Individuals with heightened anxiety vulnerability tend to preferentially attend to emotionally negative information, with evidence suggesting that this attentional bias makes a causal contribution to anxiety vulnerability. Recent years have seen an increase in the use of attentional bias modification (ABM) procedures to modify patterns of attentional bias; however, often this change in bias is not successfully achieved.This study presents a novel ABM procedure, Emotion-in-Motion, requiring individuals to engage in patterns of attentional scanning and tracking within a gamified, complex, and dynamic environment. We aimed to examine the capacity of this novel procedure, as compared with the traditional probe-based ABM procedure, to produce a change in attentional bias and result in a change in anxiety vulnerability.We administered either an attend-positive or attend-negative version of our novel ABM task or the conventional probe-based ABM task to undergraduate students (N=110). Subsequently, participants underwent an anagram stressor task, with state anxiety assessed before and following this stressor.P=.003, Cohen d=0.87) and led to a greater increase in stressor-induced state anxiety faces in the attend-negative training condition than in the attend-positive training condition .Although the conventional ABM task failed to induce differential patterns of attentional bias or affect anxiety vulnerability, the Emotion-in-Motion training did induce a greater attentional bias to negative faces in the attend-negative training condition than in the attend-positive training condition (Our novel, gamified Emotion-in-Motion ABM task appears more effective in modifying patterns of attentional bias and anxiety vulnerability. Candidate mechanisms contributing to these findings are discussed, including the increased stimulus complexity, dynamic nature of the stimulus presentation, and enriched performance feedback. We operate in a complex and dynamic world in which we are continuously confronted with an ever-changing stream of perceptual information. The limited capacity of our cognitive system means we can only attend to certain information, while other information is filtered out. Such filtering does not operate in the same manner across all individuals; however, it has become clear that there is a relationship between such attentional selectivity and individual differences in emotional vulnerability . SpecifiThe observation that ABM tasks delivered in the laboratory can exert a beneficial impact on anxiety responses to stressor has led a number of researchers to investigate whether extended exposure to such ABM training can reduce anxiety dysfunction in real-world settings. The most frequently used ABM procedure is based on the dot-probe task . In thisAlthough such encouraging findings highlight the potential clinical benefits of ABM procedures, it is important to recognize that in a number of ABM studies, the intended attentional training procedure has failed to affect emotional vulnerability -16. OverIn recent years, some investigators sought to adapt existing ABM procedures to make them more engaging by gamifying them. Gamification refers to the use of game design elements in nongame contexts . SeveralExisting gamified training protocols have been variants of the original probe approach; however, all these protocols share in common the limitation that they seek to train attentional selectivity using very simple static displays that typically present only 1 or 2 stationary emotional stimuli ,32-36. Tmotivating feedback or goal metrics, in the form of real-time visual performance feedback or points [elaborate displays or a game-shell to increase intrinsic motivation [goals-directed learning, which directs players to particular goals to increase targeted skills  [These studies did, however, incorporate different gamification and other (nongame) elements to enhance engagement with the tasks to improve their effectiveness in modifying attentional bias ,38. Somer points ,35 or blr points . Others tivation ,35,39-43rmation) . Howeverelaborate display gamification element, as it presents a large number of stimuli that dynamically move across the whole screen. The task also incorporated the gamification elements of motivating feedback through a game-by-game high score that participants were encouraged to try to beat as well as the element of goals-directed learning, as participants were explicitly instructed to track 1 particular emotional expression. The task was built upon the same principles as the original probe-based ABM task, in which the training condition was designed to increase attentional bias toward positive information, and the task performance would be improved to the extent that participants adopted a more positive attentional bias [The objective of this study was to develop and evaluate a novel candidate ABM procedure designed to modify attentional selectivity within a task setting that, like most real-world contexts, requires participants to selectively distribute attention while processing a complex and dynamic emotional environment. The task required participants to search for and track 1 particular target stimulus presented on screen among multiple moving distractors, based on its emotional valence. This screen display thus realizes the nal bias . SimilarOur primary aim was to evaluate the capacity of this new candidate ABM procedure, which we have labeled the Emotion-in-Motion task, to induce a group difference in selective attentional responding to negatively and positively valenced information and to causally impact anxiety vulnerability, as evidenced by the strength of state anxiety responses to a controlled laboratory stressor. We also delivered the conventional probe-based ABM procedure to a separate cohort of similar participants under equivalent laboratory conditions. This conventional probe-based ABM task does not include any of the gamification elements introduced in the Emotion-in-Motion task. Specifically, there is no elaborate display (only 2 static images are presented), no motivating feedback after each block , and no goals-directed learning (participants are simply instructed to discriminate the identity of a probe). We chose to compare our novel Emotion-in-Motion task with the probe-based ABM task, as this is the procedure most commonly used in studies aiming to modify patterns of attentional bias ,18,21.This study design will enable us to determine (1) whether both the conventional probe-based ABM task and this new, complex, dynamic Emotion-in-Motion ABM task produce a group difference in attentional bias in line with the allocated attentional training condition, (2) if both tasks prove capable of so modifying attentional bias, whether the Emotion-in-Motion ABM task impacts attentional bias to an equal or greater degree than does the conventional probe-based ABM task, (3) whether both the conventional probe-based ABM task and this new, complex, dynamic Emotion-in-Motion ABM task serve to induce a group difference in anxiety vulnerability as a function of allocated training condition, and (4) if both tasks prove capable of so influencing anxiety vulnerability, whether the Emotion-in-Motion ABM task impacts anxiety vulnerability to an equal or greater degree than does the conventional probe-based ABM task.A total of 129 undergraduate students at the University of Western Australia completed the study. In line with previous research, participants who did not show an elevation in state anxiety in response to the intended stressor were excluded before analyses . This leP>.05).Overall, 55 participants completed the conventional probe-based bias training and assessment tasks. These participants were randomly assigned to either an attend-positive or attend-negative training condition. Participants assigned to these 2 conditions of the probe-based tasks did not differ significantly in age, trait anxiety scores, or gender . These 2 groups did differ significantly in gender ratio, P=.03, with a higher proportion of males in the attend-negative condition than in the attend-positive condition. Consequently, we considered gender ratio as a covariate in our analyses of the data, which provided reassurance that observed effects of this experimental manipulation remained evident when this group difference in gender ratio was accounted for.Overall, 55 participants completed our novel Emotion-in-Motion bias training and assessment tasks. Participants were randomly assigned to either an attend-positive or attend-negative training condition. Participants in these 2 conditions of the Emotion-in-Motion tasks did not differ significantly in age or trait anxiety scores  even when the face presented within changed, as long as the emotional expression of the face presented remained the same . At random intervals, the emotional expression of a target face would change in addition to its identity, at which point this ceased to be the target rectangle. At that same moment, 1 of the other rectangles would assume a face depicting this emotion, and thus identifying it as the (new) target rectangle. At these points, participants had to quickly find the new target rectangle and start tracking it.The Emotion-in-Motion ABM task consisted of 8 3.5 min blocks or behind the rectangle (to not obscure the face presented within) and remained hidden as long as the participant kept it on target.At the start of a block, each face remained constant for the first 2000 milliseconds. Thereafter, individual faces within a rectangle switched to a different identity (but same expression) randomly at any point between 1 and 2000 milliseconds throughout the block. Within each block, the target rectangle switched  60 times, at random intervals of 5 to 10 seconds. All 8 rectangles moved with different randomly determined trajectories, at a randomly determined speed of between 30 and 50 pixels per 100 milliseconds. Thus, although the rectangles moved at different speeds, each rectangle’s speed was constant within a game. The rectangles bounced off the screen edges and other stimuli they contacted at an angle of reflection that matched their angle of incidence. The target rectangle was never indicated; however, when the mouse cursor was correctly located in the position of the current target rectangle, this cursor disappeared The onset of each block was preceded by a 3-second countdown presented in the center of the screen. At the end of each block, participants were presented with a tracking score , a switching score , and a total score for that block (generated by combining the tracking score and the switching score). The screen also displayed the participant’s highest prior  score. Participants were instructed that they would play several games of this task and were encouraged to beat their current high score in each successive game.The training contingency was removed from the Emotion-in-Motion training task to create the assessment task used to reveal the impact of this training on attentional selectivity. Thus, participants were required to track a rectangle displaying a face with a happy expression (among 7 rectangles displaying faces with angry expressions) on half of the blocks and to track a rectangle displaying a face with an angry expression (among 7 rectangles displaying faces with happy expressions) on the other half of the blocks. This assessment task delivered 12 short blocks, each of which contained 5 target switches, resulting in a total of 60 target switches across the assessment task. In 6 of these blocks, the target rectangle displayed a face with an angry emotional expression, and in 6 blocks, the target rectangle displayed a face with a happy emotional expression. The order of these block conditions was randomly determined, with the constraint that a maximum of 2 consecutive blocks could have a target with the same valence. Each block started with a 5-second countdown.To obtain a measure of attentional bias to negative information, an attentional bias index (ABI) was computed by subtracting the average tracking score a participant obtained in blocks where targets were happy faces from the average tracking score the participant obtained in blocks where targets were angry faces. Therefore, a higher positive score on this index reflects greater attention to negative information, as it represents more successful tracking of angry than of happy faces.The Trait Anxiety Assessment, conventional probe-based ABM, and assessment tasks as well as the anxiety reactivity assessment task are described in Participants were tested individually in a sound-attenuated room. Once informed consent from participants had been obtained, participants were instructed to sit at a comfortable viewing distance from the computer screen (approximately 60 cm), were given instructions, and completed the first assessment task. After completion of the training task, they completed the original assessment task again. Next, participants completed the anxiety reactivity assessment task containing an anagram stressor task preceded and followed by a measure of state anxiety. At the end of the session, participants were debriefed about the purpose of the study. The entire experimental session lasted about 1 hour. This study was approved by the University of Western Australia’s Human Research Ethics Committee, protocol RA415243.The criteria to identify outliers are described in Application of the outlier criteria led to the exclusion of 4 participants (2 in the attend-positive training condition). To examine whether the conventional probe-based training task was capable of modifying attentional bias, a mixed-methods analysis of variance (ANOVA) was performed with the within-subjects factor attentional assessment point  and the between-subjects factor training condition . The ABI scores obtained by participants who completed this conventional probe-based training task served as the dependent variable.F1,48<1, nor of training condition, F1,48=2.246, P=.14. Most importantly, the critical interaction between attentional assessment point and training condition fell short of significance, F1,48=3.018, P=.09, η2p=.059.Results showed neither a significant main effect of attentional assessment point, F1,50=4.602, P=.04, η2p=.084, subsumed within a higher-order interaction of attentional assessment point x training condition, F1,50=5.629, P=.02, η2p=.101. At pretraining, there was no significant difference between the ABI scores obtained by participants in the attend-positive training condition and participants in the attend-negative training condition, F1,50<1. In contrast, at posttraining, participants in the attend-negative training condition showed significantly higher ABI scores as compared with participants in the attend-positive condition, F1,50=9.903, P=.003, Cohen d=0.87. Although the change in attentional bias from pre- to posttraining fell short of significant for participants in the attend-negative training condition, t25=−1.162, P=.26, Cohen d=0.229, there was a significant change from pre- to posttraining for participants in the attend-positive training condition, t25=2.114, P=.045, Cohen d=0.415. Overall, this pattern of results confirms that the 2 training conditions exerted a differential impact on attentional bias to negative information, and the direction of the observed attentional training effects was as expected. When controlling for the gender, by adding this as a covariate, this interaction between attentional assessment point and training condition remained significant, F1,43=4.393, P=.04, η2p=.087.Application of the outlier criteria led to the exclusion of 3 participants (1 in the attend-positive training condition). To determine whether our novel Emotion-in-Motion attentional training procedure was effective in modifying attentional responding to negative information, the ABI scores obtained by participants who completed this task were subjected to a mixed-design 2x2 ANOVA that again considered the within-group factor attentional assessment point (pretraining assessment vs posttraining assessment) and the between-group factor training condition (attend-positive training vs attend-negative training). This analysis revealed a significant main effect of training condition, The state anxiety scores obtained using the analog mood scale given before and after the final anagram stressor are shown in F1,48=159.991, P<.001, indicating that state anxiety increased from before the anagram stressor  to after the anagram stressor . However, neither the main effect of training condition, F1,48=1.664, P=.20, nor the critical interaction between state anxiety assessment point and training condition, F1,48=.378, P=.54, η2p=.008, were significant.Application of the outlier criteria on participants included in the attentional bias assessment analyses led to the additional exclusion of 1 participant (in the attend-positive condition). To examine whether the 2 training conditions had a differential impact on anxiety reactivity, state anxiety scores were subjected to a mixed-methods ANOVA with the within-subjects factor state anxiety assessment point (prestressor assessment vs poststressor assessment) and the between-subjects factor training condition (attend-positive training vs attend-negative training). Results showed a significant main effect of state anxiety assessment point, F1,46=125.99, P<.001, η2p=.73, again reflecting the fact that state anxiety increased from before the stressor  to after the stressor . This main effect was now subsumed within a significant two-way interaction of state anxiety assessment point and training condition, F1,46=4.39, P=.04, η2p=.09. When controlling gender, by adding gender as a covariate, this interaction between state anxiety assessment point and training condition remained significant, F1,43=4.638, P=.04, η2p=.097.Application of the outlier criteria on participants included in the attentional bias assessment analyses led to the additional exclusion of 4 participants (2 in the attend-positive training condition). The same 2x2 ANOVA as reported above was conducted on state anxiety scores to examine whether in participants who completed the Emotion-in-Motion training procedure, the 2 training conditions had a differential impact on anxiety reactivity. This analysis revealed a significant main effect of state anxiety assessment point, t tests revealed that immediately following the attentional training procedure but before the anagram stressor experience, participants who had received the 2 training conditions did not differ in their levels of state anxiety, F1,46=.31, P=.58, η2p=.01. Participants in each Emotion-in-Motion attentional training condition responded to this stress manipulation by displaying an elevation in anxious mood state . However, the magnitude of the elevation in state anxiety evoked by this stressor was significantly attenuated in those participants who had received the Emotion-in-Motion attend-positive attentional training compared with those participants who had received the Emotion-in Motion attend-negative attentional training condition . Thus, those participants who had been exposed to the Emotion-in-Motion task training contingency designed to reduce attentional bias to negative information subsequently came to display relatively attenuated elevations of anxious mood state in response to the anagram stressor experience compared with participants who had been exposed to the training condition designed to increase attentional bias to negative information. In addition, the elevation in anxiety in the positive training condition of the Emotion-in-Motion task  was significantly smaller than the elevation in state anxiety in the positive training condition of the conventional probe-based training task , t47=2.35, P=.02, Cohen d=0.67.Follow-up The objective of this study was to develop and evaluate a novel ABM procedure intended to systematically alter selective attentional responding to emotional information in a complex and dynamic task environment. Our results showed that our novel Emotion-in-Motion training procedure succeeded in modifying patterns of attentional bias, as intended. Moreover, the participants who were allocated to the attend-positive condition of the Emotion-in-Motion attentional training task showed reduced anxiety reactivity to the subsequent lab-based stressor as compared with participants who were allocated to the attend-negative condition of this task. These results suggest that our novel attentional training task appeared capable of modifying both patterns of attentional bias and causally influencing anxiety vulnerability.A subsidiary aim was to permit comparison with the conventional probe-based attentional bias training procedure. Under equivalent laboratory conditions, the conventional probe-based attentional training approach failed to induce differential patterns of attentional bias, and the 2 probe-based training conditions did not lead to participant differences in anxiety reactivity to the subsequent stressor. In recent years, several studies (including 3 out of our lab) have reported similar failures of the conventional probe-based attentional training task to successfully modify patterns of attentional bias ,47-51; tIn reflecting on the reasons for the capacity of our novel Emotion-in-Motion paradigm to induce differential patterns of attentional bias, under conditions where the conventional probe-based training did not, several candidate factors can be considered. First, the Emotion-in-Motion task presents 8 stimuli simultaneously, whereas the conventional probe task displays only 2 stimuli. There is some evidence that attentional bias is more pronounced when assessed using visual displays that contain more stimuli ,53, but A second candidate factor that could have contributed to the findings observed with the Emotion-in-Motion approach is the dynamic nature of the stimulus presentation. In the Emotion-in-Motion task, all stimuli move dynamically around the display, whereas in other attentional training paradigms, stimuli are presented in a static manner. It is possible that the dynamic nature of Emotion-in-Motion enhanced concentration and engagement with the task, thereby increasing its capacity to deliver the intended attentional bias change. In future research, the potential contribution of this dynamic component could be examined by contrasting task variants that employ the present dynamic approach with variants that instead present the same number of stimuli in static grid.A third candidate reason for its efficacy may be the provision of enriched performance feedback in the Emotion-in-Motion task compared with the rudimentary trial-by-trial error feedback given in the conventional probe-based attentional training task. Moreover, block feedback in the Emotion-in-Motion task was encouraging, whereas trial-by-trial feedback in the probe-based task penalized participants for making errors by delaying the next trial for 3 seconds, which may have elicited increased negative mood. Block feedback of the type delivered in the Emotion-in-Motion task has been shown to enhance learning in simple repetitive tasks , whereasMoreover, in the conventional probe-based training task, images depicting different emotional expression of the same identity were paired, whereas in the Emotion-in-Motion task, each image depicted a different identity. As such, participants performing the probe-based training only needed to discriminate emotional expression on the same person, whereas participants performing the Emotion-in-Motion tasks needed to discriminate emotional expressions between different identities. There is some evidence to suggest that emotion classifications are affected by variations in identity . It is, An additional difference between the 2 training procedures concerns participants’ responses. The tracking response required in the Emotion-in-Motion task is continuous, whereas the probe task only requires a response every couple of seconds. It is likely that as a result, participants in the Emotion-in-Motion task spend more time attentionally engaged with the target valence  as compared with participants in the conventional probe task. However, it is also possible that this continuous response would be harder to sustain over time as it is more motorically demanding. As such, future research may usefully examine the acceptability of this response format in multi-session training designs. It is also relevant to note that in the Emotion-in-Motion task, the mouse cursor only disappears behind the target rectangle. As such, it is possible that participants could ignore the content of the rectangle and simply see on which rectangle the mouse cursor would disappear. Although given the speed and complexity of the task this strategy is unlikely to have occurred, importantly, this strategy would have reduced the efficacy of the attentional training. Future research could, therefore, evaluate whether modification to the task  would further increase the effectiveness of the task. It will be important to establish, however, whether such modifications that render participants’ awareness of the position of the cursor more uncertain cause unwanted frustration or disorientation in participants.It is important to consider the potential limitations of this study. One such limitation is that the capacity of the Emotion-in-Motion training task and the capacity of the conventional probe-based training task to modify attentional bias were each established using a different method of assessing attentional bias. For both training tasks, the assessment approach involved delivering the same task but with the training contingency removed. This design critically allows for comparable demonstration of near transfer across the 2 training tasks. However, it does preclude direct comparison of attentional bias change observed in response to each of these 2 candidate attentional training approaches. It is possible, for example, that the assessment version of the Emotion-in-Motion task is more sensitive to individual differences in attentional bias than the probe-based attentional bias assessment task . If this is the case, then the results of this study could be explained by the Emotion-in-Motion training task producing a greater modification of attentional bias, the Emotion-in-Motion assessment task more sensitively assessing group differences in attentional bias, or both. Future research could circumvent this limitation by employing the same attentional bias assessment approaches for all ABM tasks under evaluation.A second potential limitation is that this study was carried out on an undergraduate nonclinical participant sample. Although this design allowed us to examine whether the Emotion-in-Motion procedure can induce differential patterns of attentional bias and consequently test the causal impact of these differential patterns of attentional bias on anxiety vulnerability, it does limit conclusion concerning either the acceptability or the efficacy of our novel Emotion-in-Motion ABM approach when used with a clinical sample. Although the complex and dynamic nature of the Emotion-in-Motion task can be expected to enhance face validity of and engagement in the task, future research using clinical cohorts will be necessary to determine whether this novel ABM task is more acceptable to patients than the conventional probe-based training task.It is also important to consider some potential limitations of using gamification for bias modification. Some of the potential drawbacks are discussed by Boendermaker et al . These aIn the meantime, we hope that the Emotion-in-Motion task, which this study has shown to be capable of modifying attentional bias to emotional information and altering anxiety vulnerability as indicated by anxiety reactivity to a stressor, will be of interest and potential value to researchers investigating the potential anxiolytic benefits of directly manipulating maladaptive patterns of attentional bias. To facilitate further research using this task and to encourage independent replication of the findings of this study, we made our Emotion-in-Motion task software freely available . While w


Medication of acute episodes of mood disorders has changed over the last decades following the results of randomized clinical trials.The aim of this study was to analyze medication prescribed at discharge from two psychiatric wards. We focused on hospitalization as one of the best opportunities to start prophylaxis.We examined retrospectively the clinical records of 357 patients hospitalized for mood episodes in two psychiatric wards in the Cagliari area (SPDC-1 and SPDC-2) between 1 January and 31 December 2016. We focused on the psychotropic medication prescribed at discharge from the hospital.Most patients were discharged with antipsychotics (86%) and/or benzodiazepines (89%). Combined medication was frequent, including various co-administration of first-generation and/or second-generation antipsychotics (26% of patients), or antipsychotics combined with mood-stabilizers (51% of patients). There was a preferential prescription of first-generation antipsychotics in SPDC-1, and of second-generation antipsychotics in SPDC-2. Prescription of lithium was significantly more frequent in SPDC-1.Although the treatment was in line with randomized clinical trials, the choice of individual psychotropic agents differed significantly between the two wards. Different prescription attitudes can have consequences on the long-term outcome of patients discharged from the hospital after an acute mood episode. In particular, second-generation antipsychotics and anticonvulsants have progressively substituted first-generation antipsychotics and lithium in the treatment of acute episodes of bipolar disorder . A serieAs reviewed by Perugi and Fornaro , severalThe debate is not merely academic because prescription choices can influence public health and have a relevant socioeconomic impact.Our group has been involved in research and clinical work of mood disorder over the last four decades. In particular, CB and AB started their professional career in the late 1970s, when lithium therapy was introduced in Sardinia, and have experienced the changes in both diagnostic instruments and medication armamentarium. We have noticed that prescription attitudes have apparently resulted in a decline in the use of lithium in Cagliari area, despite the growing literature evidence of its superiority in specific circumstances. The lack of initiatives from local institutional agencies to explore this phenomenon prompted us to undertake this study, with a secondary aim of contributing to the global field beyond the local region.The principal aim of this study was to analyze the psychotropic medication prescribed at discharge from two psychiatric wards to the patients hospitalized for mood (affective) episodes over one calendar year. We focused particularly on lithium therapy, based on the consideration that a hospital stay is one of the best opportunities to start (or reinforce) prophylaxis.2We examined retrospectively the clinical records of the patients hospitalized for affective episodes ICD-10 F30-F39) in two psychiatric wards in the Cagliari area between January 1 and December 31, 2016. According to the reform introduced in Italy in 1978, SPDC (Servizio Psichiatrico di Diagnosi e Cura = Psychiatric Diagnosis and Care Service) provides for the needs of patients requiring medical treatment involving a stay in the hospital, both in the case of voluntary admissions and in that of compulsory treatment [ F30-F39 31. The duration of stay in the hospital ranged between one day and approximately six weeks. The mean duration was longer for SPDC-1.Demographic and clinical characteristics of the 357 patients studied are shown in Table 1). In particular, SPDC-1 patients were diagnosed more often with “classical” bipolar disorder (F30-F31), whereas SPDC-2 patients were more often diagnosed with unspecified (F39) or schizoaffective (F25) disorders.The two wards differed in the proportion of ICD-10 diagnoses Fig. (The great majority of patients were discharged from the two psychiatric wards with antipsychotics (306/357 = 86%) and benzodiazepines (317/357 = 89%). At discharge, apart from benzodiazepines, there was an extensive prescription of combined medication, including 92/357 (26%) cases with various combinations of first-generation and/or second-generation antipsychotics, and 181/357 (51%) cases with antipsychotics combined with mood-stabilizers.2). A slightly non-significant higher proportion of patients from SPDC-1 was discharged with antidepressants, probably reflecting the slight (non-significant) greater proportion of depressive episodes in SPDC-1 .With regard to specific classes of drugs, there was a preferential prescription of first-generation antipsychotics in SPDC-1 and a preferential prescription of second-generation antipsychotics in SPDC-2 Fig.  or as individual compounds Table 2. In 25 cases, treatment at discharge included anticonvulsants that are not officially approved in Italy for mood disorders .Prescription at the discharge of anticonvulsants officially approved for mood disorders  was similar between the two SPDCs, either as a class Fig. (2) and Table 2.The attitude of prescribing lithium at discharge differed significantly between the two SPDCs Fig. . Of the 102 patients with a history of prior suicide attempts, lithium was prescribed at discharge to a greater proportion of patients from SPDC-1 than SPDC-2 .In particular, of the 53 patients who had already been treated with lithium before the index episode, a greater proportion of those hospitalized in SPDC-1 was discharged again with lithium compared to those in SPDC-2 . These results confirm that, even if regulatory recommendations are followed with regard to acute treatments, there may be wide variations in treatment choices, that may have consequences in terms of public health and have a relevant socioeconomic impact .This survey of acute mood (affective) episodes requiring hospitalization in two psychiatric wards was undertaken to test the current prescription attitude in the “real world”, compared to the context of treatment guidelines and randomized clinical trials. The latter has resulted in the approval of several new drugs  by regulatory agencies from different countries. Prescription patterns have changed over the last two decades in favor of newer medications. This shift, however, may not be completely based on evidence with regard to the therapeutic phases that follow an acute episode  approved lithium in 1970 and the neuroleptic chlorpromazine in 1973. Other medications have long been used in acute mania but not necessarily approved by regulatory authorities. Paradoxically, haloperidol, which is not FDA-approved has been used as a comparison treatment in many modern trials of acute mania, as reviewed by Cipriani and colleagues . Haloper4.2Another example of the discrepancies between the real world and randomized clinical trials is clozapine, which has been used as monotherapy or as add-on therapy in particular in the case of treatment-resistant mania, but whose effects were addressed by uncontrolled trials alone , 17.Clozapine was prescribed to a small proportion of patients from this study (approximately 5%), supposedly those with treatment-resistant episodes. One reason for clozapine's limited use is its side-effect profile , and its association with the serious adverse effect of agranulocytosis . The lat4.3Carbamazepine was prescribed to less than 10% of patients discharged from SPDC-1 and SPDC-2. Carbamazepine has been used in the treatment of various phases of bipolar disorder since the early 1980s  but was There have been some studies comparing carbamazepine to lithium in the maintenance phase of mood disorder, finding it somewhat less effective , 26. In versus its real-world effectiveness in managing bipolar disorder” [The relatively small number of patients (3%) treated with lamotrigine at discharge from SPDC-1 and SPDC-2 can be attributed to the probable excess of patients with predominant manic than depressive episodes. Moreover, lamotrigine did not demonstrate efficacy in four out of five placebo-controlled clinical studies in the acute treatment of bipolar depression , as oppoisorder” , 34.Focusing on the challenging problem of bipolar depression, Ketter  stated tOther anticonvulsants, such as gabapentin, topiramate, oxcarbazepine, and pregabalin, were also prescribed to some patients included in this survey, even if their efficacy has not been established by controlled studies -38.4.4Between 2000 and 2004, there has been an “explosion” of controlled trials that resulted in FDA approval for acute mania of the second-generation antipsychotics olanzapine, risperidone, quetiapine, ziprasidone and aripiprazole [4.5As the acute response to treatment was beyond the scope of this survey, we focused on medication prescribed at discharge from the hospital. In fact, a hospital stay is one of the best opportunities to start (or reinforce) prophylaxis of mood disorders. Prophylaxis should be taken into account because mood disorders are often recurrent, affect the quality of life, and are associated with long-term increased mortality particularly due to suicide .e.g. relapses versus recurrences) and their therapy  [Unfortunately, controlled studies do not generally test long-term effectiveness because of the elevated costs of such trials. There are even discrepancies in the definitions of phases of mood disorders  . It mustCipriani and colleagues  in theirThe results from our “real world” survey indicate that, even if the medication has been prescribed in compliance with the indications of regulatory agencies for acute episodes of mood disorders, the prescription attitude has differed significantly among hospital clinicians serving at the two psychiatric wards studied. Individual specialists make their own decision about the patients under their care.e.g. promazine or quetiapine) were prescribed to most patients to control agitation, irrespective of diagnosis. Supposedly, sedative drugs were maintained at the time of discharge because the task of tapering or withdrawing them was left to the outpatient specialists once the agitation improved. With regard to benzodiazepines, it must be underscored that, in the 1980-1990s, prior to the advent of Divalproex and second-generation antipsychotics, adjunctive treatment with lorazepam or clonazepam was studied for acute mania in the US. For example, one double-blind study [Some choices might have been influenced by the current trend to limit hospital stays as much as possible . This could be one of the reasons why benzodiazepines and more sedative first-generation or second-generation antipsychotics  and recent (2000-2003) lithium versus placebo trials, as well as those assigned to alternative mood stabilizers  and receIn an evaluation of real-world long-term outcome, lithium has recently been found as the most effective mood-stabilizer in a study of the comparative effectiveness of pharmacologic treatments in the prevention of psychiatric re-hospitalization in a Finnish nationwide cohort of 18,018 patients with bipolar disorder followed up for a mean of 7.2 years .Similar conclusions were reported by a recent systematic review of non-randomized controlled observational studies . Maintenvs 50.8; p = 0 .017). We can speculate that the different professional experience has influenced both the diagnostic and the prescription attitude: perhaps SPDC-1 clinicians have been prone to diagnose patients with classical nosographic entities compared to their younger SPDC-2 colleagues who have often concluded for unspecified or for schizoaffective disorders. Similarly, the older SPDC-1 clinicians have preferred first-generation antipsychotics and the younger SPDC-2 clinicians have preferred second-generation antipsychotics. We can hypothesize that experienced clinicians keep their long-lasting habits and are less prone to novelty.The significant differences observed between SPDC-1 and SPDC-2 warrant some interpretation. Both wards are located in the same hospital and derive from the division of one single SPDC occurred in 2008. There was a tendency to keep the original staff of the old SPDC in SPDC-1, while the SPDC-2 personnel was new. Indeed, the mean age of the psychiatrists serving in 2016 differed between SPDC-1 and SPDC-2  and movement disorders (in particular akathisia). Comparative trials with medicines other than haloperidol and lithium are few, so the precise position of aripiprazole in therapy remains unclear .Aripiprazole appears to have a superior tolerability profile when used as maintenance treatment. Side effects like a headache, insomnia, and extrapyramidal side effects, such as tremor and akathisia may be treatment limiting in some cases . AripiprBesides tolerability of the acute and the immediately following phase, that have been addressed by controlled trials, the choice of clinicians should consider potential adverse events associated with longer-term treatment in the real world. For example, the aforementioned recent Finnish nationwide cohort study examined hospitalizations owing to physical illness  for 7.2 years . In the It must be underscored that the 7.2-year follow-up could not reveal any adverse event associated with longer-term exposure, including the decline in renal function observed in patients treated with lithium for decades , 62.Acute treatment with valproate has commonly been associated with nausea, vomiting, diarrhea, sedation, and tremor. Long-term treatment can also induce alopecia and polycystic ovarian disease. Hepatic and pancreatic toxicity have been reported, as well as an unusual encephalopathy associated with hyperammonemia. Dealberto reviewed fourteen cases of encephalopathy in the psychiatric setting published between 1995 and 2005: Ammonia concentrations varied across the reported cases from normal to ten-fold the upper limit of the normal range . Raja an5Sometimes, prescription attitudes in the real world are not completely evidence-based. Some differences can be attributed to the aforementioned variations across national regulatory agencies. One of the potential factors may be the increasing influence of American psychiatric literature worldwide.In the 1980s, before the advent of second-generation antipsychotics and divalproex, ICD-9 was the official diagnostic system in use but DSM-III have also introduced in Italy along with its new concept of affective disorders (including those with mood-congruent and mood-incongruent psychotic features). Acute mania was generally treated with first-generation antipsychotics and/or benzodiazepines, based on severity. Lithium or carbamazepine were also frequently prescribed.DSM-IV and ICD-10 were introduced in the 1990s. The first controlled Divalproex study for mania was published in 1994 , and theMeanwhile, the efficacy of lithium was questioned particularly by Joanna Moncrieff in a series of papers published in very influential British and American psychiatric journals. She strongly challenged the validity of the pioneering trials of lithium prophylaxis in bipolar disorder -71. She Thereafter, numerous studies and meta-analyses clearly demonstrated the efficacy/effectiveness of lithium, which has been used as a comparator in modernly designed trials.Moreover, with regard to longer term outcome, it must be underscored that in the only random assignment study, valproate was generally less effective than lithium as maintenance treatment in bipolar disorder , 73, andIt must also be underlined that in the 2000-2010s, as commented by Bauer and Gitlin , aggressAlthough the treatment of acute mood disorders may be in line with randomized clinical trials and treatment guidelines, rates of prescription of individual psychotropic agents may differ significantly, as in the two psychiatric wards in this study. It must be underscored that different prescription attitudes can have relevant consequences on the long-term outcome of patients in terms of effectiveness, tolerability and mortality, as suggested by observational studies carried out in the “real world”.


In addition, we demonstrate the usefulness of the inferred functional landscape to investigate the cellular role of both well-characterized and novel RBPs in the context of human diseases.The coordination of the synthesis of functionally-related proteins can be achieved at the post-transcriptional level by the action of common regulatory molecules, such as RNA–binding proteins (RBPs). Despite advances in the genome-wide identification of RBPs and their binding transcripts, the protein–RNA interaction space is still largely unexplored, thus hindering a broader understanding of the extent of the post-transcriptional regulation of related coding RNAs. Here, we propose a computational approach that combines protein–mRNA interaction networks and statistical analyses to provide an inferred regulatory landscape for more than 800 human RBPs and identify the cellular processes that can be regulated at the post-transcriptional level. We show that 10% of the tested sets of functionally-related mRNAs can be post-transcriptionally regulated. Moreover, we propose a classification of  This indicates that the regulation of mRNA transcripts is key to achieve coordinated protein synthesis. Indeed, it has been shown that sets of transcripts coding for functionally related proteins are bound by common regulatory molecules, such as RNA-binding proteins (RBPs) and/or non-coding RNAs, thus forming the so-called RNA regulons4.While transcription contributes to coordinated gene expression in time and space, several studies highlighted the discordance between levels of mRNAs and protein production6. In mammalian cells, several sets of related mRNAs are part of RNA regulons as well, e.g., histone mRNAs bound by the stem-loop binding protein (SLBP)7, transcripts involved in inflammation regulated by the RBPs ELAVL1, HNRNPL and TTP8, those implicated in DNA damage response and regulated by the RBPs BCLAF1, ELAVL1 and THRAP310 and mRNAs coding for cell cycle and proliferation factors bound by Dead end protein homolog 1 (DND1) and Pumilio 1 (PUM1) proteins9.Early protein-RNA interaction mapping studies in yeast demonstrated that many RBPs bind specific mRNAs coding for proteins involved in the same biological process  or that are cytotopically related 11. However, even though RNA regulon perturbations can lead to the onset of neurological diseases and cancers in human14, the control of these regulatory circuits exerted by RBPs is rather sketchy16, therefore calling for further scrutiny.As this regulatory phenomenon has been observed in different species, RNA regulons represent a conserved feature of the post-transcriptional regulation in eukaryotes18 allowed to identify thousands of protein–RNA interactions. However, these studies have focused on the binding ability of a reduced number of established RBPs in a few cell lines18, indicating that the protein–RNA interactions space is largely unexplored. Moreover, thanks to the recent development of RNA interactome capture technologies, the catalogue of RBPs has dramatically increased (e.g.24). Importantly, many of these RBPs lack a known RNA-binding domain and their role in RNA biology has not been characterized yet22. In this context, large-scale computational prediction of protein-RNA interactions can provide a better coverage of the protein-RNA interaction space and improve our understanding of post-transcriptional regulation.A deeper understanding of post-transcriptional regulation is subordinate to the availability of experimentally verified protein-mRNA interaction data. Over the last years, studies based on high-throughput methods to detect RNA molecules bound by RBPs, such as RNA immunoprecipitation and CLIP-based techniquesWhat is the extent of the regulon theory at the coding transcriptome scale? What are the cellular functions regulated at the post-transcriptional level? Can RBPs be classified based on the regulation they exert? To answer these questions, we inferred the functional landscape of the post-transcriptional regulation mediated by the human RBPs, by assessing the RNA regulon theory at different levels of organization of the cellular processes, such as biological pathways and protein complexes. For this, we developed and applied an original large-scale approach to identify cellular processes post-transcriptionally regulated by RBPs, using both experimentally identified and predicted human protein–RNA interactions combined with protein-protein interaction network data and statistical analyses. We showed that the post-transcriptional regulation of functionally-related mRNAs by RBPs concern 10% of the groups that we tested in the regulatory landscape. Furthermore, we identified 3 groups of RBPs possibly regulating these groups of functionally-related mRNAs by using different molecular strategies.3, an RBP can regulate a given biological process by binding a substantial fraction of mRNAs encoding the proteins involved in that process. We therefore expect to detect a statistically significant over-representation of mRNAs bound by the RBP among groups of functionally-related coding transcripts. To determine the extent of the RNA regulon theory across all human biological processes, we gathered the transcripts encoding proteins involved in the same biological process or pathway, taken from four datasets representing different levels of organization of the cellular functions, and collectively named hereafter “functional units” (FUs): (i) 1846 manually curated protein macromolecular complexes from the CORUM database25; (ii) 874 functional modules detected in a human protein-protein interaction network using the OCG algorithm, which decomposes a network into overlapping modules based on modularity optimization26; (iii) 300 pathways described in the KEGG database27; and (iv) 1627 pathways from the Reactome knowledgebase28 . We computed the ratio of interacting vs. non-interacting transcripts for each functional unit with every RBP and assessed its significance to be higher or lower than expected by chance by performing a two-sided Fisher’s Exact test  tested in only two cell lines18. Since (i) we aim at investigating a comprehensive set of RBPs including the non-canonical ones, and (ii) the depletion phenomenon observed for both RBPs and FUs could be explained by a lack of coverage of the investigated network, we generalized our analysis to a large computationally predicted network of biophysically possible and biological context-independent interactions between 877 RBPs and 13,984 mRNAs (see below). In doing so, we expectedly circumvented the limitation of our scrutiny by missing interaction data. The results obtained on the experimental RBP–mRNA network . We generated more than 12 million protein-mRNA interaction predictions, of which 3.2 million show high interaction propensity score (catRAPID score ≥ 50) (see Methods) between the 877 RBPs and ~87% of the initial coding transcripts . With the standard catRAPID score cutoff36 , RBPs are predicted to interact with 3176 mRNAs on average (26% of the tested mRNAs)   , using the m29 Fig. . This toOverall, to the best of our knowledge, we have generated the largest predicted human RBP–mRNA interaction network to date.Next, we applied our approach Fig.  to inferThe first important outcome of our functional analysis is that, based on the detection of a significant functional enrichment, we could assign at least one potential target FU to 468 RBPs for which eCLIP interaction data is not available yet , indicating that these RBPs exclusively display a binding preference for a number of FUs. A second category accounting for 427 RBPs shows both significant enrichments and depletions of their predicted targets among functional units  suggesting that they bind the mRNAs of certain functional units and avoid those of others. Finally, the third category contains 211 RBPs that display only significant depletions  within functional units, illustrating that some functional units avoid RBP binding Fig. .i.e., the D-RBP set). This discrepancy between our predicted regulatory landscape and the results obtained on eCLIP data suggests a possible influence of the chosen catRAPID interaction propensity threshold .On the other hand, from the perspective of the FUs, we observe a mirrored situation. Most functional units  are exclusively enriched in targets of at least one RBP, thus possibly regulated at the post-transcriptional level through the binding of those RBPs. Few functional units, namely 27 , are both enriched and depleted in RBP predicted targets, indicating that they may be regulated by the binding of certain RBPs and the avoidance of others. Finally, 50 functional units  show only significant depletions thereby indicating that their mode of post-transcriptional regulation consists uniquely in the avoidance of RBPs binding Fig. . The com37 (see Methods). Importantly, we found the three distinct categories for both RBPs and functional units, with the M-RBP set being involved in a similar fraction of the significant functional enrichments and depletions  those functional units that can be regulated at the post-transcriptional level and that account for 10% of the tested FUs, and (ii) the RBPs responsible for such regulation. The results obtained on both predicted and experimental RBP–mRNA interactions suggest that both FUs and RBPs can adopt several possible regulation strategies and should be classified accordingly.Overall, this two-step statistical analysis allowed us to define the potential post-transcriptional regulatory landscape of numerous cellular processes by identifying The classification of RBPs in distinct groups based on the functional analysis of their interactors motivate us to assess whether the RBPs have distinct functional and sequence features as well as system-level properties  and depletions  compared to those of the E-RBP  and the D-RBP  sets respectively. This suggests that the more numerous RBP group in our classification (M-RBP set) can potentially regulate the larger number of FUs . Indeed, Beckmann and colleagues annotated RBPs into four classes: (i) established RBPs ; (ii) RBPs carrying a characterized RNA-binding domain (RBD); (iii) enigmRBPs, which are proteins found to bind RNA but lacking a canonical RBD and with no previous evidence of involvement in RNA fate; (iv) RNA-binding enzymes, which have an RNA-independent metabolic activity.Second, we checked whether RBPs of the three groups were characterized by an over-representation of different types of RBPs according to a previously proposed functional classification38. Interestingly, we observed that the RBPs binding predominantly mRNAs are more frequent in the E-RBP (82%) compared to the M-RBP (66%) and D-RBP (64%) sets. Indeed, in the latter two sets we observed a higher fraction of ribosomal proteins and RBPs binding small RNAs . The E-RBP set has a slightly higher disorder , which is otherwise enriched in enigmRBPs  Fig. . In the 38. In a compendium of 58 human tissues (see Methods), we did not observe any statistically significant difference among the three groups . Increasing evidence indicates that the activity of RBPs can also be regulated by PTMs43. We collected the modification site data for seven PTM types from the PhosphoSitePlus database44 (see Methods) and mapped them onto the RBP sequences of the three groups. We found that RBPs of the E-RBP set have a significantly lower PTM density , whereas the 50 D-FU units show significant depletions only among the interactors of 499 RBPs (average number of RBPs per unit: 21.5). The 27 functional units in the M-FU groups are enriched among the targets of 74 RBPs (average number of RBPs per unit: 3.7) and depleted among the interactors of 600 RBPs (average number of RBPs per unit: 45.8). These results underline the importance of RBP avoidance as a possible mode of regulation.45. Among the FUs related to chromatin organization and transcription activation, we found SWI/SNF-containing complexes and distinct forms of the Mediator complex from CORUM, as well as several network modules  and pathways  involved in energy production. Interestingly, these results corroborate the known post-transcriptional regulation of the mitochondrial components55.Additional enriched FUs are related to cellular processes localized in the mitochondria. Indeed, we find that several FUs have a large number of enrichments, including the large subunit of the mitochondrial ribosome from CORUM, four Reactome pathways related to mitochondrial translation as well as complexes  or epithelium 57. Our results indicate that these RBPs could potentially regulate the fate of a regulon made of the olfactory signaling mRNAs.M-FU units are involved in several signaling pathways. Indeed, we found that two pathways related to olfactory signaling (one from KEGG and the other from Reactome) are depleted in interactors of around two-third of the tested RBPs. However, they are exclusively enriched in those coded by the ERAL1, G3BP1, G3BP2, MKRN2 and TUFM genes, all expressed in brain tissues, according to Human Protein Atlas−16) and M-FUs , respectively  are related to viral and bacterial infections, whereas the other disease functional units are linked to immune-related, neurological and metabolic disorders  we found 20 disease-related pathways from the KEGG database. The majority of them . These units are enriched in interactors of the PRKC apoptosis WT1 regulator protein, an RBP encoded by the PAWR gene , which it has been implicated in mRNA splicing in cancer cells58. Furthermore, the pyrimidine metabolism pathway is also enriched among PAWR predicted interactions. Interestingly, it has been shown that intracellular pathogenic bacteria–such as Salmonella, Shigella and Bordetella –can modulate several host cell metabolism pathways for their own benefit, including nucleotide biosynthesis59, indicating a potential role of PAWR in the post-transcriptional regulation of genes involved in bacterial infection response.We also found common RBPs among FUs-related to bacterial infection as well  some FUs may be partially overlapping  or redundant, therefore leading to an overestimation; (ii) the choice of a strict catRAPID threshold for the prediction of protein–mRNA interactions, as well as the catRAPID restriction on transcript length, may have led to an under-estimation of the number of potentially regulated FUs. Moreover, as by construction, our statistical approach detects regulation events by considering a pairwise combination of FU and RBP, ignoring possible combinatorial and/or dynamic regulation modulations that could involve several RBPs16, the regulon prevalence could have been underestimated. Indeed, the analysis carried out on the eCLIP data provides a higher proportion of regulated FUs , thus suggesting that the underestimation is the most plausible scenario.By studying both types of data, we detected statistically significant over- and under-representations of the mRNAs bound by the RBPs among the functionally related coding transcripts. First of all, these results allow an estimation of the prevalence of the regulon theory. Among the 2977 functional units that we tested, comprising protein complexes, network modules and pathways, 10% have been found possibly regulated in the predicted functional landscapes. This result can be affected by two contrasting factors: Second, the different patterns of enrichments and depletions for the RBP binding to functional unit transcripts revealed by our analysis lead to a post-transcriptional landscape shaped by the RBP-mRNA interactions. It reveals that 57,2%  of the 877 tested RBPs regulate FUs by possibly binding to their mRNAs whereas 72%  do so by being avoided, therefore indicating the prevalence of this latter RBP regulatory mode. On the other hand, the groups of functionally related mRNAs  appear to be regulated through binding rather than through avoidance of the RBPs . Notwithstanding this, 90% of the FUs do not appear as being regulated by a particular RBP. Indeed, promiscuous RBPs interacting with most cellular mRNAs and FUs interacting with those RBPs are not expected to be detected as significantly enriched by our approach since the spread of the RBP targets precludes the detection of a statistically significant signal. This could be the case for 18% of the RBPs (164 RBPs) and 90% of the FUs (2677 FUs) for which no statistical signal has been detected.(i) the presence of RBP binding in the case of RBP targets enrichment, (ii) its avoidance in the case of depletions, or (iii) presence or avoidance of binding, when both enrichments and depletions are observed for a given RBP. Indeed, whereas some RBPs (the E-RBP set) appear to act exclusively through their binding to the mRNAs of the FUs , some others (the D-RBP set) are excluded from binding by having less targets than expected by chance among the mRNAs of the FUs . Finally, for other RBPs (the M-RBP set), both strategies, presence and avoidance of binding are observed.We observed 3 different patterns of enrichments and depletions for the RBP binding of functional unit transcripts. These patterns may reflect different possible FU molecular regulation strategies by the RBPs, involving catRAPID identifies RNA-protein interactions, the ‘presence’ is the physical ability for an RBP to regulate the FUs through its binding, independently of the binding status itself, bound or unbound, which may change with conditions. Conversely, the ‘avoidance’ is the physical inability for the RBP to bind, e.g., because of the lack of binding sites. As well as the ability, the inability to bind can lead to a regulation event.What does the ‘presence’ and the ‘avoidance’ of RBP binding represent? As 60 that coding sequences are evolutionarily constrained to avoid certain RBP binding motifs in order to prevent inappropriate interactions that could impair, for instance, their correct mRNA processing. Such avoidance of regulatory elements has also been observed for target sites of microRNAs within 3′UTRs61 and to limit spurious transcription binding sites62. Our striking observation that some functional units could contain the information to not interact with certain RBPs could therefore represent a cellular regulatory mechanism per se, calling for further investigation. However, as the repertoire of experimentally identified mRNA-binding proteins is constantly increasing63, we cannot exclude that some of the D-FUs can be regulated by a RBP not present in our dataset.Interestingly, the observed depletion, or avoidance of binding, could represent a molecular mechanism that limits inappropriate binding, which could interfere with correct gene expression. Indeed, it has been recently proposed by Savisaar and Hurst64 and several signaling and structural proteins. In addition, RBPs in this group have a significant low density in PTM sites, which can regulate, for instance, RNA binding or dictate the subcellular localization of a given RBP43. Altogether, this suggests that this set of RBPs contains putative multifunctional proteins whose RNA binding activity, which represents one of their possible molecular tasks, can be potentially modulated by a not yet identified molecular signal.We further studied the properties of the RBPs belonging to the three sets and found that several features can distinguish them. For instance, the E-RBP set is characterized by an enrichment in enigmRBPs that lack canonical RBDs and for which a role in RNA biology has not been established so far. Among the 29 enigmRBPs in the E-RBP set, there are 8 metabolic enzymes, including the moonlighting protein Leukotriene A-4 hydrolase (LTA4H)65, ELAVL167 and FMR1 proteins68. Moreover, RBPs in the M-RBP group, as well those in D-RBP, show a wider range of binding preferences among RNA biotypes compared to the E-RBP set, which comprises a high fraction of RBPs binding preferentially/exclusively mRNAs. Overall, our analysis indicates that RBPs in E-RBP group have distinct features that discriminate them from the two other groups. Consequently, further experimental studies are needed to identify the in vivo RNA interactors of RBPs in the E-RBP set (only 4 have been tested with the eCLIP technology) and, in the case of the enigmRBPs, decipher their role in mRNA fate.Conversely, the M-RBP set is enriched in RBPs with canonical RBDs showing a significantly higher PTM density compared to the E-RBP set, consistent with the current knowledge that the function of established RBPs is modulated by post-translational modifications, as in case of SR splicing factorsAltogether, our analyses defined a post-transcriptional regulatory landscape occupied by functionally related mRNA differently regulated by RBPs, thereby allowing us to provide a novel classification of the RBPs. This classification may help understanding the regulatory of action of the continuously increasing number of newly discovered RBPs.18 gathering 159 experiments for 112 RBPs. We mapped BED peak coordinates referencing the GRCh38 human assembly to Ensembl v82 coding transcript models using BEDTools intersect v2.1769 with flag –wa. Interactions from replicates and different cell lines were pooled. To have an interaction set comparable to catRAPID predictions, interactions involving transcript isoforms were mapped to the corresponding coding gene and counted as one. Doing so, we obtained a final list of 131,366 experimental interactions between 112 RBPs and at least one transcript encoded by 11,647 genes.We retrieved interaction information from the ENCODE enhanced CLIP (eCLIP) dataset25; 873 functional network modules, defined as groups of proteins densely connected through their interactions and involved in the same biological process, detected by the OCG algorithm26 on a human protein binary interactome built and annotated as previously described72 , which provides Ensembl identifiers for annotated genes. The genes/proteins annotated in the OCG network modules were mapped to the corresponding Ensembl v82 gene identifiers through the Ensembl BioMart service. We restricted subsequent analyses to complexes, modules and pathways having at least 5 and no more than 500 genes/proteins .We built a wide compendium of 4646 functional units and processes by gathering annotations from different sources: 1846 manually annotated human protein complexes from the CORUM databasefu,int is the number of FU-annotated mRNAs that interact with a given RBP, mRNAfu,no-int is the number of FU-annotated mRNAs that do not interact with a given RBP, mRNAno-fu,int is the number of mRNAs that interact with the given RBP but that are not present in the FU, and mRNAno-fu,no-int is the rest of mRNAs in the interaction space. We assessed the significance of the enrichment/depletion ratio by performing a two-sided Fisher’s Exact test. P-values were corrected for multiple testing using the Benjamini-Hochberg procedure and we considered as significant only those enrichments/depletions with a corrected P-value below 0.05. We used annotated mRNAs in the eCLIP interaction space as statistical background.To assess the enrichment/depletion of FU-annotated mRNAs interacting with RBPs in eCLIP dataset, we computed, for each functional unit, the log2-transformed - ratio of FU-annotated mRNAs among RBP interacting and non-interacting transcripts as:et al.22 and their corresponding amino acid sequences from the UniprotKB human reference proteome74 (May 2016). We downloaded the human coding transcriptome cDNA sequences  from Ensembl v8275 (September 2015).We collected a list of 1217 human RBP protein-coding genes identified by mRNA interactome capture from Beckmann 22 the following annotations: whether a role in RNA biology is known, presence or absence of a recognized RNA-binding domain according to the classification proposed in Castello et al.19, whether it has been categorized as ‘classic’ metabolic enzyme . Those RBPs lacking a recognized RNA-binding and with no established role in RNA biology are labelled as enigmRBP22.For each RBP in our dataset, we gathered from the original articlecatRAPID omics algorithm29, which allows large-scale predictions between transcript and protein sequences, to compute the interaction propensities between human RBPs and coding transcripts. Due to catRAPID computational constraints, we selected mRNA sequences between 50 and 1200 nucleotides of length, as well as protein sequences between 50 and 750 amino acids. Around 72% of the RBPs (877 proteins) and 57% of the human coding transcriptome  respected the length criterion. To avoid functional biases in subsequent analyses, we further reduced sequence redundancy among mRNAs  by selecting, for each protein-coding gene, the longest transcript as the representative sequence. Doing so, we retained 13,984 transcripts coded by ~63% of the annotated protein-coding genes in Ensembl v82 . We then predicted more than 12 million protein-RNA interactions between 877 RBPs and 13,984 mRNAs.We used the standalone version of 36, we considered as interacting all RBP-mRNA pairs with a catRAPID interaction propensity score of at least 50 and non-interacting all those with a score below 50. For each functional unit in the compendium, we computed the log2-transformed ratio of the FU-annotated mRNAs among RBP predicted interacting and non-interacting transcripts and assessed its significance as described above for the analysis on eCLIP interaction dataset. As RBPs are predicted to bind to many mRNAs, we further evaluated the number of enrichments/depletions expected by chance in each dataset by shuffling protein labels among functional units 1000 times. Only RBPs having a significantly higher number of enrichments/depletions than expected by chance  were kept.To assess the over- and under-representation of the functional units among RBP predicted interactions, as done previously37 (GSEA) using annotated mRNAs in a given functional unit as gene set. We selected as significant only those enrichments  or depletions  with a false discovery rate (FDR) < 0.05 based on 1000 gene set permutations. In both tests, we used annotated mRNAs in the catRAPID interaction space as statistical background.In a second approach, we carried out a Gene Set Enrichment Analysis76 (both long and short predictions) and DISOPRED377. An amino acid was considered disordered if its probability score was greater than 0.4. We calculated the RBP sequence low complexity using the NCBI segmasker application, which is based on the SEG algorithm78, using default parameters. For each RBP, we computed the fraction of the number of predicted disordered and low complexity amino-acid residues divided by the sequence length.We computed protein residue disorder propensity using the stand-alone version of two state-of-the art disorder prediction algorithms: IUPred44, which stores data for seven different PTMs: acetylation , methylation , O-GalnAc (2115 sites in 476 proteins), O-GlcnAc (420 sites in 166 proteins), phosphorylation , sumoylation (7932 sites in 2500 proteins) and ubiquitination . We extracted PTM data for the RBPs and computed their PTM densities as the number of PTM sites over the sequence length.We collected post-translational modification (PTM) information for 18,030 proteins from PhosphositePlus56. We considered as expressed 10,579 protein-coding genes with a qualitative expression level of at least ‘low’ and a reliability score equal to ‘approved’ or higher. For each protein-coding gene, we computed the expression breath as the fraction of tissues in which the given gene is considered as expressed over the total number of tissues present in the Human Protein Atlas .We downloaded protein expression data in human tissues based on immunohistochemistry from the Human Protein Atlas (version 18)post hoc Dunn Test, which performs multiple pairwise comparisons between the individual distributions . The network in Fig. 79.Distributions of disorder propensity and low complexity content fractions, PTM densities and tissue expression breath ratios were compared by using a two-sided Kruskal-Wallis test (significance level = 0.05), a non-parametric analysis of variance method. In case of a null-hypothesis rejection, we applied a All data generated or analyzed during this study are included in this published article and its supplementary information files. The predicted protein-RNA interactions are available from the corresponding authors on reasonable request.Supplementary InformationDataset 1


Maternal nutrition during pregnancy and breastfeeding is important for the healthy growth and development of the fetus and infant.This study aimed to evaluate the long-term effects of a maternal milk supplementation (MMS) in conjunction with a breastfeeding support program on breastfeeding practices including duration of any breastfeeding and exclusive breastfeeding and child neurodevelopment outcomes at 30 months old.We followed up the offspring of 204 Vietnamese women who completed a randomized controlled trial where the intervention group received MMS with a breastfeeding support program from the last trimester to 12 weeks postpartum while the control group received standard care. At 30 months postpartum, information on child feeding practices was collected and child neurodevelopment was assessed by the Bayley Scales of Infant and Toddler Development (Bayley-III).There was no significant difference in the duration of any breastfeeding (ABF) from birth between the groups. However, the intervention group had longer exclusive breastfeeding (EBF) duration (p = 0.0172), higher EBF rate at 6 months (p = 0.0093) and lower risk of discontinuing EBF (p = 0.0071) than the control. Children in the intervention group had significantly higher Bayley-III composite scores in the domains of cognitive (p = 0.0498) and motor (p = 0.0422) functions, as well as a tendency toward better social-emotional behavior (p = 0.0513) than children in the control group. The association between maternal intervention and child development was attenuated after further adjustment for birth weight but not EBF duration, suggesting that improvements in child development may be partially attributed to the benefits of prenatal nutrition supplementation on birth outcomes.MMS with breastfeeding support during late pregnancy and early postpartum significantly improved EBF practices. The intervention was also associated with improvements in neurodevelopment in children at 30 months old. Optimal maternal nutrition during pregnancy and breastfeeding is critical for the healthy growth and development of the fetus and infant, which lays the foundation for their long-term health in later life. However, poor nutritional status in both mother and her child remains prevalent in low- and middle-income countries ,2. SinceNumerous studies have shown positive effects of maternal supplementation during pregnancy and breastfeeding on maternal, fetal, infant and child health outcomes ,4,5,6,7.While relatively more studies have evaluated the short-term outcomes of maternal supplementation, research is limited on the long-term effects including postnatal growth and development in the offspring ,19. Few We recently conducted a randomized controlled trial to evaluate the effects of maternal milk supplementation(MMS) in conjunction with a breastfeeding support program in the last trimester until 3 months postpartum on birth outcomes, short-term breastfeeding practices and child growth in comparison to the standard care in Vietnamese mothers . The finThis was an observational study involving mothers and their children at 30 months old as a follow-up to a prospective, randomized, open-label, parallel-group, multicenter trial that they participated in. The study design and participants’ characteristics of the randomized controlled trial (RCT) have been described in detail previously [2 took part in the RCT across four northern provinces in Vietnam including Ninh Binh, Ha Nam, Hai Phong, and Thai Nguyen. Mothers who were randomized to the intervention (n = 114) received two daily servings of MMS  up to 12 weeks postpartum and 4 breastfeeding education and support sessions [In brief, a total of 228 singleton Vietnamese mothers aged 21 to 35 years at 26 to 29 weeks of gestation with a pre-pregnancy body mass index (BMI) <25 kg/msessions . The conWhen the children were about 30 months old, all mother and child pairs who completed the RCT were invited to participate in this observational study. Children with a diagnosis of severe developmental disorders e.g. cerebral palsy which required medical intervention were excluded from this study. Written informed consent was obtained from each mother and the study was approved by the Independent Ethics Committees of the National Institute of Nutrition (NIN) and the Ministry of Health in Vietnam. The original trial and the present study were registered at clinicaltrials.gov with registration codes NCT02016586 and NCT02913638, respectively .The primary outcome was duration of any breastfeeding (ABF) from birth. At 30 months postpartum, the mothers were asked to fill out a questionnaire, which was used to capture information on the age of the child (in months/weeks) in which the mother stopped breastfeeding. In addition, the questionnaire also captured information on the age of the child (in months/weeks) in which the mother introduced non-breast milk substitutes including infant formula and liquids such as water or intake of solids in mothers who had exclusively breastfed from birth to 12 weeks postpartum. For mothers who had stopped EBF by 12 weeks postpartum, dietary record information of the infant’s intake of breast milk and non-breast milk including formula, juice, water and complementary foods collected during the intervention study was used to assess EBF from birth. The World Health Organization (WHO) definition of exclusive breastfeeding “the infant receives breast milk as the only source of nutrition (including milk expressed or from a wet nurse), with no other liquids or solids except for oral rehydration solution, vitamin drops, minerals and medicines” was used in this study . Any breChild feeding practices after completion of the RCT through the first year included timing of introduction of semi-solids or solid foods, and practice of fortifying child’s food using vegetable oil during food preparation.Neurodevelopment of the children was measured using two validated tools, namely the Ages and Stages Questionnaire, Third Edition (ASQ-3) and the Bayley Scales of Infant and Toddler Development, Third Edition (Bayley-III). ASQ-3 is a parent-completed, child-development screening test that assesses the child’s communication, gross and fine motor, problem solving and personal social developments. Bayley-III is a widely recognized comprehensive tool to assess the development of children aged 1 to 42 months and is often regarded as the gold standard for measuring the neurodevelopment of children. It covers five main domains of the child’s development including cognitive, language, motor, socio-emotional and adaptive behavior . The two2) was calculated from the measured weight and height. Weight-for-age and height-for-age were expressed as sex-age-specific z-scores using the WHO Child Growth Standards [Maternal and child anthropometric measurements were performed at the follow-up visit by the research staff of the NIN who were trained on standardized methods for collecting the measurements. The measurements included weight and height of the mother and child and the child’s mid-arm circumference at the follow-up visit. Weight was measured with subjects wearing light clothing and no shoes (Horse Head Brand TZ-120) and recorded to the nearest 0.1 kg. Standing height was measured using a stadiometer (Horse Head Brand TZ-120) and recorded to the nearest 0.1 cm. Child mid-upper arm circumference was measured from the upper left arm using a flexible, non-stretchable tape (Seca 201) and recorded to the nearest millimeter. BMI , was used to evaluate the cognitive stimulation of the child’s environment and the emotional relationship between mother and children .The sample size was calculated based on the duration of ABF from birth prior to the study. A previous 30-month cohort study (n = 681) showed that the median duration of ABF was 9 months with an interquartile range (IQR) of 5 to 12.5 months . Based oContinuous variables were presented as mean ± standard deviation (SD) and categorical variables were presented as absolute numbers and percentages. Group difference was examined using Student's t-test or Wilcoxon rank-sum test for continuous variables, as appropriate. Categorical data between the groups were compared using Chi-squared test. Analysis of covariance (ANCOVA) was used as a primary analysis to assess the impact of intervention on breastfeeding duration and development outcomes, controlling for child’s age, gender, study site and other potential confounding variables such as household income, parents’ highest education level, smoking, mode of delivery, children’s anthropometrics, mother’s BMI, home environment score, etc. Confounding factors with a p-value below 0.3 in the univariate analysis were included in the multivariate analysis. The normality assumption was checked using quantile-quantile (q-q) plot and Shapiro-Wilk test that declared non-normal if found significant (p < 0.001). Wilcoxon rank-sum test was conducted if the residuals were non-normally distributed. Cox’s proportional Hazard Ratios (HR) and 95% confidence interval (CI) were estimated for discontinuing EBF. As a secondary analysis, multiple logistic regression models were used to estimate the Odds Ratios (OR) of getting above-median Bayley scores, controlling for study site, child age, gender and other covariates including sociodemographic, anthropometric and home environment factors. The OR estimates do not approximate risk ratios when outcomes are common.The positive effects of the intervention on child development may be mediated through having better birth outcomes and prolonged EBF duration, which may, in turn, promote later cognitive and psychomotor development. Therefore, we conducted a regression-based mediation analysis  in whichAll the statistical analyses were performed using SAS . All statistical tests were two-sided. A p-value <0.05 was considered statistically significant and a p-value <0.10 was considered marginally significant.6 Vietnamese Dong (VND), which is about 5,645 USD. None of the mothers reported smoking during pregnancy and the follow-up until 30 months postpartum. All women reported to be either non-alcohol drinkers or having alcoholic drinks <1 day/month, except for one in the control group who reported daily consumption of alcohol. Only first-time mothers were recruited in the RCT; hence all children were the first live births. There were no significant differences in mothers’ BMI, children’s weight, height and mid-arm circumference at 30 months postpartum .Four mother-child pairs were lost to follow-up in each study group . There wThe average age when complementary foods were introduced was around 24.5 weeks for both groups (p = 0.9392) . PrematuMean developmental scores of children at 30 months in the intervention and control groups are shown in Similar effects were shown in the multiple logistic regression using dichotomized outcomes at the median values of developmental scores . CompareWhen the developmental performance was assessed using ASQ-3, no significant differences were detected in all 5 domains of child development, except for a tendency toward higher fine motor score in the intervention group compared to the control group  . In the The present study compared the long-term effects of a maternal milk supplementation (MMS) in conjunction with a breastfeeding support program on breastfeeding practices and child neurodevelopment outcomes at 30 months old. Our results showed minimal differences in ABF between the intervention and control groups. However, the intervention had sustained effects on post-intervention breastfeeding practices including prolonged EBF duration, increased EBF rate at 6 months and reduced risk of early EBF cessation in Vietnamese mothers. In addition, the intervention was associated with improved cognitive, motor, and socio-emotional development in the offspring at 30 months old.Mothers who received MMS and breastfeeding support during late pregnancy and early lactation were found to have a prolonged duration of ABF (74 weeks versus 72 weeks), although not statistically significant. Furthermore, the intervention significantly improved long-term EBF behavior in our study. This finding is especially important for Vietnam, a country where breastfeeding practices remain suboptimal ,39,40. DFurther to this, there is limited evidence that has examined the effect of breastfeeding support and dietary intervention on both EBF and ABF. Most previous studies in this area focused on either EBF or ABF as one of the outcomes ,43,44. IThe present study showed that the intervention during perinatal period had minimal influence on child growth in terms of weight, height, and mid-arm circumference at 30 months old. In a double-blind controlled trial among 747 nutritionally at-risk Indonesian women, children born to mothers who received a high-energy supplement  daily in the last trimester of pregnancy showed better growth in weight and height than those born to mothers who were given a low energy supplement  ,47. HoweThe intervention in our study was significantly associated with improved cognition, motor, and social-emotional behavior assessed using Bayley-III in the offspring at age 30 months. Two potential mechanisms, not mutually exclusive, have been suggested to account for the neurodevelopmental impact following nutrition supplementation early in life: (1) directly affecting brain development and function by increasing nutrient supply and (2) indirectly affecting children’s experiences and behavior by interacting with the environment .First, both macro- and micro-nutrients provided in our intervention improved maternal nutritional adequacy, which may have supported the structural and functional development of the brain during gestation and infancy. Evidence from animal and human research suggested that nutrient deficiency during pregnancy and lactation adversely affected neuronal cell growth, pruning, myelination, and nervous system formation within regions of the brain, all of which resulted in impaired motor functioning, memory, learning, and higher order cognition ,48. ManySecond, breastfeeding has long been suggested to positively influence the maternal bond and/or infant attachment, although this hypothesis has not been well-supported largely because of limited data in this area . In our Our hypothesis is partially supported by the few available intervention trials conducted in the 1960s that involved nutritional supplementation during pregnancy and lactation. In a double-blind RCT in 225 Taiwanese women, infants born to mothers who consumed a high calorie-protein liquid supplement  had significantly higher Bayley-II motor score at 8 months than those born to mothers who consumed a low-calorie no-protein placebo  . HoweverBrain structures and functioning develop sequentially. Structurally, the cerebellum (important for motor control) develops from early prenatal life and continues to the postnatal period while the striatum (coordinates multiple aspects of cognition) develops rapidly during the late gestational and early postnatal period ,52. MyelThis study has several strengths, including its multicenter structure, randomization of treatment, high participation rate, blinding of the outcome assessors, objective assessment of child development, and the thorough consideration of potential confounders.Nevertheless, our study has several limitations. First, this was an observational follow-up study and part of the data was collected retrospectively. Information on breastfeeding practice and child feeding behavior at 30 months postpartum was therefore subjected to recall bias. However, the literature suggests that maternal recall provides reasonably valid and reliable estimates of breastfeeding initiation and duration, especially when the length of the recall period is short (≤3 years), which is the timeframe of collecting breastfeeding information in our study . On the Second, while we have used two different development assessment tools including ASQ-3 and Bayley-III, the two scales showed differences in detecting some developmental domains. The discrepancy is consistent with a previous study which had compared the validity of the two scales and found poor agreement . ASQ-3 iThird, although both Bayley-III and ASQ-3 were translated into Vietnamese and used in previous studies in Vietnam ,59, neitIn summary, maternal milk supplementation from the last trimester to the first 12 weeks postpartum, coupled with a breastfeeding support program was significantly associated with prolonged EBF duration, increased EBF rate at 6 months, and reduced risk of early EBF cessation. The intervention also significantly improved child neurodevelopment in the domains of cognitive and motor functions at 30 months of age, when compared with the current standard care. The benefits of the maternal intervention on child neurodevelopment were partially mediated by higher birth weight but not EBF duration. This suggests that the prenatal nutritional supplementation may have played a more important role than the additional breastfeeding support given during the postnatal period in promoting neurodevelopment of the offspring. Future studies with a larger sample size are needed to examine the longer-term impacts of maternal nutritional supplementation on child growth and development.S1 Table(DOCX)Click here for additional data file.