worked without reference to the other pairs.The sequence of activities within and between sessions was carefully structured and controlled. Within a session, activities were sequenced to provide variety between music-making and language. Between sessions, the more complex skills needed for later work built on those already learned. Each activity’s basic procedures were first taught and practiced within a well-defined structure until participants were confident, then variation and free work were introduced. When appropriate, underlying principles were briefly discussed, especially in later sessions. Both authors took part, dividing leadership of activities according to their primary skill, music therapy (CF) or speech and language (SH).Musical activities in early sessions encouraged sensing a regular pulse, counting rests between beat sequences of various lengths, listening to and following each other’s dynamics, following a leader, remembering and reproducing simple rhythmic and/or melodic motifs, and so on. Later sessions introduced more complex rhythms and melodies, and more opportunity for free improvisation, including improvisational singing using vocables, and ensemble work based on 12-bar blues. Thus, at first motifs were provided and there was much imitation, whereas by the end there was a fair amount of improvisation within prescribed conceptual limits.Language games focused on rhythmic properties of language and conversation. The same principles of progression were used as for the musical activities: language work was initially easy with little or no need to think about content, which was either provided  or fairly automated  as described in section “Differences in methods for Turn-taking and Synchrony groups.” Later, content became more important, but was still kept simple and predictable so that fluent timing could be achieved.Many activities involved both music and language, to differing degrees. Most obviously, songs, raps, and chants contain both musical and linguistic elements. More inventively, as described below, in several activities normally classed as linguistic, such as reciting a poem, the focus was on maintaining rhythm, ‘melody’ and body movement. Conversely, several musical activities explicitly imitated rhythms and melody of specific utterances, and (for the Turn-taking group) the type of prediction and turn-taking typical of natural conversation.Eye of the Tiger (Survivor) and Rather Be (Clean Bandit). Raps were initially made up by creating rhythmically interesting motifs from names of football teams, cars, singers, pop groups, etc. each participant choosing and chanting a different name (and hence rhythm) repeatedly. The chants strongly emphasized the words’ particular rhythm, e.g., Mánchester Uníted, or Mán Mán Mánchester, Mánchester Uníted. The group chanted simultaneously or as a fugue etc, with the Music Therapist controlling individual entries and exits to create an exciting sound. Later sessions used well-known songs/raps, usually Prince of Bel Air, and/or limericks, participants following the words on the flip chart as needed. Participants could choose to accompany rapping with clapping and/or percussion instruments ad lib., and the Music Therapist accompanied on keyboard.All songs were familiar at that time (2016): 4White Winter Hymnal, as well as by the Music Therapist on keyboard.As the sessions progressed, simple activities  were dropped and more complex ones introduced. The transition between simple and complex began in Sessions 3 and 4; it was of course gradual and came earlier for some types of activity than others, and for groups whose initial concentration, understanding and behavior allowed faster progress. Once a song or rap was mastered, the words were accompanied by actions and body percussion modeled on Pentatonix’ Activities were designed so that, as far as possible, each turn-taking activity had a synchrony equivalent matched in structure, cognitive and physical demands . The timSynchrony groups emphasized keeping a steady pulse, starting and stopping together, following dynamics, and (in later sessions) leading vs. following. These methods amounted to working in fairly standard ways for British music ensemble work, though with more emphasis on improvisation than might occur in most non-therapy situations.Turn-taking groups did all the above activities, and in addition ‘passed’ their turn to others while performing the same or similar activities, and interjected ‘backchannels’  in ways modeled on natural conversation. Initially, turns were passed in predictable sequence around the group but in later sessions, the current performer chose who to pass to at random, looking and nodding subtly at the next recipient just as they gave up the turn. Thus everyone not performing the main turn had to predict when the current performer would choose to pass the turn on, and be ready to respond with appropriate timing.Synchrony groups emphasized starting and stopping together, and keeping together with a steady pulse while playing or talking. Participants practiced looking at and listening to a leader, and in later sessions they were taught how to lead such that everyone else joined in or stopped simultaneously, and to adjust their loudness so that an improvising soloist could be heard above the background ensemble  without having to think about content or ‘being right,’ allowed more individual freedom because it was easily understood, typically competently executed, and widely enjoyed. So the instruction might be to have an argument and resolve it. The ‘words’ are number sequences (of a length suitable for expressing a particular viewpoint) beginning at 1, so the current talker just starts with the next number in the sequence, and may produce a single number, quite a long string, or something in between; most interlocutors produced a wide range of lengths during the course of a ‘conversation.’uh-huh or right and gesturing with the fist in between phrases someone else was singing, bidding at the right time in response to an auctioneer’s patter, and getting the timing of pauses and uh-huh right in receiving directions on how to get from one place to another in the school  aligning short responses in time with another person’s longer ones. Examples are: shouting Turn-taking in music followed the same principles, and we developed musical analogs of some of the language games. For example, we imitated the ‘directions’ game with balaphons: a soloist improvised continuously while one or more people provided one- or two-note interjections at phrase boundaries when they judged it to be appropriate , cell 6TTime spent on specific activities varied somewhat between groups for several reasons. The dynamic within each group was unique, and individuals within groups also differed from one another. Girls’ groups were generally quieter and more compliant than boys’, but each group had individual exceptions. Additionally, groups differed slightly in their competence at some activities, enjoyed them to differing degrees, or preferred different types of content for the same basic activity . Over all sessions, however, every effort was made to give all individuals the same general experiences and time spent on them.In sum, members of both groups acquired similar motoric skills, and learned to listen, be creative and to perform confidently in front of their peers. The Synchrony group also learned to maintain a good rhythm, to lead and to conform (follow). Their leading required more self-reliance than sensitivity to peers. The Turn-taking group acquired the more subtle rhythmic and gestural skills associated with turn-taking in successful conversations and ensemble improvisation in music. These skills required attention to and accurate prediction of another person’s behavior, together with tight timing of a coordinated but distinct response.The main issue of interest is whether the Turn-taking group changed positively more than the Synchrony group. This is reflected in the difference between psychometric test scores obtained before and after the sessions, hereafter called Change scores. Also of interest is whether girls responded differently to the treatments than boys. Interpretation of Change scores requires us to also ask whether there were differences between the groups before the sessions started, and whether observed changes were significantly different from zero.p > 0.05), as were most Self-Esteem scores. Five Self-Esteem subscores were not normally distributed (p < 0.03). Of these, Defensiveness After the sessions was skewed toward 0 . This skew indicates that participants answered honestly. We assume their honesty generalizes across all self-reports.Shapiro–Wilk normality tests were conducted using R (shapiro.test) on Before, After, and Change scores for every test. TEQ and RMIE scores were all normally distributed . Since in tests for normality it is more conservative to make no correction for multiple tests, this result can also be taken as encouraging the use of non-parametric statistics.The non-normality of the other four self-esteem distributions encouraged supplementary use of non-parametric statistics for these cases: Academic and Social Before subscores; Parental/home and Social After subscores. Finally, the Change score for General Self-Esteem just missed the standard value for acceptance (U tests (Wilcoxon tests for independent samples) in R . One-sample t-tests were used to test whether mean change scores differed significantly from zero, using two-tailed p levels, although since positive change was predicted, one-tailed tests could be justified.For completeness, therefore, and as a reliability check, all results underwent the following analyses: Before, After and Change scores for Treatment, Sex, and their interaction, using 2 (Treatment Groups) × 2 (Sex) between-groups univariate ANOVA conducted in SPSS (v22), with additional one-tailed Mann–Whitney p ≤ 0.05 was adopted, and  results with p-values of about 0.07 are flagged as marginal and discussed when the pattern of data suggests they merit attention. Bonferroni-corrected post hoc tests were used to explore interactions when patterns of change indicated they were needed  However, observed p levels were such that correction would not change conclusions.All tests were completed satisfactorily with one exception: the RMIE After score for one Turn-taking girl. Prolonged noise in the testing area by a visiting school seriously distracted her, and it proved impossible to test her later. The disruption being unexpected, the data point was deemed missing ‘completely at random’ , p. 433.x axis) and After (y axis) scores, shows that the present results compared satisfactorily with reported norms.The 16-item TEQ has minimum and maximum scores of 0 and 64 respectively, with extreme scores unlikely. In the populations of 18-year-old Canadian undergraduates on which the test was normed, mean scores were around 45 (SDs around 7.5). F = 1.643, p = 0.216, W = 96.0, p = 0.991], but as expected girls scored slightly higher than boys . There was no interaction between Treatment group and Sex (p > 0.9). The Turn-taking group’s mean change score was also significantly greater than 0 , whereas the Synchrony group’s was not . There was no difference between the sexes in amount of change . The pattern of x = y line  reports mean scores of about 20 (SD 2.4) for 19 typically developing 10–12 year old children: 20.2 (SD 2.4) for 9 males, 21.0 (SD 2.4) for 10 females . Scores F 4.99, p = 0.038, ANOVAs of Before scores showed a significant interaction between Treatment group and Sex , and for the Turn-taking group alone . The Synchrony group alone did not improve significantly .F = 3.08, p = 0.096, F = 0.60, p = 0.464, p ≥ 0.3; After Treatment group p > 0.8, Sex p > 0.08).Turn-taking boys improved disproportionately. With a mean After score of 16 (SD 2.58), they reached almost the same skill level as the other three groups [means 17–19 (SDs 2.17–5.00)]. Consequently, the Treatment group × Sex interaction, significant in the Before analysis, was reduced to non-significance in ANOVAs for both Change . As detailed below, the Turn-taking group’s positive Change was significantly different from zero, whereas the Synchrony group’s negative change was not.Amongst the Change scores, only General self-esteem , which mW = 38.0, p = 0.073). Academic: Turn-taking mean 0.09 (SD 2.02), Synchrony mean –1.0 (SD 2.24), W = 40.0, p = 0.091. t-tests and one-sample Wilcoxon tests. Only the Turn-taking group’s General subtest was significantly different from zero , which is of course in the predicted positive direction. All the rest fall far short of significance .Changes from zero were also assessed, using both two-tailed one-sample In summary, Turn-taking significantly enhanced General self-esteem, had unreliable (weak) positive effects on GQ self-esteem, and no effect on the other subtests. Synchrony experiences had no significant effect on self-esteem.Defensiveness assesses honesty of answering, so (unlike the other tests) a low Before score, and/or a negative change over time, are desirable. Defensiveness scores were typically low on first testing: means of 2.64 (SD 1.69) and 2.73 (SD 2.01) for Turn-taking and Synchrony respectively, both well below the limit allowed for answers to the rest of the questionnaire to be deemed honest .F = 9.015, p = 0.008, t(20) = 0.007, two-tailed Mann–Whitney U, p = 0.009].The Treatment group × Sex ANOVA of the Before scores showed no significant main effects but a strongly significant interaction , perhaps due to a floor effect for School2, whose Before scores were already low. The Change scores reflect the fact that, although the After scores maintained the same pattern as the Before scores, the differences between subgroups were smaller in the After scores, resulting in no significant main effects or interactions in separate Treatment groups × Sex, and Treatment groups × Schools ANOVAs.Changes in Defensiveness were largely as expected: zero (2) or negative (15) for 17 of the 22 participants, and positive for the remaining 5. In summary, School1 children were more defensive than School2 children before the study, but by the end they reduced that difference sufficiently to produce statistically non-significant differences.This study shows that practice in turn-taking while doing group activities in music and language can improve psychometric test scores on empathy, and to some extent self-esteem, amongst 12–14 years old who struggle in social situations (but do not have notable autistic traits).The greatest measured benefit was for empathy. Empathy increased in self-report (TEQ) and in accuracy of reading emotions from a person’s eyes (RMIE). For both tests, the absolute degree of change is significantly greater than zero for Turn-taking but not Synchrony groups. Synchrony groups as a whole did not change significantly, despite their activities being similar except for the Turn-taking element. However, for the behavioral RMIE, low scorers benefited more from the program than high scorers, regardless of Treatment group, though practice in turn-taking benefited most participants too.p = 0.073). Non-significant trends in the other tests patterned consistently in favor of greater benefit from activities that include turn-taking. When the training has been relatively short, it is perhaps a lot to expect large changes on a test with high test–retest reliability. Nonetheless, it is interesting and reassuring that the most reliable effect was in General self-esteem, rather than, e.g., Social or Academic, which would take time for the testee to accumulate evidence for.Turn-taking enhanced self-reported self-esteem (CFSEI) less reliably than it enhanced empathy, but nonetheless encouragingly. The only statistically significant change in CFSEI scores was in the General subtest. For this, the Turn-taking group improved relative to their scores on first testing and relative to the Synchrony group. For the Global Quotient, the Turn-taking group improved marginally better than the Synchrony group  being livelier, the only sex difference that affected the Treatment group results was for RMIE. Low-scoring boys improved disproportionately, which, given the study design, affected the overall group statistics. The hypothesis was developed by drawing novel connections between the literatures of music, rhythmic entrainment, and the phonetics of conversation. The intervention program involved four treatment groups and lasted 6 weeks. Group size was only six, since participants needed individual attention to maximize both learning and enjoyment. Consequently, this first test was necessarily small-scale. The results encourage further investigation with more participants and a wider range of vulnerable populations, and perhaps more sessions. For example, although Details may not replicate due to study size, but since the results are consistent overall, the general patterns seem likely to. For example, while improvement in self-report measures need not necessarily result in behavioral changes, and can presumably be fudged (notwithstanding high test–retest reliability), improvements in a measure like the RMIE seem likely to be less subject to testee bias, and more likely to generalize to measurable benefits in social situations. Thus these results seem worth following up with larger groups and more sessions.Put more generally, turn-taking modeled on natural conversation and applied in music and language games takes time to learn and is a more subtle investigative manipulation than, for example, performing music or not, or acting with shared rhythm or not. So we would expect our turn-taking manipulation to take time to show measurable benefits in empathy and self-esteem. This is especially true given that as far as possible the activities were identical except for the turn-taking element. In sum, although greater change would have been more convincing, the measured effects may be about what can be expected with a short program and small sample sizes, especially given the sensitivity of these children to difficulties in their everyday lives . These caveats on the reliability of the results noted, the following sections explore their potential.Although no mean Synchrony group changes were significantly different from zero, that they were consistently negative is puzzling. There is no evidence it is due to skewing of means by outliers. Yet benefits were both predicted from the literature on effects of shared rhythmicity . This section describes and explains the distinctive elements.Two novel contributions of the present study are, first to combine language, music, and ‘cross-over’ activities such as rap in ways that emphasize their common elements, and second, to control activities so that a specific hypothesis is tested, yet in a setting that in all other respects is natural, thus making the methods suitable for therapeutic interventions. These qualities distinguish the present study from others demonstrating social and personal benefits of joint music-making [see section “Introduction,” also This mixture of rigorous hypothesis-testing in multiple, structured sessions in which the emphasis is on spontaneous behavior rather than scripted interaction, required many aspects of normal experimental control to be relaxed. For example, it was essential to keep participants engaged during each session and eager to return to future ones. Likewise, everyone must understand the instructions and want to contribute. Consequently, in contrast for example with This was a small-scale ‘proof of concept’ study conducted within a small budget. Replication would be advantageous with blind psychometric assessment, and controls on who delivers the program—ideally, more than two leaders and more groups, to remove effects of leader(s). Leaders probably cannot be kept ignorant of the hypothesis, for in our experience, they must understand what activities are or are not possible within each Treatment group. But distributing leaders across groups should minimize unwanted leader effects. Objective measures of behavioral change would be preferable to self-report.Ideally, replication should widen the age range through to old age. The literature on benefits of joint music-making finds similar effects across the life span, and specific content is easily adjusted to individual interests and abilities. Pilot work showed our methods appealed to older vulnerable teenagers and typical adults. Indeed, compared with younger teenagers, older teenagers were more sensitive to, and better able to manipulate, musical and especially conversational timing and poetic meter. Children younger than about 12 years may benefit less. Rhythmic stability is commonly established only after puberty, and some conversation work demands a good sense of pragmatics. So despite toddlers and young children engaging in turn-taking, the program might need significant modification to work within the rhythmic and cognitive abilities of children younger than about 12, and even then benefits might be small.The range of individual vulnerabilities may also be wide: music therapists have suggested the program could help people who have depression, including post-natal, teenage parents at risk of neglecting their infants, perpetrators of domestic abuse, some prisoners and ex-prisoners, anorexics, adopted people (many of whom suffer low self-esteem), and carers of people who have dementia.While the present study combined music and language work, future research might examine effects of turn-taking practice in the two domains separately. An informal impression was that more advanced language work was sometimes tiring and/or less immediately engaging for all. While one would not want to omit language work  from this type of program, and it was usually easy to increase interest and engagement, language work is not suitable for everyone, so it would be worth assessing the merit of turn-taking in structured, interactive musical activities alone, and of linguistic turn-taking games alone.This study’s results suggest that interaction that requires looking, listening, predicting and turn-taking seems to particularly encourage the development of empathy, and possibly self-esteem. Music seems most likely to help develop empathy when it involves activities that encourage individual rather than synchronous group performance, yet within groups, tightly entrained with others’ performance, and emphasizing attention toward other people, prediction and mutual cooperation. This is not an aspect of music-making that has received attention in the literature on the personal and social benefits of joint music-making, although it is taught in some spheres of Western music-making, including jazz and chamber music. The present study suggests that it is also achievable in musically less-skilled interactive group improvisation. Players need to sequence and coordinate relatively independent playing, and hence to sensitively predict and respond to another person’s intentions and wishes. Note, however, that our musical turn-taking emulated conversational turn-taking: it was explicitly modeled on how conversational turns work, rather than on turn-taking and turn-taking signals typical of jazz and other ensemble playing.We have hypothesized that music enhances empathy and self-esteem by different routes. Turn-taking may enhance empathy directly via enhanced interpersonal understanding and sensitivity, due to frequent temporally-coordinated reciprocal action, while joint synchrony may enhance personal well-being and hence prosociality directly, from which enhanced empathy develops indirectly later.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the School of Humanities and Social Sciences, University of Cambridge. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin, and by the young participants themselves.SH developed the study concept, negotiated with institutions, designed the experiments and many of the games, conducted the statistical analyses, lead the linguistic activities in each session, and had primary responsibility for writing the manuscript. CF contributed to design conceptualization and negotiation with institutions, session design and the content of musical activities, led session delivery, and contributed to the manuscript. Both authors worked together to assess the games’ validity and feasibility before use, and their success and any improvements immediately after each session.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


M = 12.54, SD = 1.72; 49% girls) at risk for developing psychopathology. First, participants self-reported on their overall use of different ER strategies. Next, the previously validated Attentional Breadth Task (ABT) was used to measure visual attention breadth toward emotional information. No evidence was found for the relationship between 2 specific ER strategies  and visual attentional breadth for neutral, positive and negative emotional information. Surprisingly, “distraction” was associated with visual attentional narrowing, which was unrelated to the valence of the emotion. These unexpected results indicate the multifaceted relationship between trait ER, distraction specifically, and visual attentional breadth for emotional information. Future research, especially in younger age groups, could further elaborate on this research domain.Attentional deployment is currently considered as one of the most central mechanisms in emotion regulation (ER) as it is assumed to be a crucial first step in the selection of emotional information. According to the broaden-and-build theory, positive emotions are associated with attentional broadening and negative emotions with attentional narrowing toward emotional information. Given that ER strategies relying on attentional deployment  have the possibility to influence positive and negative emotions by (re)directing one’s attention, there could be an association with one’s attentional scope. The current study investigated the association between the general (trait) use of three specific ER strategies and visual attentional breadth for positive, negative, and neutral information in a selected sample of 56 adolescents ( ER is often defined as motions” . Given ts affect .dysregulation has become a topic of increasing interest as it is linked to 75% of the diagnostic categories of psychopathology described by the Diagnostic and Statistical Manual of Mental Disorders   . Becauses adults .autonomously regulate basic emotions  through the use of behavioral strategies   . As cognmselves) . Althougmselves) . Given tmselves) .strategies  situation modification or the ability to influence emotions by changing external, physical characteristics of the situation, (3) attentional deployment or the process of directing the attention in a particular way within a situation in order to influence the situation-specific evoked emotions, which can take many forms including physical withdrawal of attention , internal redirection of attention  and external redirection of attention , (4) cognitive change or the ability to influence emotions by reappraising the emotion-eliciting situation, and lastly (5) response modulation or the ability to influence physiological and/or behavioral responses associated with the emotion. It is generally assumed that problems in ER can occur in each of the phases in the ER process  in clinically anxious children compared to children with no or lower levels of anxiety symptoms . HoweverGiven that both deficits in attentional deployment and ER more broadly are associated with psychopathology, studies unraveling the association between attentional in an emotional context and ER strategy use can provide crucial information for both research and clinical practice . NeverthCommon trait ER strategies that predominantly rely on attentional deployment, and may therefore be associated with one’s attentional scope in an emotional context, are cognitive reappraisal, rumination and distraction .Cognitive Reappraisal is generally categorized as an adaptive ER strategy and refers to the way in which a positive perspective or reinterpretation of the situation can decrease negative affect and/or increase positive affect . In geneRumination is considered a maladaptive ER strategy in which there is a repetitive focus on (negative) emotions, its causes, as well as its consequences . RuminatDistraction represents the intentional shift in attention from one’s negative emotions toward an external situation or stimulus  and has attentional breadth)  . A broadbreadth) .In this theory it is stated that specifically the attentional scope related to negative and positive emotions has different effects on both cognition and psychophysiology. Studies have shown that positive emotions  are associated with a more broadened attentional scope, which is considered a protective factor against stressful events . Negativbroadening . Based on the aforementioned line of thought and existing literature  use of specific ER strategies and the attentional scope in youth (9–15 years) in an experimental study. The broaden-and-build theory of M = 12.54, SD = 1.72; 49% girls) were recruited  ranging from 1 to 29 was found. Since the current study is part of a larger project . After obtaining informed consent, participants were asked to fill out a self-reported trait emotion regulation  and a depression  questionnaire using an online platform hosted by the Research Unit on a computer of the treatment center (see measures below) prior to a lab-study that took place on the same day. On the day of testing, adolescents were instructed to first explore the lab with the aim of familiarizing themselves with the lab-setting. As previously mentioned, the current study was part of a larger experimental study, took approximately 45 min per participant and consisted of several phases. Adolescents completed an attentional breadth task (see measures below) after watching a white screen for 3 min, which served as a neutral baseline to prevent experiencing negative affect at the start of the experimental task . After cFragebogen zur Erhebung der Emotionsregulation bei Kindern und Jugendlichen (FEEL-KJ); , in which a higher score means the ER strategy is more often used. In addition, a total score can also be calculated for the total use of adaptive, maladaptive, and external ER. The FEEL-KJ is considered reliable and valid with an acceptable to good internal consistency over all subscales . Furthermore, an acceptable to good test-retest reliability was reported with correlation coefficients between 0.76 and 0.90 ;  was usedand 0.90 . In the To measure fluctuations in affect during the lab study, the intensity of positive and negative affect was obtained through Visual Analog Scales (VAS). Youth scored their feelings of happiness, sadness, anxiety, frustration, boredom, and anger on a scale from zero (not present) to a hundred (very present) . This waVisual attentional breadth in relation to centrally presented emotional stimuli was measured using an experimental task . The tasWhen the central Emoji appeared, 16 gray dots with a diameter of 2 cm simultaneously surfaced in two concentric circles around the Emoji see . In totaParticipants were instructed to (1) correctly identify the valence of the presented central Emoji , and (2) localize the black circle that appeared in one of the 16 gray dots. For both responses there was no time limit. After the presentation of the central Emoji and target stimuli, a first screen appeared and participants were asked to identify the valence of the central Emoji see . After gThe task consisted of eight practice trials with a 250 ms presentation time, followed by eight practice trials with a shorter 68 ms presentation time. The test phase itself consisted of 144 trials with six types of trials randomly presented in three blocks of 48 trials each. The type of trials were based on the distance of the target stimuli to the central Emoji, as well as their valence .For the analyses of the attentional breadth task, all trials were deleted in which the central Emoji was incorrectly identified, to make sure participants also maintained attention to the centre of the screen during the task. The dependent variable was the number of correctly identified target stimuli (black circle). Only trials with correctly identified central Emoji were included. Performance on the attentional breadth task was examined by performing a 3 Valence  × 2 Distance (far vs. close) mixed ANOVA with the accuracy rates, i.e., number of correctly localized peripheral targets as the dependent variable.Thereafter we calculated an Attentional Narrowing Index (ANI) by subtracting the proportion of correctly identified targets in the far trials from the proportion of correctly identified targets in the close trails for positive trials (posANI), neutral trials (neuANI), and negative trials (negANI) separately. Although our primary interest lays in attentional broadening, we calculated ANI scores to keep the outcome variables consistent and to enable comparison with the original task used in previous research . Higher n = 15) were excluded from further analysis if the number of deleted trials for any of the different trial types was more than 50%  = 33.90, p < 0.001. Follow-up Wilcoxon’s signed-rank tests showed that participants made less errors when identifying neutral compared to positive stimuli , as well as neutral versus negative stimuli  when the target stimulus was presented close to the central stimulus. No difference between negative and positive stimuli was detected .A non-parametric Friedman’s ANOVA was performed as the data on the percentage of deleted trials was non-normally distributed. The ANOVA test revealed that if the target stimulus was presented close to the central stimulus, there were significant differences in the accuracy for identifying the central stimulus depending on the the emotional valence, χ2  = 34.85, p < 0.001. Again, follow-up Wilcoxon’s signed-rank tests showed that participants made less errors when identifying neutral compared to positive stimuli , as well as neutral versus negative stimuli  when the target stimulus was presented far from the central stimulus. No difference between negative and positive stimuli was detected .A second ANOVA test revealed that, if the target stimulus was presented far from the central stimulus, significant differences occurred in accuracy for identifying the central stimulus depending on F  = 67.80, p < 0.001, Mdiff = 5.20, SE = 0.63, p = 0.001), and a main effect of Valence, F  = 6.19, p = 0.003, Mdiff = 0.78, SE = 0.335, p = 0.025) or negative , but not when the target stimulus was positive compared to negative . Finally, no significant 3 Valence  × 2 Distance (Far vs. Close) interaction effect was found, F  = 0.096, p = 0.909, The 3 Valence  × 2 Distance (Far vs. Close) mixed ANOVA on the accuracy rates of target detection see  yielded F  = 4.86, p = 0.010, M = 7.69, SE = 1.09) compared to girls  when the target stimulus was positive  = 6.45, p = 0.015, ps > 0.188.Next, we added age and gender to the model in order to check whether these variables significantly affected the overall task performance. First, a significant Valence  × Gender interaction was found, tive see . Second,F  = 5.09, p = 0.030, The effects pertaining to the self-reported ER strategy variables  in the 3 Valence  × 2 Distance (Far vs. Close) revealed a significant Distance (Far vs. Close) × FEEL-KJ-DIS, t-test was conducted to examine fluctuations in affect before and after completing the experimental task. There was a significant difference in frustration before  versus after  the task; t(39) = –3,894, p < 0.05, in anger before  versus after  the task; t(39) = –2,996, p < 0.05 and in boredom before  versus after  the task; t(39) = –2,492, p < 0.05. Suggesting that youth were left significantly more frustrated, angry and bored after completing the ABT.A paired-samples broadening for emotional information. Second, no evidence was found for the relationship between trait “rumination” and attentional narrowing for emotional information. Third, trait “distraction” was associated with overall visual attentional narrowing for emotional information. These rather unexpected findings indicate a multifaceted relationship between ER and visual attentional breadth for emotional information. Future research, especially in younger age groups, is needed to further elaborate on these findings.The current study examined the relationship between the general use of specific ER strategies  that rely on attentional deployment and visual attentional breadth for negative, positive, and neutral emotional information in youth at risk for developing psychopathology. To measure ones’ specific ER strategies on a trait level, the FEEL-KJ was used . To measIn the current study, the role of attention was approached by the broaden-and-build theory in which it is stated that negative and positive emotions have different effects on the attentional scope . Positivpositive affect is reported by previous studies that examined this ER strategy form a state perspective  have already been mentioned. Future studies could also consider using  studies . During  studies . Based oFrom a clinical perspective, upcoming research on training ER provides evidence that learning to use specific adaptive ER strategies improves the ability to influence affect  . In the To our knowledge, the current study is the first to investigate the relationship between specific emotion regulation strategies and visual attentional breadth in youth at risk for developing psychopathology. Based on the broaden-and-build theory of positive emotions, it was hypothesized that adaptive ER facilitates positive affect and is therefore related to attentional broadening for emotional information. In contrast, given that maladaptive ER maintains negative affect, it was hypothesized that there would be an association with attentional narrowing . Three eThe raw data supporting the conclusions of this article will be made available by the authors upon reasonable request.The studies involving human participants were reviewed and approved by Committee of Medical Ethics UZ Gent. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.EB collected all the data. EB and M-LV analyzed the data. All authors were involved in writing the manuscript and had final approval of the submitted and published versions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


N-(4-hydroxyl phenyl) maleimide enters between the flakes as an intercalating agent and participates in the Diels–Alder reaction as a dienophile to increase the interlayer spacing of graphite. Then, graphite is exfoliated into graphene with the aid of ultrasound. The exfoliated product is hydroxyl phenyl functionalized graphene with a thickness of 0.5–1.5 nm and an average lateral size of about 500–800 nm. The exfoliation process does not involve any acid or catalyst and would be a safe and environmentally friendly approach. In addition, the exfoliated graphite shows high resveratrol adsorption capacity, which is ten times that of macroporous resins reported in the literature. Thus, the method proposed herein yields exfoliated graphite with high resveratrol adsorption capacity and is of great significance for the mass production of graphene for practical applications.Scalable preparation of graphene with high adsorption capacity is an important prerequisite for fully realizing its commercial application. Herein, we propose an environmentally friendly route for exfoliation of graphene, which is established based on the Diels–Alder reaction. In our route,  Graphene, a honeycomb-structured two-dimensional material with C=C bonds, is formed from sp2-hybridized carbon atoms. Owing to its intriguing properties, graphene has shown broad application prospects in many fields. Although graphene was first prepared by Geim in 2004 , the comWalimbe  proposesThe Diels–Alder reaction is one of the common chemical reactions involving conjugated alkenes. In 2012, Robert  made simN-(4-hydroxyl phenyl) maleimide (4-HPM) was employed to exfoliate graphite into graphene. 4-HPM is a maleimide derivative with a bulk phenolic substituent on the N side, which may act as an effective molecular wedge to promote exfoliation when the C=C in the 4-HPM molecule undergoes the Diels–Alder reaction with the diene moiety of the graphite. In our previous work  cycloaddition reaction a. The coin 4-HPM . Therefoin 4-HPM . The intin 4-HPM  and Mohain 4-HPM . The datXPS was employed to further analyze the elemental compositions of graphite and exfoliated graphite compounds. −1, a strong G peak at 1580 cm−1, and a 2D peak at 2708 cm−1, which were consistent with the data in previous reports [D/IG, of carbon material is used to determine the ratio of sp3 to sp2 carbon atoms, which is a measure of the number of defects in the material. As the reactions’ number increases, ID/IG value  method in the ref. . With thTo investigate the adsorption capacity, the exfoliated graphite was used as an adsorbent of resveratrol. The result showed that the resveratrol adsorption onto exfoliated graphite followed the pseudo-second-order kinetics, and the equilibrium data fitted well with the Langmuir isotherm model . AdditioIn summary, an ultrasound-promoted one-pot Diels–Alder reaction strategy was developed to chemically exfoliate graphite into hydroxyl phenyl functionalized graphene using 4-HPM as a dienophile. The exfoliated graphite compound with a thickness of 1–3 layers of carbon skeleton was obtained. The as-obtained graphene had an average lateral size of about 500–800 nm. During the heating–ultrasonic irradiating process, 4-HPM acts as the intercalating agent, but also as the dienophile to participate in the Diels–Alder reaction. Consequently, the interlayer spacing of graphite grows and finally leads to exfoliation of graphite into phenolic functionalized graphene. On the one hand, the facile preparation method provided in this study does not require any acid or catalyst and has the potential to be amenable for mass production. On the other hand, the exfoliated graphite has excellent pH sensitivity owing to the introduction of 4-HPM. In addition, the exfoliated graphite shows high resveratrol adsorption capacity (292.4 mg/g), which is 10 times that of macroporous resins reported in the literature. Thus, the method proposed herein yields exfoliated graphite with high resveratrol adsorption capacity and is of great significance for the mass production of graphene for practical applications.


This work shows the contributions of carbon and the different nanostructures present, as well as the impact of functionalizing graphene with oxygen and nitrogen, and the effects of including conducting polymers within the hybrid materials. Different mammalian neural growth models differentiate the roles of the substrate material in absence and in presence of applied electric fields and address optimal electrodes for the future clinical applications.Nanostructuring nanocarbons with IrO Carbon plays a unique role in electrochemical catalysis and energy storage systems or sensors. Its contribution to conductivity and its intrinsic capacitance has been explored in capacitors and batteries, alone and in combination with other components . In partFrom a biological point of view, carbons have been shown to be good substrates for cell growth ,7, and i+ ions and in turn enhance neuron excitability . Wh. Wh34]. x-eG hybrid. It can be observed that within days, dendrites fill the void space, being the biggest change around day 2.Furthermore, additional models for repair may be clarifying the role of electrode materials. Focusing only on neural repair, a physically created scratch in a stable neuron cell culture is a very representative model for injury and wound repair, in absence and in presence of applied electric fields. x [x-graphene hybrids, with one order of magnitude larger CSC are used, however, even for the same charge delivered, a larger repair is observed in both the cathode and the anode side. Furthermore, since CSC values are so much larger, longer electrostimulation times are also possible, suggesting a longer available electrostimulation time for implanted electrodes based on these materials.When an electric field is applied, however see , the resx . When thConcerning the electrochemical cell configuration used to achieve such electric field application, there are several ways to enhance the global cell charge capacity during electrostimulation. Using asymmetric electrochemical cells where the anode has been previously reduced allows for an additional global charge delivery of the cell. In such a case see , the finx, although the calculated depletion of extracellular Na+ ions is very small. Additionally, and more relevant, is the possible side reaction involving dissolved O2 reduction, that may generate oxo radicals. No evidence exists yet to elucidate which of those mechanisms is the significant one, although is clear that the effect is much lower for these hybrids than for Pt electrodes.Furthermore, comparing neural repair in the anode and cathode sides in any of the possible combinations of electrodes reported , the anox-eG hybrid electrodes, while in the case of Pt electrodes lower repair below the spontaneous one. The repair in the case of the hybrid is also above the lower CSC polypyrrole-PEDOT-lysine results, resulting in a significant outcome for the new electrodes [In either case, as quantified in ectrodes .x-nanocarbons, containing nanotubes, graphene or graphene oxide, that allow much larger charge delivery, without damaging effects, and with proven regeneration effects in very short times in the nervous system.Electrostimulation electrodes find a new family of nanostructured coatings, IrOx environment allows a significant wide span of oxidation states for nanocarbons, from labile oxygen in exfoliated graphene to nitro graphene, and with that a wide range of redox properties that contribute to the large CSC values found. Significantly enough, when considered per carbon atom, exfoliated pristine graphene is the nanocarbon that contributes more to the charge capacity and also the one that allows a larger reproducibility and impact in regeneration.From a fundamental point of view, it is remarkable that the IrOx-graphene hybrid, and while increasing their CSC even more, result in non-biocompatible materials.Additional components that may favor softer and mechanically appropriate electrodes, like conducting polymers, decrease the compatibility for primary neurons, although are optimal for a more realistic model of astrocytes-neurons cocultures. In particular, the polymer forms around other components, yielding to their encapsulation, and isolation from the electrolyte media, and decreasing the charge capacities to those of the polymer alone. N-doped graphenes, on the other hand, may reach the nitro-graphene state when part of the IrOx-nanocarbon hybrids are unique at present, in modulating the redox range available, but also in the nanostructure found, which is governed mostly by the nanocarbon structure, and the interaction with the iridium oxohydroxide. A reinforced concrete -type nanostructure or a millfeuille nanostructure are found, that are stable over more than a thousand cycles, and with similar charge capacities for acting within the range of potentials are for aqueous and biologic solutions.Thus, IrOx [From a more applied point of view, the resulting impedance of coatings derived from them are significantly lower and favor electrostimulation minimizing greatly the detrimental effects found for pure metals. In all cases, such conductivity also allows for bipolar electrochemical use, without electrical contact, as already proven for IrOx , and worx. In turn a new range of charge delivered in a biological system is possible. Regeneration is being observed in short times, and with very long times of stability of the electrodes. That, in turn, offers new clinical applications in electrostimulation, beyond the individual graphene or IrOx components.In conclusion, microstructural and chemical factors along with dipolar interactions determine the nanostructure achieved and the final electrochemical properties that have allowed the creation of new biocompatible materials based on nanocarbons and IrO


Taken together, this microplate-based method is convenient, economical, and rapid in locating antibody epitopes as well as interaction sites of two proteins.Trans-active response DNA-binding protein (TDP-43) is a multifunctional regulatory protein, whose abnormal deposition in neurons was linked to debilitating neurodegenerative diseases, such as amyotrophic lateral sclerosis, frontotemporal lobar degeneration, Limbic-predominant age-related TDP-43 encephalopathy, and Alzheimer’s disease with a secondary pathology. Several reports showed that TDP-43 proteinopathy as a comorbidity can form aggregates with other pathological proteins. The co-deposition of alpha synuclein and TDP-43 inclusions was previously reported in glial cells and by observing TDP-43 proteinopathy in Lewy body disease. In this study, it was hypothesized that alpha synuclein and TDP-43 may co-aggregate, resulting in comorbid synucleinopathy and TDP-43 proteinopathy. A solid-phase microplate-based immunoassay was used to map out the epitopes of anti-TDP-43 antibodies and locate the interaction of TDP-43 with α-synuclein. A region of the low complexity domain of TDP-43 (aa 311–314) was shown to interact with full-length α-synuclein. Conversely, full-length TDP-43 was shown to bind to the non-amyloid beta component of α-synuclein. Using in silico sequence-based prediction, the affinity and dissociation constant of full-length TDP-43 and α-synuclein were calculated to be −10.83 kcal/mol and 1.13 × 10 Trans-active response DNA-binding protein 43 kDa) (TDP-43) is a regulatory protein linked to various roles in transcriptional repression, mRNA splicing, and regulation of protein translation. It was discovered as an HIV-1 expression regulatory element that functions by binding to trans-active response (TAR) elements ,3,4. Str kDa -42, and to investigate interactions between myofilament proteins ,19. Here®, Waltham, MA, USA), 0.5 to 1.0 mg peptide were dissolved in an appropriate volume of dimethyl sulfoxide  to make a stock solution of 10 mg/mL. These peptides were then diluted to a final concentration of 5 µg/mL in phosphate buffer saline (PBS) solution with 10 mM EDTA. One hundred µL of each peptide solution was then transferred to a corresponding well of a maleimide-activated plate , and incubated overnight at 4 °C.Twenty-three non-overlapping peptides that cover the complete amino acid sequence of TDP-43 , and 43 After washing all coated wells three times with PBS with 0.05% Tween-20 (PBST), 100 µL of 10 µg/mL of L-cysteine  dissolved in PBS was added to all wells and left to incubate at room temperature for 1 h to block remaining exposed maleimide groups. After washing three times with PBST, 100 µL of 0.1 µg/mL of interacting recombinant protein to be tested was added next and left to incubate for 1 h at 37 °C. In mapping the epitopes of the antibodies for each peptide, no recombinant protein was added, and the antibodies were directly added instead on to the coated wells . After wThree µg/mL of α-synuclein and TDP-43 diluted in PBS were coated on separate polystyrene plates , and incubated overnight at 4 °C. The plates were then washed three times with PBST and blocked with 200 µL of 3% bovine serum albumin  diluted in PBS for 1 h at room temperature. After washing, 100 µL of serially diluted  of recombinant TDP-43 and α-synuclein in PBST were applied on to α-synuclein and TDP-43 coated wells, respectively. The plates were then left to incubate for 1 h at 37 °C. After washing, 100 µL of 0.1 µg/mL of biotinylated TDP-43 antibodies  diluted in PBST with 10% BlockAce, and anti-α-synuclein antibodies  were applied on wells treated with TDP-43 and α-synuclein, respectively, and left to incubate for 1 h at 37 °C. After washing, the wells were treated with 100 µL of HRP-conjugated streptavidin diluted 10,000 times in PBST with 10% BlockAce and left to incubate for 30 min at room temperature. After washing, chemiluminescent substrate  was added to achieve greater sensitivity. The resulting luminescence was then measured using a Victor 3 multilabel reader.d) of two protein sequences. The sequences of aa 310–329 . The differences were considered significant at Three antibodies were selected from a systematic literature review of 671 anti-TDP-43 antibodies featured in publications from 2006 to 2014 . Ab-2 2. TDP-43 Using the same microplate-based approach, the TDP-43 peptides were separately treated with three different recombinant proteins: albumin, tau, and α-synuclein. Results showed that only full length α-synuclein produced a significant reaction in the well coated with peptide 17 (aa 310–329), a region in the LCD of TDP-43 . To bettTo investigate the region in α-synuclein which full length TDP-43 interacts with, 43 overlapping peptides of α-synuclein that shift every three residues were coated on to the wells and treated with full length TDP-43. Amino acids 61–76 of α-synuclein showed the highest reaction with TDP-43 . Interesx-axis), charge at neutral pH (y-axis), and the generated absorbances (z-axis) of the proteins that bound to the peptides was generated for TDP-43  to confirm this interaction. Moreover, the affinity prediction of ISLAND is sequence-based and other factors outside this sequence may affect the calculated parameters. It was stated that affinity prediction based on the peptide sequence is not satisfactory, and development of effective and practical methods in this domain is still an open problem [Using ISLAND, the binding affinity and the k problem . HoweverResearch on the comorbid interactions of TDP-43 with other pathological biomarkers is growing, and epitope mapping techniques are particularly useful in further characterizing interactions in these comorbidities. Here, a microplate-based immunoassay was used to determine interaction sites between TDP-43 and α-synuclein. A significantly high concentration of full-length α-synuclein was found to bind with aa 310–329 of the LCD in TDP-43. Using overlapping peptides that cover this region, MNFG residues (aa 311–324) were common to peptides that resulted in a high signal of bound α-synuclein. Thus, not only do TDP-43 CTFs initiate the formation of cytoplasmic inclusions by aggregating amongst themselves, but they can potentially create hybrid TDP-43/α-synuclein fibrils by interacting indiscriminately with all α-synuclein species. In contrast, a significant amount of full-length TDP-43 was found to bind with the NAC region of α-synuclein. Most importantly, co-incubation of human neuroblastoma SH-SY5Y cells with heterotypic fibrils composed of TDP-43 LCD and α-synuclein showed increased cellular toxicity when compared to SH-SY5Y cells incubated with either homotypic TDP-43 LCD or α-synuclein fibrils, reinforcing the pathological significance of this interaction .Though other approaches such as NMR spectroscopy can better resolve these interactions, the self-aggregation of some regions can mute chemical peak shifts that indicate interaction sites. Separating the proteins as shorter peptides and testing binding for each peptide separately circumvents this problem. However, this approach can be limited when the actual site of interaction happens to be fragmented between multiple peptides and overlapping peptides in the suspected region must be designed and synthesized to resolve the interaction site. The length of each peptide also has to be estimated well so it can contain an optimal number of residues for binding to be observable. Finally, it is worth noting that the results generated herein employed recombinant proteins, thus, it may be necessary to confirm their physiological relevance via cell or animal models in future studies. Still, this approach is used because it is relatively convenient, economical, less tedious, and more rapid than other approaches.


In recent years, bioprinting has attracted much attention as a potential tool for generating complex 3D biological constructs capable of mimicking the native tissue microenvironment and promoting physiologically relevant cell–cell and cell–matrix interactions. The aim of the present study was to develop a crosslinked 3D printable hydrogel based on biocompatible natural polymers, gelatin and xanthan gum at different percentages to be used both as a scaffold for cell growth and as a wound dressing. The CellInk Inkredible 3D printer was used for the 3D printing of hydrogels, and a glutaraldehyde solution was tested for the crosslinking process. We were able to obtain two kinds of printable hydrogels with different porosity, swelling and degradation time. Subsequently, the printed hydrogels were characterized from the point of view of biocompatibility. Our results showed that gelatin/xanthan-gum bioprinted hydrogels were biocompatible materials, as they allowed both human keratinocyte and fibroblast in vitro growth for 14 days. These two bioprintable hydrogels could be also used as a helpful dressing material. Hydrogels are crosslinked, insoluble and hydrophilic polymers that are capable of containing a large amount of water thanks to their porosity and three-dimensional network structure . The hydGelatin (Gel), which is produced by partial hydrolysis of collagen, displays good biocompatibility and biodegradability properties and is commonly used in medical areas, due to its great biological advantages and low cost . SeveralXanthan gum (Xnt) is a microbial-derived high-molecular-weight heteropolysaccharide that is employed in medical field and tissue engineering for its biocompatibility and gelling characteristics , as wellAn important limit of gelatin and xanthan-gum hydrogels is their mechanical weakness and dissolution behavior in solvent . It is kCrosslinking agents can be inserted into hydrogels through different methodologies, such as physical methods and the use of chemicals or enzymes . Among tThree-dimensional bioprinting is an early stage technology with important consequences and applications in the biomedical and tissue-engineering fields, as it allows the creation of 3D tissue constructs through programmed models and the distribution of the so-called bio-ink by means of the movement of a motorized stage ,29. Bio-Considering this, the aim of present study was to develop a low-cost crosslinked 3D printable hydrogel based on biocompatible natural polymers (gelatin and xanthan gum) and characterize it as a cell-growth scaffold to use for both in vitro growth of tissues and wound dressing.As reported in After the printing process, three different concentrations  of glutaraldehyde and two different immersion times (1 and 3 h) were used for the hydrogel crosslinking test. The higher concentrations of glutaraldehyde, with a longer immersion time, led to greater resistance and permanence of the print shape, which could adversely affect the biodegradability of the printed hydrogels. Therefore, in order to reduce the excessive tribological qualities (durability) ,32, and 2 square shapes obtained by extrusion bioprinting. A comparison of the different hybrid composites hydrogels showed that both 3Gel4 crosslinked and non-crosslinked have a defined shape compared to 2.5Gel3, which appears to be less viscous. These results showed that xanthan gum is a good stabilizing agent, because the hydrogel with 3 w/v% of Gel without crosslinker is able to maintain its print shape, which even improves after the immersion in GTA.The evaluation of the water content of a hydrogel is required for many biomedical applications. The percentages of water in the hydrogels were similar to each other, independently from the gelatin content . Moreovep-value 0.02543), increasing after three hours of submersion until the sixth hour, where all the prints resulted in being completely dissolved  of 3 w/v% Gel content. In The swelling tests were carried out on 2.5Gel3 and 3Gel4 printed hydrogels crosslinked and non-crosslinked. These prints containing 2.5 w/v% and 3 w/v% of gelatin, after one hour of submersion in deionized water, showed a swelling rate of 1110.25% ± 339 and 423.17% ± 47.34 respectively  and after 14 days of 24.41 ± 28.16% (p = 0.005756) in water at 37 °C . These results were compared with those obtained by morphological analysis of both hydrogels . A greater number of bonds between hydroxyl groups of the excess xanthan gum, not linked to gelatin, and water molecules cause an increase of hydration during the synthesis of the hydrogel, and therefore a lower viscosity.Gelatin is widely used in wound-healing applications ,34,35,36Furthermore, observing the images obtained under the microscope and shown in The human body is known to be largely composed of water. The main goal of this work is the 3D printing of the cell-seeded hydrogel during therapies such as skin grafts  to repai2) of lysine or hydroxylysine belonging to the gelatin. This leads to the formation of amide linkages [Furthermore, swelling is also an important property to evaluate. Swelling is the ability of a hydrogel to absorb water, gaining weight and volume . Hydrogelinkages ,44. The linkages . This prlinkages ,45.The size and number of pores, their connectivity and geometry are the main characteristics for the use of the hydrogels in biomedical applications. Indeed, the ability of cells to proliferate into a 3D structure is affected by the size of the pores, as well as is the biodegradability of the structure itself . In partThe results of the enzymatic degradation and hydrolysis test showed the biodegradability of the produced hydrogel. Although crosslinked gelatin hydrogels are soft materials, they are capable of causing irritation and reactions . For thiFinally, human fibroblast and keratinocytes were mixed with 2.5Gel3 and 3Gel4 hydrogels respectively, and a skin-like structure was printed to evaluate the biocompatibility of both materials. The images of the cell distribution and proliferation in the hydrogels collected after 1, 7 and 14 days in culture showed a good proliferation. These results are also due to the presence of numerous and small pores that induce greater cell proliferation in a 3D structure  and to tShawan et al.  reported2 squares. After preparation, hydrogels were left at 4 °C from 3 to 10 days, and they were warmed to room temperature before printing. Bio-ink syringes (3 mL) were filled with the different hydrogels and then loaded into the 3D printer. Printability trials were run at different pressures (from 10 to 20 kPa), as reported in CellInk Inkredible 3D printer  was used for the hydrogel printing. Hydrogels were printed to obtain 1 cmCrosslinking trials were performed by using different concentrations of glutaraldehyde  obtained from a 25 v/v% stock solution. In addition, for each concentration, different submersion times in the glutaraldehyde solution (1 or 3 h) were tested. The crosslinking of a hydrogel structure occurs in the post-printing process. Crosslinked hydrogels were added to cell culture Dulbecco’s Modified Eagle’s medium (DMEM) to test their stability .The macroscopic morphology of the hydrogel was observed by taking several photos, using a high-resolution digital camera (NIKON D5600). Optimal cutting temperature compound (OCT compound) was used to incorporate the hydrogels prior to frozen sectioning on a microtome cryostat, obtaining hydrogel micron slices. The optical reverse microscope  was used to evaluate the microscopic morphology of the different printed hydrogels, both crosslinked and non-crosslinked.The moisture percentage was measured as described by Shawan et al. . On day Printed hydrogel, both non-crosslinked and crosslinked, were dried at 37 °C for 48 h to perform the swelling test . Dried pAll the experiments were replicated three times, with at least three samples for each condition.I is the before soaking weight, and WF is the remaining weight after soaking and removing of the deionized water. All the experiments were replicated three times, with at least three samples for each condition.In order to evaluate the hydrolysis, crosslinked printed hydrogels were weighed (time 0) and immersed in deionized water at 37 °C. After the time point from 4 to 14 days, the weight of the crosslinked prints was taken by removing the deionized water and lightly dabbing the crosslinked prints with filter paper. The percentage of hydrolysis was obtained by using the following equation (Equation (3)):1 is the initial weight of pure ethanol, W₂ is the total weight that combines the weight of the hydrogel with the weight of the ethanol and W₃ is the ending weight of ethanol without hydrogel [The porosity of the printed hydrogels was evaluated by using the liquid displacement method . Absoluthydrogel .Furthermore, Greyscale 8-bit TIFF images of the 2.5Gel3 and 3Gel4 were processed by using ImageJ software. The ImageJ “particle analysis” function was then used to calculate the size and the numerosity of hydrogel pores. All the experiments were replicated three times, with at least three samples for each condition.The biological stability of the hydrogels was evaluated by exposing them to 0.1% collagenase type IA  to assess degradation rates . Printed2 at 37 °C. Two different cell populations (keratinocytes and fibroblasts) inserted into the two hydrogels of different porosity were used for printing a simple 3D skin-like model with a surface layer that mimics the epidermis (high cell concentration for the epithelium) and a lower layer that mimics the dermis (low cellular concentration for the connective). The 2.5Gel3 and 3Gel4 printable hydrogels were gently mixed with 0.5 × 106 fibroblasts/mL and 5 × 106 HaCaT cells/mL, respectively, using two syringes connected under a sterile cap. Small cylinders  were printed by using cell-laden 2.5Gel3 for the lower layers and 3Gel4 for the upper layers. The cylinders were crosslinked by using GTA as previously indicated. The cell-laden cylinders were washed three times with sterile PBS and incubated for 1, 7 and 14 days in DMEM 10% FBS at 37 °C in a 5% CO2 atmosphere. At the end of the incubation time, the culture medium was removed, and the hydrogels containing the cells were washed with phosphate buffered saline (PBS) 3 times, formalin fixed and paraffin embedded. Samples were then cut in 5μ slices and stained with hematoxylin/eosin; the cells grown in hydrogels were observed by using a digitizer Pannoramic Midi for microscope slides , and their number was evaluated by using the ImageJ software. All the experiments were replicated three times, with at least three samples for each condition.Primary human-derived-skin fibroblasts and human keratnocytes spontaneously immortalized (HaCaTs) were used for testing printable hydrogels biocompatibility. HaCaTs were purchased from Cell Lines Service GmbH , while human fibroblasts were a kind gift from Professor Marco De Andrea . Both cell types were grown in culture flask in Dulbecco’s modified Eagle’s medium (DMEM) containing high glucose levels supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% Penicillin–Streptomycin  in a humidified atmosphere containing 5% COp-values < 0.05 were considered statistically significant.Data were presented as mean ± standard deviation (SD). Statistical analyses were performed by using GraphPad PRISM software. Kolmogorov–Smirnoff test was applied to verify the normal distribution of data. One-way ANOVA was employed for multiple comparison, the whereas t-test was used when a comparison between two group occurred. The The hybrid composite hydrogels containing different percentages of gelatin and the same amount of xanthan gum were designed to obtain printable materials for in vitro and in vivo biomedical applications. Among all the hydrogel formulations modulating the process and the parameters of the 3D printer, the 2.5 w/v% and 3 w/v% of gelatin with 1.2 w/v% xanthan gum resulted in being printable, with a good resolution. The characterization results showed that 3Gel/4hydrogel crosslinked with 0.3 v/v% GTA presents optimal properties compared to the hydrogel with 2.5Gel3. In particular, the 3Gel4 hybrid composite hydrogel was able to maintain its print shape, to resist to the swelling and to degrade easily. Moreover, it shares a very good biocompatibility with the 2.5Gel3, and it could be used as a matrix for cell growth, for the development of in vitro tissues or also for wound dressing, allowing the host cells to gradually replace the hydrogel with a secreted matrix.


Background: Historically, idiopathic pulmonary fibrosis (IPF) was considered a chronic inflammation disorder, but this conception was reassessed in the past decades. Our understanding of the role of inflammation in IPF and its association with clinical significance remained incomplete.Methods: We downloaded mRNA expression data of peripheral blood mononuclear cells (PBMCs) from the Gene Expression Omnibus (GEO) repository. Inflammation-related genes (IRGs) expressed differently between IPF and control (CTRL) were determined. In this study, we systemically analyzed the expression of differently expressed IRGs by comprehensive bioinformatic analysis, and then investigated their potential prognostic values. The related prognostic gene expressions were verified in our cohort.Results: 110 differently expressed IRGs were identified in this study, including 64 upregulated and 46 downregulated IRGs. Three IRGs  were identified as potential hub genes for prognosis. Those genes were subsequently subjected to the construction of the prognostic models. In the results, IPF patients categorized as high risk demonstrated a poor overall survival rate compared to patients categorized as low risk. Based on this prognostic model, the area under the curve (AUC) of the survival-dependent receiver operator characteristic (ROC) for 1-year, 2-year, and 3-year survival rates was 0.611, 0.695, and 0.681, respectively, in the GSE28042 cohort. These observations were validated in the GSE27957 cohort, confirming the good prognostic effect of this model. The expression of the three genes was validated in our cohort. We also conducted a nomogram based on the three IRGs’ mRNA for quantitative IPF prognosis.Conclusion: Three IRGs  were identified as potential markers for the prognosis of IPF. Idiopathic pulmonary fibrosis (IPF), caused by an unknown reason and characterized by poor clinical prognosis, is a progressive, chronic, and irreversible interstitial lung disease (ILD) . IPF occTOLLIP (Toll-interacting protein), directly involved in inflammatory processes, resulted in reduced protein expression, which possibly contributes to an increased pro-inflammatory response observed in IPF patients  and white blood cells, are indicative factors of disease onset, progress, and prognosis of IPF . Maya etThe subject of this study was to investigate the roles of inflammation played in IPF and explore the potential clinical utility of IRGs for prognostic stratifications. Therefore, we downloaded the mRNA expression data of PBMCs and the clinicopathological data from the Gene Expression Omnibus (GEO) repository. We identified abnormally expressed IRGs between IPF and control (CTRL) using bioinformatic analysis, and systematically investigated their potential molecular mechanisms and functions. Finally, we analyzed the potential impact of IRGs on prognosis by combining the IRG expression data with clinical information.https://www.ebi.ac.uk/QuickGO/), which provides detailed annotations of genes. We downloaded the microarray expression matrix dataset of 75 IPF and 19 CTRL PBMCs with corresponding clinical data from GSE28042 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE28042). To identify differently expressed inflammation-related genes between IPF and CTRL PBMCs, and the limma R package (http://www. bioconductor.org/packages) was used to perform the negative binomial distribution method according to the absolute value of fold change (FC) (>1.3) and false discovery rate (FDR) <0.05.A total of 569 inflammation-related genes were extracted from the Gene Ontology and GO Annotations (http://string-db.org) (version 10.5) for the prediction of the PPI network. Meanwhile, Cytoscape (version 3.8.0) was applied to visualize the PPI networks. The hub genes were identified in the PPI network according to the prediction of cytoHubba plug-in with Nodes’ Scores. p values less than 0.05 were considered statistically significant.The differently expressed IRGs were submitted to the Search Tool for the Retrieval of Interacting Genes (STRING)  analysis was applied to assess the prognostic capabilities of the above model by the SurvivalROC R package.p values lower than 0.05 were set as statistical significance.45 samples of IPF patients with the corresponding prognosis from the GSE27957 dataset were used to validate the estimating results to evaluate the accuracy of the model prediction. We also performed expression validation of the three prognostic genes in our cohort by real-time PCR. We collected PBMC samples and clinical information of IPF patients in Tongji Hospital in Wuhan, China, from July 2020 to November 2020. Adult patients with clinical IPF diagnosis according to the American Thoracic Society/European Respiratory Society guideline (2011) were eligible for this study . This stA systematic analysis was carried out for the critical roles and the potential prognostic values of IRGs played in IPF. As shown in Gene network complexes were generated by STRING according to the results of differently expressed IRGs. The PPI network contained 110 nodes and 470 edges . The intS100A12), C-C motif chemokine receptor 7 (CCR7), and TNF superfamily member 4 (TNFSF4) were identified as independent predictors for IPF patients.To identify IRGs of significant correlation with the overall survival, a univariate Cox regression analysis was established based on hub IRGs. As shown in the forest map of the hazard ratio , 28 IRGsThe three hub IRGs identified from the above analysis were used for the construction of the inflammation predictive model. The risk score of each IPF patient was calculated asThen, we performed a survival analysis to evaluate the predictive effect of this model. As the result, 75 IPF patients were categorized as high-risk or low-risk according to the median of risk scores. As revealed by the result, IPF patients with high-risk scores were associated with poor prognosis compared with those with low-risk scores . A surviS100A12 was significantly increased in IPF patients, while CCR7 and TNFSF4 were decreased , and serum were increased in acute respiratory distress syndrome (ARDS) patients compared to healthy controls  reported that CCR7 expression was significantly raised in usual interstitial pneumonia (UIP) relative to biopsies from patients diagnosed with nonspecific interstitial pneumonia (NSIP) or respiratory bronchiolitis/interstitial lung disease (RBILD). CCR7 was expressed by IPF fibroblasts but not normal fibroblasts and could promote migration and proliferation of IPF fibroblasts (CCR7 abrogates pulmonary fibrosis induced by the adoptive transfer of human pulmonary fibroblasts to immunodeficient mice (CCR7 in disease. The CCR7 axis could combat the spread of cancer by trafficking of effecter cells involved in mounting an immune response to a growing tumor, while contribute to the expansion of cancer via controlling the migration of tumor cells towards the lymphatic system and metastasis (The decreased level of roblasts . Therapeent mice . The distastasis .TNFSF14 of PBMCs was associated with a worse IPF prognosis. The cytokine TNFSF14 was demonstrated to be an important factor in the development of lung tissue remodeling in the murine model, which is associated with asthma, IPF, and systemic sclerosis (TNFSF14 was upregulated in bleomycin-induced pulmonary fibrosis and is a profibrogenic cytokine (TNFSF14 as an inflammatory indicator was increased in different diseases (TNFSF14 should be validated in IPF patients.The lower level of clerosis . Rana Hecytokine . Considediseases , the serSubsequently, a risk model was established for the prediction of IPF prognosis according to multiple stepwise Cox regression analysis of hub IRGs, validated using the GSE27957 cohort. In this study, the ROC curve analysis demonstrated that the IRG signature was better fitted for diagnosis rather than predicting poor prognosis in IPF patients. A nomogram was constructed for intuitively predicting the 1- and 3-year overall survival. This prognostic model based on these three IRGs is financially cost-less and efficient for clinical use.Nonetheless, limitations were found in this prediction system. First, this prognostic model was constructed according to the GEO database; therefore, further validation in the clinical patient cohort was needed. Second, as a retrospective study, further prospective studies were needed for verification of the outcomes. Finally, functional experiments were needed to further reveal the potential mechanisms of IRGs in the future.Conclusively, this study identified three IPF prognostic IRGs based on comprehensive bioinformatic analyses in GSE datasets. Our results contributed to the understanding of the roles inflammation played in IPF pathogenesis, and therefore potentially promoted the development of markers for prognosis assessment.