The Q-cycle mechanism entering the electron and proton transport chain in oxygenic photosynthesis is an example of how biological processes can be efficiently investigated with elementary microscopic models. Here we address the problem of energy transport across the cellular membrane from an open quantum system theoretical perspective. We model the cytochrome  We have henceforth witnessed the rapid development of the field of quantum biology3, a growth that continues nowadays towards many directions at the forefront of pure and applied scientific research5.Approaching the field of biological sciences from the perspective of microscopic physical processes is extremely intriguing and yet partly unexplored in current research. Presently, photosynthesis occupies a particularly privileged position in this quest: the molecular basis of its fundamental mechanisms are actively investigated at the biological, biochemical, and biophysical levels. Additionally, the nature of the process itself is well suited to be investigated with the tools of either classical or quantum statistical physics. In fact, it involves analyzing the light-matter interaction within the cell as well as excitation and particle transport through molecular networks, together with compelling energetic considerations. It has been more than ten years now since the attention of quantum physicists was triggered by remarkable spectroscopic results reporting possible evidence for quantum coherent processes in light harvesting complexes10, one of the most relevant theoretical achievements in the field lies in the application of the theory of open quantum systems to model biological processes featuring a single or a few excitations embedded in the complex landscape represented by a protein, a membrane, or even a cell3. Following a similar approach, we hereby focus on the electron transport chain, which represents the second stage of the photosynthetic machinery, immediately connected to the primary light-absorbing processes. Several early applications of open quantum systems (OQS) theory to such crucial particle and charge transport processes at the molecular level are known in the literature18, including investigations of coherence11, correlations15 and non-Markovian effects14. Building on an OQS-based kinetic model originally proposed by Smirnov and Nori17, here we develop a simple but accurate Markovian theory of the Q-cycle. The latter is a remarkable biochemical mechanism that efficiently conserves energy stored in excited electrons to increase the number of protons per electron that are pumped against the concentration gradient across the cellular membrane.While the significance and interpretation of many initial findings in quantum biology, most notably the origin and the effect of quantum coherences, is still actively debated17, our approach connects charge transport within the Q-cycle to the reservoir dynamics of the surrounding environment. After benchmarking our alternative model by calculating the so-called quantum yield  and comparing our results to the original ones17, we then extend the original study and provide new predictions of biological relevance: in particular, we determine the thermodynamic efficiency of the Q-cycle as a function of the external temperature, and find optimal performances around ambient temperatures compatible with physiological conditions for living cells. We further strengthen our analysis by introducing a new figure of merit combining information from the quantum yield and the transferred charge, which again shows optimality around room temperatures.In deriving the relevant rate equations describing the dynamics of this complex biochemical process, we exploit the Lindblad formalism to describe single-particle transfer reactions on a microscopic level. Consistently with previous derivations20, this theoretical work potentially provides a deeper and more complete understanding of many bioenergetic aspects at the molecular level.We emphasize that even though no genuine quantum effect such as entanglement or coherence-enhanced transport is expected to play any relevant role in the Q-cycle itself, our work confirms that the standard tools of OQS theory and quantum statistical mechanics prove to be very well suited for the physical study of microscopic biochemical transport processes, giving adequate control over the relevant parameters and leading to very natural results with potential biological and experimental relevance. Complemented by alternative mathematical approaches21. These photosystems are typically assisted by light harvesting complexes (LHC) during the photon absorption and exciton migration stage. The two photosystems are actually successive stages of the same global energy transfer chain in which the energy contained in high energy chemical bonds fuels the transfer of protons  between different molecular species such as quinone, semiquinone, and quinol.In Fig. 37. Referring to Fig. 17: Under CEF conditions, each 3840. A complication arises here, since the single Marcus rates from two individual states explicitly depend on their energy difference. Therefore, we shall distinguish between the cases in which other charged molecules are present or not, for example in all the transitions involving shuttle electrons. This is precisely the effect of the Coulomb interaction terms, which contribute to make some specific transitions more or less favorable. We thus introduce projectors on the states of the system, We now solve for the dynamics of this model by explicitly resorting to the theory of OQS applied to the full density matrix of the complete system, 38: indeed, the dynamics of the diagonal elements of the density matrix, namely the electron and proton populations in the possible basis states, evolve according to a relaxation equation of the form 3817, the present formalism is based on the complete density matrix of the system and retains the linear structure typical of quantum mechanics, even at the stage where it is reduced to a set of rate equations. Therefore, it fully captures all possible correlation effects  without further assumptions. In particular, it conserves the total number of particles in electron and proton transfer reactions. Moreover, since the full master equation gives in principle access to the dynamics of the quantum mechanical coherences, the formalism could be easily extended to include a richer phenomenology when applied to different contexts.As already anticipated, from Eq. 17. Here we keep such an approach, thus including a stochastic differential equation (SDE) to describe the position of the PQ shuttle, whose diffusive commuting inside the rather dense lipidic membrane is modeled as an overdamped Langevin-Brownian motion i.e. the inverse of the diffusion coefficient times the temperature, 2117.A very peculiar element of the description of the In this section, we present some of the most significant results we obtained by performing extensive numerical simulations of the hybrid quantum and stochastic model discussed above . Notice that all the simulations presented in this work can be performed on a standard Personal Computer. The dynamical behavior of the system was investigated by varying some physically meaningful external parameter, such as the electrochemical gradient of protons across the membrane, the static surface potential, or the temperature. For each parameter under individual scan, the relevant range of variation was empirically identified and divided into a number of steps. For each one of these steps, i.e. for each individual value of the parameter under scan, six independent stochastic diffusion trajectories were simulated, all with the same set of parameters and initial conditions. Each trajectory covered 17. In all the simulations, any parameter not involved in the scan was kept fixed at a default value chosen consistently with the relevant literature17, in such a way to guarantee a consistent comparison of the results for benchmarking our theoretical method. All such values are reported in the Methods section. First, the electrochemical gradient difference for protons, 4117, which have been extracted from Figs 17, also explicitly shown in Fig. A few benchmark simulations were performed in order to validate our formalism with respect to previous resultsWe now go beyond previous studies by analyzing the effects of temperature variations. Temperature is directly included in the definition of many quantities affecting the dynamical behavior of the model, and influences both electron and proton transfer reactions as well as the stochastic motion of the In scheme I, the temperature was varied while keeping fixed 17, this dependence takes the form In scheme II, we held the pH gradient fixed while changing the temperature. This time, the Finally, we can apply the formal general definition for thermodynamic efficiency, as given in Eq. The Q-cycle mechanism is a relevant energy conversion process entering the electron transport chain in oxygenic photosynthesis, but its complex biochemical nature, difficult to capture with theoretical approaches, has to date limited the development of analytic and numerical models. Building on a microscopic description based on an open quantum system formalism, here we have started to bridge the gap between a first principles dynamical evolution of elementary charges and physiologically relevant conclusions on a macroscopic level. First, we were able to reproduce one of the most interesting effects associated with the Q-cycle, namely the bifurcation of electron flows: upon net transfer of two protons from one side (stroma) to the other (lumen) of the cellular membrane, only one of the two electrons simultaneously present on the charge carrier gets consumed, while the other goes through a recycling path within the In general terms, it is remarkable that the Q-cycle mechanism is highly conserved, since not only it is found in the thylakoid membranes of photosynthetic organisms, but also in the inner mitochondrial matrix, where it serves as an energy-conserving proton-pumping mechanism in the 19, some phenomenological observations, mainly obtained while studying the response of the mitochondrial 44. In principle, these hypotheses could be theoretically tested by extending the present model to include an external control over the allowed transitions, and better detailing the individual chemical mechanisms of such processes.In perspective, the present model is intended as a first step towards a full description of the 18, it is certainly worth pointing out that the Lindblad approach could easily be applied to many other biologically relevant examples. On one hand, the conceptual simplicity and modularity of the resulting model could guide the design of experimental investigations in which, e.g., the stoichiometry of the electron and proton transport can be assessed and compared with predictions45, keeping all the relevant parameters and external conditions under control. On the other hand, the markovianity and weak-coupling assumptions, which are naturally embedded into the Lindblad form of the master equation, could be violated in some cases, e.g. when the interplay between particle or energy transfer events and the evolution of the protein scaffold is stronger14, or when the relaxation time of the environment is comparable to the relevant quantum dynamics46: under such conditions, our proposed formalism may serve as a guide towards non-trivial extensions, e.g., to non-Markovian quantum master equations47 or hierarchical approaches48. Finally, we cannot neglect that the interplay between biology and its formal analysis is bidirectional: in this respect, the elementary CEF formulation adopted here might inspire new possible routes to artificially engineer coupled electron-proton translocations.On the technical side, we highlight that our work clearly demonstrates that a Markovian open quantum systems formulation is sufficient to explain the Q-cycle as a naturally emerging process, given the general structural and physical parameters defining the protein complex environment. Moreover, such formalism is certainly suited for the exploration of more genuinely quantum effects at the mesoscale. Indeed, it is worth reminding that quantum mechanical features are naturally embedded into the description at a fundamental level: this is reflected in the linear structure of the resulting set of rate equations, which in principle preserve all possible quantum and semiclassical correlation effects and could readily be extended to situations in which the dynamics of quantum coherences plays a non-trivial role. Given the close similarity of the working principles of the Here we provide further details on the mathematical structure of the model described in the main text, together with the techniques that were used to derive and solve the resulting set of equations.17 and constitute the basis for our original developments based on an open quantum systems formalism. The explicit expression of the full system Hamiltonian can be written as Here we explicitly report all the Hamiltonian terms entering the microscopic model of the Q-cycle discussed in the main text and further details concerning the stochastic motion of the The asymmetric electrostatic surface potential, varying linearly with position inside the membrane, can be modeled as The master equation for the quantum evolution was derived by using techniques from the theory of open quantum systems. The source (Fd) and drain (Pc) terms are realizations of the well known case of a single two-level system in contact with a thermal reservoir whose creation and annihilation operators obey anti-commutation rules. The proton reservoir terms are similar in spirit but distinguish the possible configurations of electrons and protons on the 49 featuring the two binding sites coupled by a tunneling interaction and connected to the surrounding molecular vibrational environment. Marcus transition rates are obtained by applying perturbation theory and Fermi’s golden rule while assuming a Debye form for the bath spectral function 50 for details).which are essentially an adapted version of the well known spin-boson Hamiltonian17, here the fully linear structure of Eq. 11, matrix exponentiation to compute the exact time evolution for arbitrary times 17) to allow for a direct comparison. In particular, electron and proton binding energies and electrochemical potential ranges are consistent with typical redox potentials for the relevant electron transport chains and 34 and with experimental measurements of membrane potential and pH gradient in molecular structures closely related to the 37. The values and temperature dependence of the surface potential M. laminosus34, while other structural parameters such as the overall typical dimensions are retrieved from biological and crystallographic characterizations of the 36. The effective drag coefficient 51. Finally, the remaining parameters that are specific to our model, such as tunneling matrix elements (17, where they were optimized for the Q-cycle performances at fixed temperature to reproduce the typical reaction speed and yield.The dynamics of the system was numerically simulated with an original Python code.The coupled quantum and stochastic system equations (see Eqs mentclass2pt{minim16.As an explicit example, we show in Fig.


The profession of the health-care providers (HCPs) influences their recommendations to the patients. Conversely, interdisciplinarity seeks to challenge such differences, so that the patient receives one single and consistent therapeutic message. Some studies also suggest associations between HCPs life habits and recommendations. Our hypotheses were (1) that despite interdisciplinary work, the profession remains a predictor of recommendations and (2) that HCPs who are more physically active recommend more activity. Three clinical vignettes were presented to a group of experts of low back pain (LBP) (guidelines), and 20 physicians, 22 physiotherapists, and 23 nurses to assess how they evaluate the symptoms and pathologies of LBP patients and how much work and physical activity they recommend. Physical activity was assessed with accelerometers and questionnaires. Some interprofessional differences remained present within an interdisciplinary team. The nurses were more restrictive and further away from the guidelines. The physicians were the most in line with them. The physiotherapists recommend as much physical activity, but less work activity than the physicians. The level of physical activity of the HCPs is not associated with their recommendations. To ensure a clear and unique message, educational actions may be undertaken to promote the biopsychosocial model and clarify the guidelines. Interdisciplinarity has become a key concept in health care. It allows a comprehensive approach to develop consensual guidelines and recommendations . AccordiWhat is already known is that individual beliefs and life habits may affect attitude and recommendations of HCPs notably regarding low back pain (LBP) –10. LikeInterdisciplinarity is important in LBP care and rehabilitation . LBP is Our main objective was to evaluate the coherence of PA recommendations to patients within an interdisciplinary team of physicians, physiotherapists, and nurses. We postulated that if the team worked coordinately, the members of the team should give similar recommendations regardless of the professional background. Conversely, in case of a strong professional group effect, the recommendations could differ despite the interdisciplinary work.Our secondary objective was to test whether recommendations depended on PA level and personal beliefs. Specifically, the hypotheses were (1) more physically active HCPs would recommend more PA and (2) HCPs that perceived themselves as more active would recommend more PA.The study included HCPs of the Clinique Romande de Réadaptation, a teaching rehabilitation centre in the French-speaking part of Switzerland. Patients with chronic LBP represent a large proportion of the musculoskeletal patients' population . The aim of the administrated therapeutic program (4 to 5 weeks with at least 3 to 4 h of daily therapies excluding weekends) is to manage pain and to improve function, activity, and participation, including return to work , using a multidisciplinary biopsychosocial approach according to the recommended practice for patients with chronic pain .The rehabilitation centre strongly encourages interdisciplinary work and makes efforts to implement shared goal, clear role for each HCP, mutual trust, effective communication, and measurable processes and outcomes, as advanced by Mitchell et al. . Every wThe therapeutic team is mainly composed of physicians, nurses, and physiotherapists. Occupational therapists, education and vocational guidance professionals, and psychologists are also members of the team, but they are far less numerous and were therefore excluded from this study for statistical reasons. During the rehabilitation program, the physicians are responsible for the diagnosis, the prescription of medication and treatment, the coordination of the rehabilitation program, and the follow-up of the patients. The physiotherapists assume the therapeutic training, the functional tests, the reconditioning (strengthening and aerobic exercises), stretching, and manual therapy; they are specialized in the rehabilitation of the musculoskeletal system. The nurses are responsible for the assessment of the biopsychosocial complexity, for the training for self-administrated medication, for counseling for the management of activities of daily living, and upon request for the psychological support and assistance. Moreover, the nurses conduct patients' interviews at the beginning of the rehabilitation program, according to the INTERMED tool, to detect biopsychosocial complexity –36.We invited 97 HCPs working in the centre as physicians, physiotherapists, or nurses to participate in the study. Information about the study was given during the weekly teaching team meeting of each professional group. The HCPs who were absent on the day of the meeting were excluded from the study. The participants were informed of the procedures involved in the study and gave written informed consent.We conducted a cross-sectional survey in January 2015. The study consisted of clinical vignettes and questionnaires that the HCPs have to fulfil during questionnaire sessions and objective measurements of the PA with accelerometers. The accelerometers were distributed to HCPs between November 2014 and February 2015, according to their availability. The local Research Ethics Committee granted the approval to undertake the study (CCVEM 041/14).To assess the effects of the professional discipline and the level of PA performed by the HCPs on their therapeutic advices, the HCPs were asked to evaluate three hypothetical medical cases and to give their recommendations for it.Three clinical vignettes as described by Rainville et al.  were useSix different self-administrated questionnaires were completed by the HCPs to assess some variables with a potential effect on the evaluation and recommendations of the clinical vignettes.Demographic data were collected which included age, gender, working discipline, working experience, training in LBP, and LBP history (present or previous).The Pattern of Activity Measure (POAM-P) is usually administered to assess the activity pattern in chronic pain patients . POAM-P The Tampa Scale for Kinesiophobia (TSK) was originally designed to evaluate the fear of movement and/or (re)injury in patients with LBP . The TSKThe Health Care Providers' Pain and Impairment Relationship Scale (HC-PAIRS) is a questionnaire that estimates the attitudes and beliefs of HCPs about functional expectations for chronic LBP patients . The HC-The HCPs completed the validated French version of the Hospital Anxiety and Depression Scale (HADS) , 44 to mThe Intolerance of Uncertainty Scale (IUS) is a 27-item questionnaire used to assess the belief that uncertainty is unacceptable and leads to frustration, stress, and the inability to take action . Each itWe used questionnaires and accelerometers to evaluate the PA level.To assess the HCPs' habitual PA, the Baecke habitual physical activity questionnaire  was compThe participants' objective PA level was assessed during a normal working week, from Tuesday to the following Sunday (six consecutive days). The PA was measured with a validated three-axis microelectromechanical system (MEMS) accelerometer, the ActiGraph wGT3X-BT . The ActigGraph recorded body accelerations continuously (sampling rate: 50 Hz). The sensor is a small and light box (19 g) attached on an elastic belt, and that should be worn on the left hip. Participants were instructed to wear the device during waking hours, from awakening to bedtime to measure their habitual PA. The accelerometer detected when and how much the HCPs were walking. The number of steps per day was calculated and considered as a marker of the HCP's PA .For the analysis, the days with less than 12 h of acceleration data were discarded. We also discarded rest days, according to the working schedule of each participant. The Matlab software  was used for data analysis. An algorithm specifically recognized walking through signal amplitude and frequency pattern and computed the average number of steps per day performed by the subjects . The numDifferent measures were taken to avoid or evaluate the bias that could interfere. Firstly, all the HCPs of the same professional group answered the questionnaires on the same day during the same weekly meeting to avoid contamination of the data by discussions and mutual influence. Secondly, we assessed social desirability that is the tendency of the respondents to answer questions in a manner that will be viewed favorably by others. Social desirability was used to evaluate to what extent it was possible to rely on the answers as a reflection of the real behaviour of the HCPs. The Marlowe–Crowne Social Desirability Scale (MC-SDS) measures the inclination to respond in a socially desirable way . The MC-η2 was used as an effect size measure, as it can be interpreted as the proportion of variance in the dependent variable that is attributable to the professional groups. The significance level was set at p < 0.05.Data were analysed using Stata 13.1 . For each variable, the means (M) and standard deviations (SD) were calculated for the three professional groups and for all the HCPs. Analysis of variance (ANOVA) followed by post hoc Scheffe multiple-comparison tests (for continuous data) and chi-squared tests  was performed to measure the associations between the professional groups and the different variables. In addition, sev-sympt), for the average of the three questions about severity of pathology (biassev-patho), for the average of the three questions about recommendations for daily PA (biasrec-act), and for the average of the three questions about recommendations for work (biasrec-work). ICC was calculated for the average of the six questions about severity (two for each vignette) (ICCsev) and for the average of the six questions about the recommendations (ICCrec). We did not calculate the ICC for each of the four subscales separately because the number of items was not sufficient to get statistically significant ICC values.The mean responses of the experts were considered as the guideline. For each HCP, the discrepancies between their answers and the guideline were expressed with the bias (raw difference between the answer of the HCP and the guideline) and the intraclass correlation coefficient (ICC) calculated with Shrout and Fleiss form 2.1 . Bias waFor the PA (objective and perceived), the Pearson correlations between the subscores on Baecke (perceived) and the number of steps per day (objective) and the evaluation of severity and the recommendations were also determined independently of the professional groups.Ninety-seven HCPs were invited to participate during their weekly team meetings. Seventy-two HCPs gave written informed consent, which results in an overall inclusion rate of 74%. Seven HCPs were excluded due to incomplete data. Finally, 65 HCPs were considered for the analysis: 20 physicians, 22 physiotherapists, and 23 nurses. The HC-PAIRS, which was sent afterward, was answered by 56 HCPs only .p=0.09). The physicians tend to be older than the nurses (significant Scheffé's post hoc comparison). A large majority of the physicians have a special training external to the medical centre for LBP and/or pain (85% for LBP and 90% for pain). It is less the case for physiotherapists (73% and 45%) and it is much less common for nurses (22% and 35% resp.) (significant difference between the professions (p < 0.01)).The main anthropometric and professional characteristics of the subjects are summarised in Body mass index (BMI) was not significantly different among the professional disciplines. No significant interprofessional difference was found for the number of years of working experience and the prior and current LBP experience.The group of experts answered the four questions about the three clinical vignettes. η2, η2 = 0.3).Overall, the total variance explained by professions was moderate  than the physicians and the physiotherapists. Concerning PA recommendations, nurses recommended significantly less daily PA than the physicians and the physiotherapists. Concerning work recommendations, the physicians recommended more work than the nurses and the physiotherapists.sev-symp (physicians significantly different from nurses), on biasseve-patho (physicians significantly different from physiotherapists and nurses), on biasrec-act (nurses significantly different from physicians and physiotherapists), and on biasrec-work (physicians significantly different from physiotherapists and from nurses). ICCrec tended to be different (p=0.05) across the professional groups, while ICCsev was not different (p=0.24).The answers of the HCPs were compared with the experts. The bias and the ICC are presented in The main scores of the questionnaires are summarised in There were significant differences across the three professional groups for the POAM-P (avoidance score), the TSK, the HC-PAIRS, the HADS (both anxiety and depression), and the IUS.Nurses have a greater tendency to think that they would avoid activity if they suffered from LBP. For the pacing and overdoing subscores, no significant difference was found between the three groups.p ≤ 0.01). The belief that movement and PA are harmful for LBP patients was more developed by nurses. They were significantly more afraid of movement and (re)injury for their patients than physicians and physiotherapists.The TSK score was also significantly different between the professions (p ≤ 0.01). The nurses had the most biomedical approach (65 (9)), whereas the physicians the less biomedical approach (52 (7)); the physiotherapists were in between. The HC-PAIRS scores of nurses were significantly higher than those of the physicians and the physiotherapists.The HC-PAIRS score indicated significant differences between the professions in the treatment orientation (p=0.02 for both anxiety and depression). Nurses showed the most anxious and depressive symptoms while the opposite was observed for the physiotherapists.HADS scores were significantly different between the professional groups (p=0.03). The nurses tended to be the most intolerant to uncertainty (51 (14)), significantly more than physicians. The scores of the physiotherapists were once again in between.For the IU, the mean score was 47 (12) with a significant difference between the professional groups  and with the recommendations for work and daily activity . The HCPs who considered themselves as less active at work are those who considered the severity as lower and who recommended more activity.Independently of the professional group, the work index was significantly correlated with the global evaluation of severity of symptoms and pathology (p < 0.01). The physiotherapists were the most active during working days. The physicians walked significantly less than physiotherapists and nurses.Independently of the professional group, no significant relationship was found between the number of steps per day and the evaluation and recommendations for the clinical vignettes.The mean score on MC-SDS was 19.2 ± 4.3 with no significant difference between the professional groups . The cutWe aimed at investigating the influence of the profession on the recommendations given to the LBP patients within an interdisciplinary team. Our results suggest that despite interdisciplinary team working, each profession keeps its own beliefs and attitudes. Physicians thus considered symptoms and pathology as less severe than the other professions and were more in line with guidelines. Conversely, nurses diverged substantially from guidelines. In fact, they recommended more limitation of work and PA. Physiotherapists ranged generally between the two other groups; their recommendations for PA were similar to physicians' recommendations, whereas their recommendations for work were closer to those of the nurses.We observed a strong influence of professional background on recommendations and beliefs. HCPs that were less in accordance with the guidelines and gave more restrictive PA and work recommendations  had the following characteristics: (1) they had a more biomedical approach; (2) they were more likely to behave with an “avoidance” pattern; (3) they were more kinesiophobic and (4) intolerant to uncertainty, and (5) they had more anxious and depressive symptoms.Generally, our results are consistent with previous studies. Houben and colleagues have noted an association between the TSK score, the HC-PAIRS score, and the recommendations for the clinical vignettes , 10. LikThe nurses diverged substantially from the experts . They teThe physiotherapists were often between nurses and physicians regarding attitudes and beliefs. Concerning recommendations, the relevant differences with the physicians were for the severity of pathology, and the recommendations for work, physiotherapists were more restrictive than physicians. This may be because the physicians are especially trained to diagnose pathologies and to certificate medical leave . ConversThe physicians were more in line with the experts. Thus, they evaluated the symptoms and the pathology as less severe than the other professions and recommended a higher level of activity and work. A possible explanation could be that physicians are generally well trained in pain and LBP management . In addiThese results highlight the necessity to improve the educational background of HCPs that play a key role with the patients.The hypothesis that more active HCPs would recommend more PA for their patients is not supported by the results. On the contrary, we observed that the less active HCPs  recommend more PA. Physician's worktime is mostly dedicated to patients' examination and administrative work, which is not conductive for a high PA level. Likewise, physiotherapists' engagement in physical therapies makes them the most active professional group, but they do not recommend the most PA. So it is probably an effect of the profession rather than a real association between low PA level and more activity is recommended. Furthermore, Baecke's work index was negatively correlated with the recommendations. In other words, concerning PA recommendations, professional background seems dominate over personal PA practice. Previous studies suggested a possible effect of the perceived PA on the recommendations . By usinThe originality of our study resides in the inclusion of a group of experts to establish valid answers according to the guidelines. The main advantage was that the experts gave their opinion about practical cases that can be directly compared with those of the HCPs.The assessment of both perceived and objective PA is another strength of the study; in this way, the different facets of PA that may affect the recommendations were measured.Through the MC-SDS questionnaire, we take into account social desirability. The MC-SDS scores are moderate. Therefore, the other questionnaires probably reflect the real beliefs and attitudes of the HCPs. Importantly, the MC-SDS scores were not significantly different among the three professional groups. Between-group comparisons were thus not biased.A limitation of our study is that we did not observe the actual HCP's professional practice and behaviour. We only relied on their subjective opinion facing hypothetical cases (vignettes) and situations (questionnaires).The limited sample size is also a limitation. As we want to compare all HCP working within the same team, it is rather difficult to include larger sample size.Another limitation may come from the selection of the experts. The fact that the experts were either physicians or physiotherapists may limit the interpretation of the differences observed between the nurses and the others professional groups.The use of the number of steps to assess PA is also limitation because it neglects sport activities, in particular cycling, strength training, or swimming. Additionally, it would be interesting to assess the PA during days off to have a more global view of the PA with more leisure time.Finally, our study was conducted in a teaching centre, whose characteristics might not be comparable to other settings. Further studies are necessary to verify the external validity of our findings.Our main finding is that, despite working together and sharing similar regular teaching meetings, HCPs deliver recommendations that substantially differ according to their professions. With that in mind, efforts should be made to harmonize the beliefs, attitudes, and recommendations. These efforts should be especially directed to the nurses and physiotherapists whose approach of pain and treatments is more biomedical and further from the guidelines. In our opinion, a better anchoring of the biopsychosocial model must be obtained through a focused training of the new HCPs and dedicated information to medical centres. The importance of training was highlighted by Kennedy et al. who showed that the beliefs of students in medicine and physiotherapy are closer to the guideline at the end of their studies compared to the beginning . Further


Experimental economic laboratories run many studies to test theoretical predictions with actual human behaviour, including public goods games. With this experiment, participants in a group have the option to invest money in a public account or to keep it. All the invested money is multiplied and then evenly distributed. This structure incentivizes free riding, resulting in contributions to the public goods declining over time. Face-to-face Communication (FFC) diminishes free riding and thus positively affects contribution behaviour, but the question of how has remained mostly unknown. In this paper, we investigate two communication channels, aiming to explain what promotes cooperation and discourages free riding. Firstly, the facial expressions of the group in the 3-minute FFC videos are automatically analysed to predict the group behaviour towards the end of the game. The proposed automatic facial expressions analysis approach uses a new group activity descriptor and utilises random forest classification. Secondly, the contents of FFC are investigated by categorising strategy-relevant topics and using meta-data. The results show that it is possible to predict whether the group will fully contribute to the end of the games based on facial expression data from three minutes of FFC, but deeper understanding requires a larger dataset. Facial expression analysis and content analysis found that FFC and talking until the very end had a significant, positive effect on the contributions. Cooperation is one of the major foundations of economic actions. Given its essential role, a lot of research in economics focuses on how cooperation can be improved. At the heart of this research is the evidence that communication is a highly effective tool to mediate social dilemmas . A very In this paper, we predict the end-game contribution behaviour of groups in a public goods game  after a For the independently conducted content analysis, verbal conversations were first transcribed into texts. Subsequently, the texts were classified using binary content parameters, e.g., whether a specific topic  was raisThe remainder of this paper is organised as follows. Studies have shown that a large part of affective communication takes place either nonverbally or paralinguistically through audio or visual signals ,7. BesidAutomatic analysis of social behaviour, in particular of face-to-face group conversations, is an emerging field of research in several domains such as human computer interaction, machine learning and computer vision ,22,23. TResearch has shown that group behaviour in the first few minutes (3 min) is highly correlated with the decisions made and actions taken in the future ,30,31. TSome researchers implement “human assessment” methods to predict the behaviour of subjects in laboratory experiments. In such cases, individuals attempt to guess whether other people will cooperate or not in setups similar to ours ,42. ThisThe main contribution of this paper is twofold. First, we introduce the application of computer vision and machine learning to experimental economics research in order to analyse group behaviour. Second, we introduce the economics topic of free riding in groups to the behaviour analysis community. Previous research has investigated the impact of communication on cooperation behaviour in groups . Yet, soIn the setup of a public goods game, all the group members benefit from the public good, regardless of their own contribution . Within The first step in our analysis pipeline uses OpenFace  to extraFor each participant and video, a facial activity descriptor is extracted from the corresponding facial features, as described in Section 2.2 of . In brieThe GAD is used to predict group behaviour of the FFC. After we calculate the four FADs of each group, we concatenate them in all 4! = 24 permutations  to form the GADs. This way, each group conversation is represented by 24 instances and each is given the group label. This obviously increases the dataset size, which is favourable because the dataset with 127 FFC videos is quite small. The feature space dimensionality of each GAD for FF1 is 7,296, for FF2 is 4,032, for FF3 is 3,840 and for FF4 is 4,032.The (127 × 24 = 3048) GADs are classified individually. The classifier uses leave-one-session-out (24-session) cross-validation, i.e., a subject never appears in a training and test set simultaneously. Cross-validation is a very useful technique for assessing the effectiveness of our models to mitigate overfitting. We hold out the GADs of one session for testing while the rest form the training set. We train the model on the training set and evaluate it on the test set. The cross-validation process is then repeated 24 times, with each of the sessions used exactly once as the test set. In total, 24 sessions provide 24 training sets and 24 test sets. The results of test sets are averaged to form the final performance.This section introduces the parts of a larger experiment that are relevant to this paper. The collected data and the experimental setup are detailed in ,40. The This section provides the experiments and results of the group’s contributions in the public goods games after a group conversation. We conducted several experiments in order to gain insights into the contribution behaviour classification and to improve the classification accuracy. For reference, we calculate a trivial classier which always votes for the majority class of the training set. Note that this is also done within the leave-on-session-out cross-validation, providing 24 different results (depending on the class distributions of the test sets). This informed guess allows us to have a meaningful comparison with our models.The results in We investigate the influence of the number of trees on performance and training time. We train 100, 1k, and 5k trees both on FF1 and FF4. The results in In the previous experiments, the RFc is trained on all the GADs of the training set. Each group conversation is represented by 4! = 24 samples and each is given the same label see . HoweverWe calculate the optimal threshold and estimate the final performance by using nested cross-validation. The hyper-parameter search is performed with inner cross-validation, while outer cross-validation computes an unbiased estimate of the expected accuracy of the RFc with the optimised threshold. For each fold of the outer cross validation, the inner cross-validation runs on the outer cross-validation’s training set, trains 23 RFc models and tests them with a predefined set of thresholds. The threshold that yields best accuracy (mean across the 23 inner test sets) is selected as the optimal threshold. In outer cross-validation, we apply RFc with the optimal threshold to the remaining session (1/24) of the data (test set) and calculate the accuracy. In total, we get 24 results, which are averaged to form the final performance. This double cross-validation procedure provides an unbiased estimate of the accuracy for the RFc.Applying the optimal threshold increases the accuracy of the RFc (5k trees) model (using FF4) from 67.39% to 68.11%. Therefore, we continue using RFc (5k trees) with FF4 and optimal threshold.Computing the FADs for the entire video see  might reTo gain further insights into the improvements obtained by the proposed approach, we summarise the experiments for ablation studies as listed in We analysed the content of the verbal conversation by first transcribing the spoken language into texts and subsequently classifying the texts using binary content parameters, e.g., whether a specific topic  was raisIn this paper, we proposed two approaches  to investigate what influences subjects’ contributions in a public goods game utilising a dataset of 127 groups, each group with 4 participants. We automatically analyse the facial expressions of the FFC videos in three steps. First, we extract facial features from each individual face and frame by using OpenFace  see Sec. Second,Several experiments in facial expression analysis of FFC were conducted, trying to predict the full-contribution behaviour better than uninformed and informed guessing. The results show that predicting the contribution behaviour of the group based on the facial visual cues communication is challenging; the results were better than uniformed guesses and slightly better than informed guesses . We expected this task to be particularly difficult because we are trying to predict decisions that have far more hidden influences. The initial results presented are promising and we believe that a larger dataset is necessary to predict cooperation behaviour more reliably. This hypothesis is supported by the results in We initially assumed that the beginning of the FFC video was more informative when it came to predicting human behaviour, perhaps even just the first seconds. Our findings did not support this assumption, with both the facial expression analysis and content analysis approaches indicating that the last part of the FFC video was probably more informative. However, we were not able to prove this, probably due to an issue of the dataset size.We looked at the correctly classified and wrongly classified FFC video and found little difference in the behaviour between groups at the beginning of the FFC video. However, groups that do not contribute fully show less commitment later in the FFC video. Therefore, we conclude that the last part of the FFC video can be used to predict the contribution behaviour of groups more reliably because it is easier to establish whether the group is communicating well when the introductory phase has already ended. Furthermore, participants might control their facial expressions more at the beginning of communication, while their spontaneous facial expressions become visible at the end of the communication. Thus, facial expressions and head movements provide valuable information that contributes to the classification of behaviour. In short, the current results show how it is possible to predict whether all the participants in a group will contribute fully in the last period of the public goods game. Although facial expression analysis and content analysis show that the final part of FFC videos seems to be more advantageous for predicting the contribution behaviour of groups, we have no proof that the last part of FFC videos are better than the beginning of the FFC videos due to a lack of data. It is essential to use larger datasets to obtain a deeper understanding of the problem. This would allow cooperation behaviour to be predicted more reliably and our findings to be confirmed.


The elimination of canine rabies through the implementation of high coverage mass dog vaccination campaigns is a complex task, particularly in the resource-limited countries of the rabies endemic world. Here we demonstrated the feasibility of applying targeted rabies vaccination campaigns to deliver more impactful intervention campaigns in resource-limited settings using evidence and lessons learnt from other diseases. With the use of strategic rabies intervention programs, we demonstrate the noteworthy reduction of rabies cases in two very different African settings. The strategic intervention was most significantly aided by the use of a custom-developed vaccination tracking device  Data Logger) and an integrated rabies surveillance system . Our first case study, an island-wide strategic dog vaccination on Tanzania’s Unguja island, reduced the incidence of rabies by 71% in the first 16 months of implementation. In the second case study, a similar approach was applied in the metropolitan capital city of Zimbabwe and the incidence of rabies declined by 13% during the first 13 months of implementation. The methodologies and results presented here suggest that, in resource-limited settings, an optimal approach towards the elimination of dog rabies would revolve around strategic interventions, subject to the use of appropriate planning, surveillance, and vaccination tools. Rabies lyssavirus (RABV)—the type species for the Lyssavirus genus—was developed as far back as the 1880s [Rabies is one of the oldest known zoonotic diseases . Despitehe 1880s , rabies he 1880s ,4. The che 1880s ,5,6,7. Ihe 1880s . While rhe 1880s ,10. Throhe 1880s .Given the long-term commitment required, can resource-limited countries in Africa emulate the success of those countries that have to-date eliminated dog-mediated rabies? An array of studies suggest that this would be feasible, subject to national government prioritisation of the large and continued investment towards (i) the implementation of national MDV campaigns and (ii) all of the resources associated with the multi-year operational costs that are required to ensure programmatic success ,12,13,14When considering strategies that have been implemented successfully for other diseases, one key element was paramount in all the cases that we have investigated. This commonality was simply a strategic intervention approach that relied on prioritized targeting of the at-risk population as opposed to the total population. In such a strategy, some surveillance data and knowledge of the disease epidemiology is critical to focus primarily on the areas where the disease is prevalent before progressing to populations that were considered at a lower risk. The role of such a targeted intervention approach in the eradication of rinderpest and the enhanced control of foot and mouth disease (FMD) and malaria is briefly discussed below to highlight the effectiveness of the strategic approach and use this information to make a clear case for its use in rabies elimination.Rinderpest is the second disease to be eradicated . Since tSimilar to both rinderpest and the current rabies situation, FMD was eliminated from most European countries between the 1950s and 1990 through large-scale interventions and mass vaccination campaigns . These aFor malaria, vector control remains the recommended prevention method in endemic countries. Vector control should ideally target all mosquito populations in an endemic country but this is logistically infeasible. As such, the core concept of the “Framework for Malaria Elimination” is that intervention strategies should be limited to targeted campaigns that respond to areas where malaria cases are reported, with intervention being prioritised based on the reported incidence rates ,22. ThisWhile these strategic disease intervention campaigns showcased remarkable successes compared to mass intervention approaches, they would not have been successful without the ability to rapidly detect and report the location of disease incidences and subsequently apply resources in a considered manner. Indeed, the routine reporting and geo-spatial mapping of health data using digital platforms are widely implemented throughout Africa, with some diseases  often having dedicated reporting channels within endemic countries ,24,25,26The main aim of this manuscript was to provide clear evidence towards the feasibility of eliminating dog rabies through strategic interventions based on accurate and timely surveillance. To this end, we report on the development and implementation of tools and resources that have been specifically designed to facilitate the implementation of strategic dog vaccination events in resource-constrained rabies-endemic countries. In addition, we provide evidence showcasing the feasibility of this approach in the form of two distinct case studies, viz. Tanzania’s Unguja island and Zimbabwe’s metropolitan province of Harare.The Rabies Epidemiological Bulletin (REB) is a free-to-use web-based data platform that was developed in 2016 by the Global Alliance for Rabies Control (GARC) in an effort the reduce the under- or inconsistent-reporting of rabies by endemic countries in Africa, the Middle East, and Asia ,29,30. TSince becoming operational in 2016, the benefits associated with the use of the REB has resulted in its use in more than 42 rabies-endemic countries around the world, with 17 of those countries benefitting routinely from the online platform and its functionality. While the REB was initially developed to collect, collate, analyse and disseminate national-level data—and still serves that purpose for any interested rabies-endemic country—it has also been continuously improved through the development of various add-on “components” that have been designed to collect sub-national data at any administrative level. These components allow users to improve the resolution of their surveillance data by providing information at a more localised-level while also automatically aggregating the data up the administrative hierarchy—easing data reporting and improving data quality at all administrative levels . Two of The RCS component of the REB facilitates the development of interactive spatio-temporal maps that pinpoint the locations of both laboratory-diagnosed and suspect-rabies cases at a community-level by enabling the end-user to input the animal species, the diagnostic outcome, and the sample number for each diagnosed specimen. Once the metadata has been captured, each case is plotted by the system, in near real-time, on a map using either manually captured global positioning satellite (GPS) coordinates or an interactive map that can be used to identify the location within the community if no precise GPS coordinates are available. The RCS component enables end-users to upload surveillance data for any administrative level in the country after which the data is not only aggregated to the national level but also becomes usable data in the form of maps, graphs, and tables that enable decision-makers to identify rabies hotspots and high-risk areas where intervention campaigns can be implemented. The maps and graphs are all interactive, with users being able to select specific cases to reveal the exact information pertaining to that sample , eliminating the need to find the sample in the database. In addition to the maps, the data entered using the RCS component also results in a cascade of notifications being sent to stakeholders informing them of the newly captured case so that action can be taken, ensuring timely responses to outbreaks and previously unidentified cycles. As eluded earlier, this surveillance information—and the subsequent identification of hotspots—was instrumental in driving successful intervention campaigns for the various diseases that have been either eradicated or successfully controlled.Like the RCS component, the RVT component helps track mass vaccination data using maps and visuals as outputs. The RVT component enables end-users to either automatically import or manually enter the information for each vaccinated animal . Similar to the RCS component, an interactive map can also be used to obtain broad GPS coordinates if no grass-roots level data was captured with regards to the location of each animal. In addition to the maps, the data entered using the RVT component can also be used to populate graphs and other visuals that can be used to increase the government’s knowledge of its dog population and its ecology. System calculations are available to determine current vaccination coverage in terms of the estimated dog population for each area (based on the human to dog ratio), the sex and age ratios of vaccinated animals and a variety of other calculations important for actionable decisions.In summary, both the RVT and RCS component serve to provide a valuable commodity to governments by overcoming the need for timely analysis and the costly GIS experts/software required to adequately analyse the collected data . In addiIn most rabies-endemic countries, rabies vaccine distribution is monitored by comparing the number of doses of vaccine given to each vaccinator at the start of the day to the number of unused vaccines returned at the end of the day. While this approach provides a basic indication of vaccinated animals it does not provide an indication of where the vaccinated animals reside within the community nor does it allow stakeholders to effectively distribute limited resources to areas where it is needed most. To overcome these limitations and improve the resolution of the information collected during vaccination campaigns, methodologies aimed at collecting and monitoring grass-roots level data during vaccination events have recently become more prominent. Of these methodologies, mobile phone applications have been the most widely used and tested to-date ,36,37. WThe GARC Data Logger (GDL) is a labile, hand-held data collection device that was custom-developed to facilitate the rapid collection and storage of dog vaccination data. To limit the wear and tear of the device, the GDL was developed using a hard-wearing plastic casing with touch-activated buttons and indicator light-emitting diodes (LEDs) . The lacBeing independent of mobile networks, the costs associated with the GDL only pertain to the initial purchase cost (comparable with affordable smartphones), with no additional running costs needed (except electricity for charging the device between use). In addition, as the GDL is sturdy and durable—evidenced by the fact that some devices have been operational for more than 16 months without breakages or a need for replacement—no additional costs relating to repairs or maintenance are required.When designing the GDL, it was not only the device’s external features that were designed to be as simple as possible but also the data capturing process. Indeed, for each vaccinated animal, the vaccinator would answer three basic questions pertaining to the animal by touching the corresponding picture/touch-activated button, viz. (1) dog or cat, (2) male or female, and (3) adult or juvenile A. Once aTM Windows operating system but also enables the end-user to add additional campaign details to the collected data prior to downloading it. The supplementary data that can be added include the vaccine brand that was used, the vaccine’s lot number, and the specific vaccination site name, amongst others  format. By first downloading the data to a computer, the end-user decides what happens to the data in terms of data sharing and distribution. Some end-users have access to skilled GIS professionals readily available and can thus have the data analysed and subjected to spatio-temporal plotting using independent software programs. Most often though, the stakeholders running dog vaccination campaigns do not have those resources readily available and would thus not be able to analyse their data in a timely manner. In such instances, the end-user can choose to add their data onto the REB where the GDL data is collated and analysed by the RVT component before maps and visuals are automatically generated by the system. Indeed, the data exported in the CSV format from the GDL is in the exact format required to ensure that the data can be automatically uploaded onto the REB, allowing the map and other visuals to be updated in near real-time without the need for skilled GIS experts or the additional costs associated with data analysts.Downloading the data from the GDL results in the information being stored directly on the end user’s computer in a MicrosoftThe Zanzibar Archipelago, an extensive group of islands off of the eastern coast of Africa, is a semi-autonomous region of the United Republic of Tanzania, under the authority of the Revolutionary Government of Zanzibar. Based on anecdotal evidence, the Zanzibar Archipelago remained free from dog-mediated rabies until the 1990s, when the disease was detected on two of the biggest islands: Unguja island and Pemba island ,41. Of tIn 2016, GARC facilitated the establishment of a laboratory-based rabies surveillance system on Unguja island. To this end, animal health technicians were trained on the safe collection of brain stem and mixed brain samples , while lIn response to the detection of rabies cases across the island, GARC and the Department of Livestock Development started planning a vaccination programme that could be implemented with limited resources. In contrast to the “Rabies Control and Dog Management Project” that aimed to achieve sufficient vaccination coverage of the entire dog population , a strategic vaccination approach was considered. Through visually impactful outputs, the REB-generated spatio-temporal maps plotting rabies surveillance data-enabled hotspot areas to be identified. The impact of this methodology was two-fold: (1) It ensured that the vaccinators were directed to the high-risk areas; and (2) it resulted in the reduction of the amount of vaccine required as a 70% coverage was required in only the hotspot areas, equating to a smaller estimated dog population requiring vaccination .Between August 2017 and December 2018, the relatively small vaccination team consisting of 13 members, vaccinated 6174 dogs and 457 cats at selected fixed-point vaccination sites across the island with each vaccination being recorded using a GDL device . While tWhile rabies cases continued to be sporadically detected by the end of December 2018 , the numBased on the information provided here, the Department of Livestock Development of Unguja island is on track to control and eliminate dog-mediated rabies through the implementation of a second round of strategic dog vaccination in 2019/2020. If achieved, this would make Unguja island the first African region to comply with the OIE guidelines  that recZimbabwe is a land-locked country in southern Africa that has been endemic for dog-mediated rabies since the 1950s ,48,49. DDue to the limited resources and person-power preventing the vaccination of the city’s entire dog population  approach ), GARC aBetween February 2018 and March 2019, the relatively small vaccination team consisting of 10 members, vaccinated 11,699 dogs and 85 cats . Of the Despite only achieving an estimated 5% vaccination coverage of the city’s entire dog population, a marked drop in the percentage rabies-positive samples had been observed by the end of 2018 (from 46% in 2017 to 33% in 2018). In addition to the reduced rabies positivity, we calculated the odds ratio for rabies positivity between the two periods (OR: 0.637) and used logistic regression to determine that there was a 36% (95% CI: 21–48%) lower likelihood of detecting a rabies-positive sample at the end of the vaccination campaign in 2018. Furthermore, we speculate that the benefit of the strategic vaccination campaign could have been far greater had it not been due to political disturbances  and diseWith the target date for eliminating dog-mediated human rabies by 2030 fast approaching ,56, timeAs reported in this manuscript, the ability to implement strategic dog vaccination is firmly rooted in the ability to report rabies cases from the community-level with these data not only being geographically defined but also being disseminated to the responsible authorities so that disease intervention campaigns can be initiated in a timely manner. While the use of independent software programmes and manual notification channels could, in theory, provide governments with this much-needed information, the freely available RCS component of the REB has been demonstrated to provide all of the functions in near real-time while not requiring capital investments, extensive training or dedicated subject matter experts. These benefits were demonstrated in the two different case studies described here, each demonstrating how the RCS component of the REB was used to identify suspected or confirmed rabid animals before notifying the responsible authorities that the area should be earmarked for strategic vaccination. In addition to geographically defining suspect or confirmed rabid animals, strategic dog vaccination also relies on the ability to track each vaccination event  by also recording metadata that is beneficial to understanding the ecology of the dog population in question.While various methodologies have been developed to collect information during vaccination events using either paper-based forms or mobile phone applications, we specifically demonstrated the use of the custom-developed GDL, while also demonstrating how the interoperability with the freely available RVT component of the REB enabled the data to be uploaded and visualised in near real-time. This approach not only ensured that the governments were in control of their own data, but also allowed them to visualise the work done by the individual vaccinators, direct vaccinators to additional areas that were still at-risk, and closely monitor vaccine usage. In this way, the rabies programmes could be strategically overseen and controlled by a relatively small working group of professionals.While the methodology and findings presented in this manuscript have shown the value of strategic dog vaccination in resource-limited settings, all of these areas would need to persist with this approach until elimination is achieved. The significant successes demonstrated through local-area campaigns should provide strong grounds for continued government support and commitment to rabies elimination. Such commitment will be essential for any nation to achieve zero dog-mediated human rabies cases by 2030.


Imagery of facial expressions in Autism Spectrum Disorder (ASD) is likely impaired but has been very difficult to capture at a neurophysiological level. We developed an approach that allowed to directly link observation of emotional expressions and imagery in ASD, and to derive biomarkers that are able to classify abnormal imagery in ASD. To provide a handle between perception and action imagery cycles it is important to use visual stimuli exploring the dynamical nature of emotion representation. We conducted a case-control study providing a link between both visualization and mental imagery of dynamic facial expressions and investigated source responses to pure face-expression contrasts. We were able to replicate the same highly group discriminative neural signatures during action observation  and imagery, in the precuneus. Larger activation in regions involved in imagery for the ASD group suggests that this effect is compensatory. We conducted a machine learning procedure to automatically identify these group differences, based on the EEG activity during mental imagery of facial expressions. We compared two classifiers and achieved an accuracy of 81% using 15 features (both linear and non-linear) of the signal from theta, high-beta and gamma bands extracted from right-parietal locations (matching the precuneus region), further confirming the findings regarding standard statistical analysis. This robust classification of signals resulting from imagery of dynamical expressions in ASD is surprising because it far and significantly exceeds the good classification already achieved with observation of neutral face expressions (74%). This novel neural correlate of emotional imagery in autism could potentially serve as a clinical interventional target for studies designed to improve facial expression recognition, or at least as an intervention biomarker. Faces represent a critical source of visual information for social perception, conveying relevant information about identity and emotional states of others . Since tASD is a neurodevelopmental disorder characterized by deficits in the social domain which represent hallmark early characteristics . Even foImportantly, no previous study has considered the role of MI in the FE processing domain, possibly because of the challenges in identifying imagery signatures that mimic neural responses during simple observation. The perceptual strength and spatial frequency of the FE stimuli seem to be relevant to yield ASD group differences during simple visual presentation , but theAnother limiting aspect is the notion that specific processing experimental contrasts are needed to isolate effects of interest. For example, the use of blank screen baselines, before the presentation of faces, generates a non-specific contrast of face with expression against a baseline without any stimulus. Therefore, those responses comprise both the processing of low-level core aspects of the face and the specific processing of the FE. In this EEG study we used dynamic FE morphing in a virtual avatar and used its neutral expression as baseline, to ensure a FE specific contrast. This way, the neutral FE is already present in the baseline. We believe this stringent contrast provides a response specific to the processing of the FE aspects, isolating it from the simple response to the face static itself. A systematic review of EEG studies regarding FE processing in ASD conducted by MI is defined as the simulation or re-creation of perceptual experience . Most ofHere, by providing a critical link between visual observation and subsequent replay imagery, we bound MI to the FE of an avatar, in a task where the participant mentally replays the previously observed dynamic image of the avatar performing a happy or a sad FE. We believe this link between visual observation and MI of FE in others addresses both the deficits of FE processing, emotion identification and theory of mind, due to the lack of thinking from the perspective of the other present in ASD. Therefore, the concept of visually imagining others smiling recruits the faculties of expression processing and pretend play, and our experimental design allowed to study such imagery process in ASD, and to use two distinct classification approaches, based on linear and non-linear features describing brain signals, to differentiate between the disease state and normal cognition. Non-linear features consist of quantitative measures that represent in a relatively simple way complex dynamic characteristics of the EEG signals, which the traditional linear methods  are not able to capture. They have been adopted more and more frequently in EEG analysis in general and ASD biomarker research in particular , 2017.Seventeen male teenagers with the diagnosis of idiopathic ASD were recruited from the Unit of Neurodevelopment and Autism from the Pediatrics Unit from the University Hospital of Coimbra and from Portuguese ASD patient associations (Coimbra and Viseu). Since ASD is a disorder far more prevalent in male individuals, with a ratio of four males to every female, and there is accumulated evidence for sex differences in brain connectivity , only maWritten informed consent was obtained from the parents of the participants or, when appropriate, the participants themselves. The study was approved by the ethics committee from Faculty of Medicine from the University of Coimbra and was conducted in accordance with the declaration of Helsinki.WorldViz Vizard 5 VR Toolkit (development edition) using the male002 virtual avatar from the Complete Characters HD pack and its FE poses. The total duration of the experiment is about 50 min, including 15 min for scalp cleaning and placement of the EEG cap, 30 min for the experimental tasks and 5 min to clean up at the end of the session.The experiment is divided in two tasks: one of visual stimulation and one of MI requiring “mental replay” of previously observed FE, with the goal to identify similar neural signatures. The visual stimulation task and overall experiment were developed in This task consists in observing a virtual avatar performing either sad or happy FEs , for a total of 240 trials. The participants were asked to fixate the face of the avatar in the middle of the eyes and observe the expressions. A rest period was included between blocks to ensure focus and reduce fatigue throughout the experiment. A total of 120 trials per condition were recorded.The second part of the experiment consists of a MI paradigm. In this task, the participant is asked to mentally imagine the avatar performing the same types of FEs used in the stimulation part . The computer screen shows the neutral face of the avatar during the whole period, except for the instruction, when it performs the FE the participant is asked to imagine. Then, after a cue, the participant imagines the avatar performing the FE, in a period of 4 s, returning to no imagery after that period. The (c) section of Figure The experiment was conducted in a 22-inch LCD Monitor . The participants sat about 60 cm away from the screen (distance measured from the eyes to the center of the screen) and were asked to keep their eyes open and fixed on the face of the avatar. EEG data were recorded using a 64 channel actiCHamp system from Brain Products.The scalp of the participants was first cleaned using abrasive gel and then the 64 channel actiCAP cap was placed on their head. Data were recorded from 64 Ag/AgCl active electrodes (Brain Products), placed across the head according to the international 10–10 standard system. The ground electrode was placed at AFz position and the reference electrode at the right ear. The impedance of the electrodes was kept under 10 kΩ during the recordings. The electrodes were connected directly to the Brain Products actiCHamp amplifier and sampled at 1000 Hz. EEG data were recorded using the Brain Products Recorder software. For each paradigm, the individuals were informed about the respective task. The total duration of the experimental procedure (preparation + 2 tasks) was around 50 min.infomax algorithm  and go to 900 ms after the start of the expression morphing . ERPs were computed by subtracting each epoch by the mean of its baseline (from 100 ms pre-stimulus to 0) and then averaging all epochs corresponding to the same stimulus condition.Source analysis were conducted using the sLORETA toolbox . The proFor each expression and each group, we identified the peaks of the first and second ERP component for each electrode, and extracted the latencies for both peaks across the scalp. We performed the source localization of the mean activity of around those two ERP components .Similarly to the visual stimulation ERPs, for the source analysis we looked for the mean global field power in the window of 0–250 ms. The pipeline was analogous to the VEP, as well as the statistical framework.For the MI periods, we investigated frequency bands of the signal during the time window of 500–3500 ms, avoiding the contribution of the beep ERP and covering the main period of MI, because MI processes are best captured using time-frequency analysis . The freFor both ERP and frequency analysis we conducted voxel-by-voxel between-group comparisons of the current density distribution for each expression, in a way analogous to the VEP procedure.To explore the MI processes through the EEG data, we defined several features from the time, frequency, and non-linear domain. We then performed a ranking analysis and selected the best features to train a classifier to discriminate participants between groups. Features were extracted for each channel and trial by trial and averaged across all imagery trials and electrode clusters.We follow the procedure of For the time and frequency domain, we selected the signal envelope (env), Teager energy operator (teag) and instantaneous power (pow) as features. A detailed description of these features is present in Supplementary Table i), i=1, …,NXτ is the number of points (states) in the phase space.where τ is time delay, We reconstructed a 2 and 3-dimensional phase-space associated to the EEG data, and the time delay was considered to be the mean of the first local minimum from the signal’s autocorrelation (hereafter defined as lag).From the non-linear domain we extracted the spatial filling index (SFI), largest Lyapunov exponent (Lyap), correlation dimension (CorrDim), approximate entropy (ApEn) and sample entropy (SpEn) as features. We provide a detailed description of these features in the Supplementary Table The features were extracted from 3 time windows in each trial: baseline [-500 ms to 0 ms pre instruction], emotion imagery [500–3500 ms after imagery trigger] and neutral [500–3500 ms after neutral trigger]. For the emotion and neutral time windows, we used the absolute value for the non-linear features and the normalized values (subtracted by the same feature extracted from the baseline) for the time/frequency domain.All features were extracted from signals filtered at different frequency bands. Band-pass Infinite Impulse Response (IIR) filters were used as implemented in EEGLAB toolbox, for the frequency bands: θ [4–8] Hz, α [8–12] Hz, low β [12–21] Hz, high β [21–30] Hz and γ [30–40] Hz.a priori information provided by the source localization and selected only the clusters closer to the right precuneus region, namely C1, C2, C4, and C5.In order to reduce the dimensionality of the feature set, we averaged the features extracted from each electrode in spatial clusters, as defined in Supplementary Figure t-tests, using only the samples from the training set, and the features were ordered by absolute T value, from the most important to the least.We ended up with 8 different features × 5 frequency bands × 4 clusters, for a total of 160 different features. We then computed the statistical discriminative value of each feature between groups with two sample We trained a SVM with a linear kernel, for being one of the most used classifiers applied to EEG signals  and alsoWe trained the classifiers to discriminate the group of the participant, based on the feature vector extracted from his EEG data. We divided the participants into train and test sets: 80% of the cases were randomly chosen for training and the remaining 20% for testing. We repeated the procedure more than 30 times, to avoid overfitting, following the guidelines provided by To explore the relation between accuracy and the number of features used, the procedure was conducted starting with 5 features and adding 5 more features up to the total of features.We repeated the full classification procedure using the EEG signal from the neutral part of the MI task, in order to check if the results were specifically improved during over emotion expression imagery.This section presents the results of the analysis performed on the ERP responses to the visual stimulation task (observation of happy and sad FEs), which was used to identify neural signatures relevant to validate the imagery task.The ERPs obtained from the visual stimulation task present two clear independent components, the first one peaking around 300 ms and the second around 600 ms Figure . Since tFor the source analysis of the visual stimulation task ERPs we defined time-windows of 250 ms around the two component peaks of activity in the ERPs. We show the results for the first and the second ERP component, separately. The mean peak latencies used for each expression and each group is detailed in Supplementary Table The mean current source density of activity in the intervals around the component peaks showed group differences for both expressions in the first component, using voxel-by-voxel independent tests between groups, corrected for multiple comparisons at the 5% level using the SnPM method (two-tailed). Both expressions show the group differences right-lateralized and located at the superior parietal region, in the precuneus area Figure . As for This section presents the results for the MI task. We analyzed the ERP for the initial imagery period and the longer MI blocks through source analysis of the power spectrum and the analysis of several characteristics of the signal using machine learning techniques.p < 0.01, using voxel-by-voxel independent tests between groups, corrected for multiple comparisons using the SnPM method. Importantly, the same region identified group differences for both expressions. This was also the same region that was identified during visual stimulation. Accordingly, the ASD group presented higher activation in the superior parietal region , we conducted a source analysis of the defined frequency bands of the signal. A statistical significant result was found for in the imagery of sad expressions, for the theta band Figure . The ASDWe then tested whether the identified neural signatures of imagery of FEs could be identified in a data driven manner using statistical classifiers. The linear SVM and the WiSARD classifier were able to achieve high test set accuracies , with the WiSARD yielding the best accuracy of 81% with just 15 features , and no feature was significantly correlated with any of the covariates.We then focused on the top 15 features that generated the 81% of accuracy. We investigated the most selected frequency bands and clusters of these top features. Figure Here we addressed for the first time FE imagery in ASD and identified a common neural correlate of observation and MI of dynamic FEs in this condition, in the precuneus. Robust statistical classification of brain activity patterns using linear and non-linear features could also be achieved, and the identified biomarker of abnormal imagery in ASD can potentially be used as an outcome measure to evaluate clinical interventions addressing cognitive and behavioral improvement in this condition.We focused on MI of FEs in ASD as a major research target in this study. This is a very important cognitive process in the context of this disease, because mental rehearsal is very important for action perception cycles, in particular in the context emotional face recognition. MI is the process of creating a mental representation and corresponding sensory experience of an episode or stimulus without a direct external source . In the A critical aspect that renders the study of imagery difficult in ASD is that it is important to ascertain that imagery really reflects the expected visual content. We could achieve this by showing that similar neural signatures  can be found by both observation and imagery of FEs. The ERP elicited by the imagery cue did indeed reveal that source differences were very similar as compared to the ERP of the FE stimuli, with the precuneus showing higher activation for the ASD group. The right precuneus belongs to task-active networks  that areOne of the common aspects of visualization and MI of the others FEs is the need to incorporate the perspective of the other. Because we use a stringent contrast in the visual stimulation task, we expected the core processing of the face to have less weight than the perspective taking aspects task. The precuneus is one of the core regions present in the perspective taking network, as showed by The link between the precuneus and its role in FEs processing has already been demonstrated by some studies , but ourFrequency band decomposition of the MI signals showed that theta and high-beta/gamma bands explained the main group differences. The source analysis of the theta band further revealed again a higher activation of the right precuneus for the ASD group . It was already known that FEs elicited higher theta responses than neutral expressions in healthy participants  of ASD, based on the MI process. The need for diagnostic, prognostic and intervention biomarkers in ASD is well recognized. While ASD biomarkers range from genetics to clinical .We then performed a further analysis of the top 15 features selected for classification. The most representative frequency band, when using non-linear features, was the theta band, while the most discriminative features were from the time/frequency domain and high-beta/gamma frequency bands. Those bands and their relation with the precuneus have been explored in the literature by The overall use of dynamic FE morphing enabled a more realistic and ecologic approach, because the stimuli featured more realistically the daily life characteristics of social interactions than the commonly used static stimuli. Moreover, we used a specific face expression contrast . As stated by Our approach to morph the expression into a virtual avatar makes a potential bridge between dynamic FEs and rehabilitation possibilities using, for instance, virtual reality. Understanding how the FEs are processed in virtual environments opens the door for intervention solutions, where the environment is completely controlled . This isA common characteristic of most studies in the literature using EEG and observation of FEs is the use of a blank-screen as baseline for the visual stimulus , thus elOur study focused only on male subjects to avoid an effect of gender in the analysis. There is evidence for sex differences in brain connectivity in ASD which might influence the EEG analysis we conducted . The repIn conclusion, we found for the first time, a neural correlate of emotion expression imagery in ASD, which was validated as a replication of the neural signatures evoked by visual observation of specific FEs. We developed an innovative approach to study FE processing in ASD, combining visualization of dynamic FEs  and MI of FEs in others. Our results emphasize the important role of the precuneus in the ASD facial processing circuit and suggest that its increased recruitment may serve as a compensatory strategy to overcome the natural deficits in their emotional processing. Furthermore, we extracted a set of features and trained a classifier that was able to discriminate between groups with high accuracy. The features were then observed to match topographically and spectrally the group effects, and can therefore be potentially used as intervention targets.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.This study was carried out in accordance with the recommendations and Guidelines of Comissão de Ética da Universidade de Coimbra, with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Comissão de Ética da Universidade de Coimbra.MS, RM, JA, SM, GO, PC, and MC-B conceived and designed the study. MS, RM, and JA performed the experiments with the participants. SM performed the screening and psychological evaluation. MS, RM, and JA analyzed the data. MS, RM, JA, PC, FF, and MC-B discussed the results and iterative approaches. MS, RM, and MC-B wrote the paper. All authors read, contributed and approved the final manuscript.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.