There is ongoing debate on the overlap between Attention-Deficit/Hyperactivity Disorder (ADHD) and Borderline Personality Disorder (BPD), particularly regarding emotion dysregulation (ED). In this paper, we present a narrative review of the available evidence on the association of these two disorders from several standpoints. First, we discuss the unique and shared diagnostic criteria for ADHD and BPD, focusing particularly on ED. We consider the methodology of ecological momentary assessment and discuss why this approach could be an alternative and more accurate way to qualitatively distinguish between ADHD and BPD. We summarise key findings on the genetic and environmental risk factors for ADHD and BPD and the extent to which there are shared or unique aetiological and neurobiological risk factors. Finally, we discuss the clinical relevance of considering both disorders in the assessment of patients presenting with trait-like behavioural syndromes, distinguishing the two conditions and implications for treatment. In recent years, a debate has ensued over the nosological distinction between Attention-Deficit/Hyperactivity Disorder (ADHD) and Borderline Personality Disorder (BPD) . ImpulsiIn this review paper, we present a narrative description of the available evidence on the association between ADHD and BPD pertaining specifically to emotional dysregulation (ED). We start by presenting an account of the main diagnostic features of each disorder and outlining the clinical features that are common to BPD and ADHD, then summarising studies that have reported on comorbidity between the two disorders. We then review findings from studies that have measured ED in ADHD and BPD using experience sampling methods, as this provides a precise and ecologically valid way of assessing the phenomenon of ED. Finally, we discuss the extent to which there are shared genetic and environmental risks, and shared neurobiology, for the two disorders, before considering implications of these findings for treatment.ADHD is a common neurodevelopmental disorder emerging in childhood or early adolescence, characterised by a pervasive pattern of developmentally inappropriate levels of inattention and/or hyperactivity-impulsivity that lead to clinically significant functional and psychosocial impairments . The disApart from the main symptoms used to classify ADHD, ED is considered to be an associated feature supporting the diagnosis of ADHD , 12. In According to the Diagnostic and Statistical Manual of Mental Disorders (DSM–5), the diagnosis of ADHD requires six out of nine ADHD symptoms of either inattention or hyperactivity/impulsivity in childhood, and five out of nine in adults Table . AdditioThe symptom profile and severity of ADHD varies greatly between individuals, with both inattention and hyperactivity/impulsivity associated with functional impairment in multiple domains , 15. ED ADHD seldom exists in isolation and up to 90% of adults with ADHD are reported to have one or more co-occurring mental health disorders . Of thesBPD is a complex and severe mental health disorder, with typical symptom onset during adolescence and presence of behavioural precursors in childhood, persisting into adulthood . BPD is In the general population, BPD has a prevalence of around 6%  and withLike ADHD, individuals with BPD commonly present with comorbid mental health disorders. In particular, around 90% of BPD cases are reported to have co-occurring mood disorders including depression and dysthymia , along wPsychiatric comorbidity is commonly found across all mental health disorders  and is dA high prevalence of co-occurring ADHD and BPD is consistently reported in the literature. In a large in- and outpatient cohort of 372 adults with ADHD referred for ADHD assessment and treatment at a tertiary referral centre, 27.2% also met criteria for BPD assessed by the structured clinical Interview for DSM-IV II (SCID II) . SimilarIn a sample of 118 adult women from out-patient clinics seeking treatment for BPD, a high co-occurrence rate was reported: 41.5% met criteria for childhood ADHD (assessed retrospectively), and 16.1% met current criteria for the DSM-IV combined subtype, as well as meeting ADHD criteria as children . Howevern = 107) with emerging BPD drawn from a European research project investigating the phenomenology of BPD in adolescence, the prevalence of ADHD was 11%, an estimate that was not attenuated even when excluding symptoms of impulsivity accounting for possible symptom overlap . While gene-based analysis yielded two significant genes for BPD, DPYD on chromosome 1 (1.20x10− 6) and PKP4 on chromosome 2 (8.24x10− 7), no genome-wide significant association was found for any SNP [To date there have been two GWAS studies of BPD. One study assessed two Dutch cohorts (lination . Seven sificance . The othcontrols . While gr = 0.59) was found between ADHD symptoms and borderline personality traits; consisting of four subscales- affective instability, identity problems, negative relationships and self-harm [Though there is evidence for symptom overlap between the two disorders, to date only one study has explored whether this could reflect overlapping genetic influences. Using a population twin sample, high phenotypic correlation , shared environment may still play a major role through gene by environment interactions. It is therefore likely that for both disorders there are genetically driven individual differences in susceptibility to environmental stressors. The relatively high genetic correlation between ADHD and BPD is based on the correlation of trait scores in the general population, rather than diagnosed cases, but suggests a considerable degree of underlying shared aetiology that may explain the frequent co-occurrence of ADHD and BPD. Further studies are needed to investigate the genetic overlap between the two disorders, but also the overlap with specific symptom domains such as ED.Treatment approaches to ADHD and BPD are widely divergent. According to evidence-based clinical guidelines, in BPD there is limited evidence that medications reduce borderline personality symptoms, including ED, and psychological treatments are the cornerstone of treatment . In contClinical trials support the safety and efficacy of stimulants  and atomoxetine, with reductions in the ADHD symptoms of inattention, impulsivity and hyperactivity, with moderate to large effects sizes ranging between 0.4 to 0.7 in adults –95. In aIn contrast to treatment of ADHD, psychotherapy is regarded as first line treatment for people with BPD . The mosCurrently, there is insufficient data on the treatment of co-occurring BPD and ADHD. With regard to drug treatment, there have been no RCTs of stimulants or atomoxetine in BPD alone or in co-occurring BPD-ADHD cases .n = 14) reported significant improvement of BPD symptom severity (SMD = − 1.5) and aggressive impulsive behaviour (SMD = − 1.31) following treatment with methylphenidate for 12 weeks [n = 24) showed a statistically significant improvement in anger control (SMD = 0.14), motor impulsiveness (SMD = − 0.62), depression (SMD = − 1.09) and ADHD severity (SMD = − 0.5), compared to those without medication (n = 23) [There have however been only two case reports , 104 of 12 weeks . In a fo(n = 23) .n = 72 patients with ADHD, an adaption of DBT, addressing emotion regulation, depression, impulse control, stress management, neurobiology of ADHD and ADHD in relationships, DBT has therapeutic benefit for people with ADHD [n = 8 patients with ADHD, an adaption of cognitive behavioural therapy led to improvement in the same psychometric elements listed above; ES = 0.99 for the BDI, ES = 2.22 for the ADHD-Checklist and ES = 1.35 for the SCL-16 [Similarly, there are various psychotherapeutic treatments available for adults with ADHD, who are either unresponsive to stimulants and/or atomoxetine, or in need of adjunctive psychotherapy. There have been two exploratory open label studies , 108 exae SCL-16 .n = 31, n = 43 and n = 51 respectively), addressing the effects of psychotherapy (in conjunction with medication in some cases) in adult ADHD that resulted in positive outcomes on all scales measuring severity of ADHD symptoms developed and tailored to the treatment of ADHD, compared to clinical management (CM) reflecting optimal usual clinical care, with both groups randomised to methylphenidate or placebo [p = 0.003), there were no significant differences in ADHD symptoms for those receiving GPT or CM (p = 0.16). In fact, in this trial, medication proved to be superior to intensive behavioural therapy, yet the latter resulted in better outcomes when combined with medication as compared with placebo [There have also been three RCTs of cognitive therapy –111 with placebo . While m placebo .Overall, while DBT modules and other systematically tailored psychotherapies appear to be helpful in ADHD, it is not yet clear whether they improve the core symptoms of ADHD , and there is insufficient data reported for effects on emotional dysregulation in ADHD –113. ThiIn clinical practice, it should be acknowledged that the co-existence of ADHD with BPD may complicate the diagnostic process, and hinder treatment outcomes. Currently, patients with co-occurring ADHD and BPD are often seen by different specialists and provided treatments for one condition or the other, but only rarely for both. In fact, there is lack of empirical data to guide future clinical practice. Beyond the issues of differential diagnosis, there is insufficient awareness within specialist ADHD and BPD services of the potential benefits of treating the other condition. This needs to be addressed because treatment of both conditions may have positive benefits for individuals with overall better control of ADHD and BPD related symptoms and behaviours. Indeed, open clinical trials indicate the value of such a dual treatment approach.executive function deficits , as well as physical restlessness and impatience, lead to difficulties in commitment and adherence to psychological therapies [Commonly in BPD patients with co-occurring ADHD, inattention and so called herapies . For exaherapies .A further potential benefit in a subpopulation of individuals with co-occurring ADHD and BPD may be a reduction in emotional dysregulation and impulsivity following medication treatment of ADHD. Similarly, psychotherapeutic interventions may be helpful for ADHD cases with high levels of emotional dysregulation with partial or no response to ADHD drug treatments, which could be accounted for by BPD. We therefore advocate a more nuanced approach to the management of people presenting with both ADHD and BPD.An important question arising from the literature is the specificity of emotional symptoms that are seen in both ADHD and BPD. However, symptoms reflecting dysregulation emotional responses are also seen in other mental health disorders. A recent EMA study examined the dynamics of affective instability in patients with BPD compared with post-traumatic stress disorder and bulimia nervosa . Using tGiven the emerging genetic findings in relation to ADHD and BPD, and the overlap of symptoms such as ED, there may be gains from comparing the cognitive-neural underpinnings for ADHD and BPD, as well as overlapping symptom domains such as ED. At this stage, clinical trials are needed to evaluate the role of both ADHD medication and psychotherapy in the treatment of comorbid ADHD-BPD, and to identify treatment prognostic indicators. Under current circumstances, we suggest that health care professionals involved in diagnosing patients with either BPD or ADHD need to be aware of the potential diagnostic overlap and co-occurrence of these two disorders. Further, there should be sufficient clinical expertise to ensure that patient receive the evidence based treatments they require. This includes the potential benefits of drug treatments for ADHD, and psychotherapy for BPD.


Additionally, during the 48 h recovery period, pomegranate juice supplementation accelerated (p < 0.05) the recovery kinetics of the malondialdehyde (5.6%) and the enzymatic antioxidant defenses compared to the placebo condition (9 to 10%). In conclusion, supplementation with pomegranate juice has the potential to attenuate oxidative stress by enhancing antioxidant responses assessed acutely and up to 48 h following an intensive weightlifting training session. Therefore, elite weightlifters might benefit from blunted oxidative stress responses following intensive weightlifting sessions, which could have implications for recovery between training sessions.The aim of this study was to test the hypothesis that pomegranate juice supplementation would blunt acute and delayed oxidative stress responses after a weightlifting training session. Nine elite weightlifters (21.0 ± 1 years) performed two Olympic-Weightlifting sessions after ingesting either the placebo or pomegranate juice supplements. Venous blood samples were collected at rest and 3 min and 48 h after each session. Compared to the placebo condition, pomegranate juice supplementation attenuated the increase in malondialdehyde (−12.5%;  Oxidative stress reflects an imbalance between oxidant production and antioxidant responses where the former exceeds the latter. It is well documented that strenuous exercise acutely increases oxidative stress biomarkers and is accompanied by a prolonged pro-oxidant redox status following such exercise ,2,3,4. IOn the other hand, there is emerging evidence that supplementation with pomegranate juice (POMj), which is a rich source of polyphenols and other biologically active compounds  ,13, confAlthough it is established that polyphenol-rich POMj can improve numerous physiological processes in individuals placed under stress ,15,16 anThe aim of the present study was to assess whether consumption of natural POMj could reduce the immediate increase of oxidative stress responses and accelerate the recovery of such responses following a weightlifting training session. We hypothesized that the consumption of natural POMj could blunt acute and delayed oxidative stress responses following a weightlifting training session, which may help in recovering the resting levels of redox parameters at 48 h post exercises.Nine elite male weightlifters )) volunteered to participate in this study. The participants were recruited on the basis of: (i) they trained at least five sessions per week, (ii) they had at least 3 years’ experience of Olympic weightlifting,(iii) they did not have any injuries, and (iv) they did not use any antioxidant or anti-inflammatory drugs during the experimental period nor for one month before commencement of the study . AdditioOne week before the start of the experimental period, the heaviest weight lifted in a single repetition (1-RM) was assessed for each participant in each Olympic movement . ThereafBefore test sessions, participants underwent an overnight fast and were only permitted to drink one glass of water (15–20 cL) to avoid the potential confounding influence of postprandial thermogenesis ,26. AddiThe natural POMj was prepared from a fresh pomegranate fruit 48 h before the beginning of the experimentation and was frozen and stored at −4 °C. No additional chemical products were added to the natural POMj. Each 500 mL of POMj contained 2.56 g of total polyphenols, 1.08 g of orthodiphenols, 292.6 mg of flavonoids, and 46.75 mg of flavonols . PLA juin-hexane and 4 mL of a methanol/water  mixture in a 20 mL centrifuge tube. After vigorous mixing, they were centrifuged for 3 min. The hydroalcoholic phase was collected, and the hexane phase was re-extracted twice with 4 mL of the methanol/water  solution each time. Finally, the hydro alcoholic fractions were combined, washed with 4 mL of n-hexane to remove the residual POMj, then concentrated and dried by evaporative centrifuge in vacuum at 35 °C.The phenolic extracts were obtained following the procedure of Chtourou et al.  with som2 = 0.990). The optical density (OD) was measured at λ = 765 nm using a spectrophotometer . The concentration of o-diphenolic compounds in the methanolic extract was determined by the method of Dridi-Gargouri et al. [o-diphenolic content was expressed as milligrams of GA equivalent per kilogram of POM . The OD was measured at λ = 370 nm, using the same spectrophotometer.The determination of the total phenolic compounds was performed by means of the Folin-Ciocalteau reagent using the method described by Gargouri et al. . The toti et al. . The tot2O. At zero time, 0.30 mL 5% Sodium nitrite (NaNO2) was added to the flask. After 5 min, 0.30 mL 10% Aluminium chloride (AlCl3) was added. At 6 min, 2 mL (1 mol·L−1) NaOH was added to the mixture. Immediately, the reaction flask was diluted to volume with the addition of 2.40 mL of double-distillate H2O and thoroughly mixed. Concerning the absorbance of the mixture, pink in color, it was determined at 510 nm versus prepared water blank. As for the total flavonoids of fruits, they were expressed on a fresh weight basis as mg/100 g catechin equivalents. It is worth noting that the samples were analyzed in triplicate.Total flavonoids were measured by a colorimetric assay developed by Gargouri et al. ; 1 mL al2O2 concentration [2O2 was added at low concentration (0.2 M) to a sample with CAT, this enzyme catalyzed the transformation of this substrate to oxygen and water. To check the activity, a kinetic curve had to be measured for 30 s at λ = 240 nm using a molar extinction coefficient of 43.6 cm−1M−1 to know the amount of H2O2 eliminated [ε = 6.22 × 103 cm−1M−1. One CAT or GPX enzymatic unit (units) was defined as the amount of the enzyme that catalyzes the conversion of one micromole of substrate per minute. Malonaldehyde (MDA) was measured according to procedures described by Wong et al. [Blood samples (7 mL) were collected from a forearm vein. Samples were placed in an ice bath and immediately centrifuged at 2500 rpm and 4 °C for 10 min. Aliquots of the resulting plasma were stored at −80 °C until analysis. To eliminate inter-assay-variance, all samples were analyzed in the same assay run. All assays were performed in-duplicate in the same laboratory with simultaneous use of a control serum from Randox. UA and Tbil were determined spectrophotometrically  using uricase and diazonium methods, respectively, and using commercial kits  with an intra-assay-coefficient of variation of 0.5% [ntration . When H2iminated . To deteiminated . This meg et al. . Plasma 2) for the ANOVA analysis to assess the potential practical significance of the findings. Pearson correlation was used to assess the correlation between the responses of MDA and the antioxidant markers. Statistical significance was set at p < 0.05 and data are presented as mean ± SD unless otherwise stated.All statistical analyses were performed using STATISTICA 10.0 Software . Normality of the data distribution was confirmed using the Shapiro-Wilks-W-test. To analyze the effect of POMj supplementation on the biological responses during training sessions , a two-way analysis of variance ANOVA (2 levels (supplementation (PLA and POMj)) × 2 levels (training-session (Pre and Post)) with repeated measures) was employed. To analyze the effect of POMj supplementation on the recovery kinetics of the selected parameters, a one-way ANOVA was utilized. When significant main effects were observed, Tukey’s honest-significance-difference (HSD) post-hoc tests were conducted. Effect sizes were calculated as partial eta-squared (ηpp = 0.0002) and POMj conditions (p = 0.0006). However, the rate of increase was lower (−12.47%) after POMj supplementation compared to that of PLA supplementation (p < 0.01). Similarly, biomarkers of enzymatic and non-enzymatic antioxidants increased pre to post training session completed with both the PLA  and POMj  supplements. However, the rates of increase pre/post training session were enhanced after POMj supplementation . For all these parameters , a significant training-session  and POMj  effects were observed. Additionally, significant interactions (Training session × supplementation condition) were observed for MDA (p = 0.02), CAT (p = 0.04), and UA (p = 0.03).The oxidative stress responses immediately (3 min) after the weightlifting training sessions are presented in p = 0.004 for MDA, p = 0.008 for CAT and GPX, p = 0.005 for UA, and p = 0.007 for Tbil) and POMj  conditions. However, the rates of decrease were higher (p < 0.05) following POMj supplementation compared to that of PLA supplementation .p > 0.05). However, following PLA supplementation, the resting values of the non-enzymatic antioxidants  were recovered after 48 h, but the values of MDA and the enzymatic antioxidants  remained elevated after the same period .Moreover, after supplementing with POMj, the 48 h recovery period was sufficient to recover the resting values of all parameters  and non-enzymatic antioxidant markers with the highest correlation registered between MDA and UA . With regard to the delayed response of MDA during the 48 h recovery period, the change in the rate of lipid peroxidation was only correlated  and GPX .rrelated  to the eThe aim of the present study was to investigate the effect of supplementation with a natural POMj on the acute and delayed oxidative stress responses following a weightlifting training session. The main results showed that, compared to PLA, the consumption of POMj 48 h before and during the training session enhanced the recovery kinetics of the acute and delayed oxidative stress responses. Specifically, there was: (i) a reduction in the immediate increase of the MDA (−12.47%), (ii) an increase in acute antioxidant responses , and (iii) an acceleration in the delayed recovery kinetics of MDA (5.63%) and the antioxidant markers  with POMj compared to that of PLA supplementation.+), catecholamine production, as well as muscle damage, and inflammation post-intensive exercise have been also suggested to contribute to the increased oxidative stress [The present study reported a significant training-session effect on the oxidative stress parameters  with higher values following a training session compared to pre-training session values in both experimental conditions. These findings are in line with previous studies reporting increased oxidative stress biomarkers following weightlifting exercises ,4,8. Exee stress ,3,7,34. e stress ; excessie stress , which we stress ,38. Thesr = 0.7 and 0.6, respectively) during the training session in the present study. Therefore, it could be suggested that the antioxidant activities of UA and CAT have the highest effect on reducing the acute MDA response immediately following intensive physical exercise completed after POMj supplementation. The apparent efficacy of POMj as an antioxidant might be attributed to its effectiveness as a polyphenol donor [In this study, POMj supplementation showed a significant impact on the acute post-exercise MDA and enzymatic and non-enzymatic antioxidant responses. Specifically, a lower rate of increase in MDA and greater rate of increase in antioxidant markers were observed immediately post training in the POMj trial compared to the PLA trial. Previous studies in sedentary participants have demonstrated a potent antioxidant effect of POMj on neutralizing reactive oxygen species (ROS) ,17,18,39ol donor .r = 0.6 and r = 0.5, respectively), it seems that enzymatic antioxidants have a greater potential to mitigate the delayed oxidizing effects of ROS exposure. In contrast, results from the present study indicate that POMj supplementation during the recovery period facilitated the return of the post-training-session values of all parameters to their resting concentration levels. Indeed, the results showed that the rates of decrease of MDA, CAT, and GPX were higher  with POMj compared to that of PLA supplementation. The more rapid recovery kinetics of lipid peroxidation and antioxidant markers following high-intensity resistance exercise with POMj supplementation confirms the potent antioxidant properties of polyphenol-rich POMj reported previously [After PLA ingestion, only UA and Tbil returned to resting levels 48 h following training with MDA, CAT, and GPX remaining elevated compared to the resting values. These observations confirm previous results showing that a 48 h recovery period lowers MDA levels and antioxidant responses compared to values observed immediately post-training, but was not sufficient to completely recover lipid peroxidation and enzymatic antioxidant parameters to resting levels ,4. Howeveviously ,27. Indeeviously .2+, Cu2+) involved in free radical production [Although the exact mechanisms underlying the antioxidant effects of POMj are not entirely understood ,21, thesoduction ,45. Otheoduction , enhanceoduction , the mododuction , and recoduction ,45. HoweDespite our findings suggesting that POMj supplementation has the potential to blunt oxidative stress responses following weightlifting exercise, it has been previously reported that the acute oxidative stress response following such exercise sessions is greater in the morning compared to the evening session (+12%) . TherefoIn conclusion, the results of the present study extend on previous studies reporting improved protection against oxidative stress in healthy and diseased conditions after POMj consumption by indicating that POMj supplementation can reduce the acute oxidative stress response to an intensive session of resistance exercises by enhancing antioxidant responses and accelerating the recovery kinetics of oxidative stress markers. These findings about reduced oxidative stress following POMj supplementation might have implications for recovery of performance following intensive resistance training.The present study is the first to demonstrate a potential protective effect of polyphenol-rich POMj supplementation on the degree of lipid peroxidation induced by intense weightlifting exercises, as inferred from a lower circulating MDA content. However, it should be acknowledged that since exercise has been shown to provoke oxidative modifications to several biological components  , and sinUsing the same protocol of the present this study, a recent study reported an increase in the total (+8.3%) and maximal (+3.3%) load lifted during a weightlifting training session, and a lowering in markers of muscle damage and the delayed onset of muscle soreness 48 h after the weightlifting training session after POMj supplementation compared to that of placebo supplementation . Therefo


Objective: This study assessed which moderators influenced the effectiveness of a low-intensive behavioral teacher program for children with symptoms of Attention-Deficit/Hyperactivity Disorder (ADHD).Methods: Primary school children (N = 114) with ADHD symptoms in the classroom were randomly assigned to the intervention program  or control group . Multilevel regression analyses assessed differential treatment gains of the intervention program in terms of ADHD symptoms and social skills. Moderators included demographic characteristics , severity and comorbidity of problem behavior , social functioning, and classroom variables .Results: Results revealed larger program effects for older children and children from highly educated families and smaller beneficial effects for children with comorbid conduct or anxiety problems.Conclusion: The intervention program seems more beneficial for highly educated families and children without comorbid problem behavior, but more intensive treatments appear necessary for children facing additional challenges.ClinicalTrials.gov registration number: NCT02518711 There is a large body of evidence indicating that children with attention-deficit hyperactivity disorder (ADHD) benefit from behavioral teacher programs  comprised primary school children (6–13 years) who displayed high levels of ADHD symptoms in the classroom and took part in an earlier study assessing the effectiveness of the PR program  receiving the PR program (44 classrooms of 30 schools), or the waitlist control group  that could receive care as usual . Teachers of the participants in the waitlist control group received the PR program after having participated in this study.The study sample . Demographic characteristics and outcomes on screening measures are displayed in Table 1.Inclusion criteria were (a) high levels of teacher-rated ADHD symptoms (>90th percentile) based on the Hyperactivity/Impulsivity and/or Inattention scale of the Disruptive Behavior Disorders Rating Scale , with the single exception of the element ‘provide the child with three compliments after a reprimand is given’ which was used on average 61% of the time (SD = 0.37). Most teachers (81%) reported adequate or good implementation of the universal reward system during the entire intervention, with the remaining 19% reporting inadequate implementation in 1 or 2 weeks during the course of the entire 18 weeks. Teachers reported to have used all elements of the DRC adequately most of the 18 weeks . Only 15% of the teachers consulted the helpdesk during study participation.Teachers reported to have used all elements of the universal program for at least 85% of the time during the 18 intervention weeks  was administered . The SSRModerator variables were measured prior to the start of the intervention to assess which factors moderated the effectiveness of the PR program.The demographic characteristics used as moderators in the current study were age (in years), gender (using boys as reference group), and parental educational level. Parental educational level was measured as the average of both parents’ educational level using an adapted version of the Dutch educational classification system  and severity of conduct problems and internalizing problems at baseline was assessed with the SDQ . Each SDQ scale  consistsr = 0.47 and r = 0.44; The moderators related to social functioning were teacher-reported social skills as measured by the SSRS (see section “Outcome Measures”) and peer acceptance. Peer acceptance was measured through peer ratings, in which participants and their classmates were asked to indicate how much they liked each classmate on a 5-point rating scale  were assessed using the same instruments as described above. Hence, the SDQ (teacher version) was used to measure conduct problems and internalizing problems, the SCAS as measure of self-reported anxiety and the Teacher SSRS and peer ratings as measures of social skills.The moderating classroom variables used in current study were teaching experience (in years) and class size .o), 6 weeks after start of the PR program (T1), and after 18 weeks at the end of the intervention (T2). For the children in the intervention group, the SWAN was also administered in week 9, 12, and 15 after start of the intervention to monitor progress and to adjust the intensity level of the individual program . Although the universal program was used in the entire classroom, only children displaying ADHD symptoms in the classroom were included as participants in this study. Dependent variables were measured at three time points: at baseline . If an interaction involved the SWAN ADHD scale (measuring ADHD symptoms), the term ‘relatively high’ and ‘relatively low’ levels of ADHD symptoms were used to describe children scoring at one of the extreme ends of the ADHD continuum. Alpha-level was set at 0.05.The moderating effects of comorbid problem behaviors  were assessed by looking at three-way interactions between group and all possible pairs of comorbid problem behaviors . Similar three-way interactions were used to assess whether the level of social skills in combination with other problem behaviors  moderated treatment gains on social skills. In case of significant three-way interactions, Table 2. The three-way interactions assessing the moderating effects of the program are not displayed in Table 2, but the significant three-way interactions are visualized in Figures 1 and 2.Results of the multilevel regression analyses, assessing what moderators influenced the effectiveness of the PR program, are displayed in b(SE) = -0.50(0.11), p < 0.001 for the SWAN and b(SE) = -0.87(0.35), p = 0.017 for the SDQ ADHD). With regard to the SWAN, moderating effects were found for Age and Parental educational level, indicating that the intervention effect on ADHD symptoms was more beneficial for older compared to younger children (b(SE) = -0.12(0.05), p = 0.036) and for children from highly educated families compared to lower educated families (b(SE) = -0.37(0.10), p < 0.001). No significant moderating effects were found for any of the other variables on ADHD symptoms as measured by the SWAN.Before assessing which moderators influenced the effects of the PR program on ADHD symptoms as measured by the SWAN and the SDQ ADHD scale, primary analyses confirmed positive intervention effects on ADHD symptoms when controlling for baseline ADHD symptoms (b(SE) = 0.04(0.02), p = 0.014). To illustrate this interaction, post hoc analyses were conducted to assess treatment effects for four different groups . Results of these post hoc analyses  suggest that children with relatively high levels of ADHD symptoms  and high levels of anxiety problems (>18 on the SCAS that ranged from 1 to 78) did not profit from the PR program in terms of ADHD symptoms (b(SE) = 0.34(0.63), p = 0.603). Significant treatment gains were found for children with relatively high levels of ADHD symptoms but low levels of anxiety (b(SE) = -1.52(0.56), p = 0.013), meaning a reduction of almost one standard deviation on the SDQ ADHD scale . No significant treatment effects were found for the other two groups . No other significant moderating effects were found on ADHD symptoms as measured by the SDQ ADHD.Results on the SDQ ADHD did not reveal a significant moderating effect for Age nor for Parental educational level. The SDQ results did reveal a significant moderating effect for comorbid anxiety problems, suggesting smaller program effects for children with high levels of both ADHD symptoms and self-reported anxiety (b(SE) = 3.26(1.26), p = 0.003). Results revealed a significant moderating effect for peer acceptance (b(SE) = -2.47(0.87), p = 0.005), indicating that the intervention was more effective for children who were less accepted by peers at baseline. Furthermore, results also showed a higher effectiveness of the PR program for children with low levels of both conduct problems and teacher-rated social skills (b(SE) = 0.16(0.06), p = 0.014). This finding was confirmed by post hoc analyses assessing treatment effects for four different groups  by dichotomizing both variables through median split . Post hoc results showed largest treatment gains for children scoring low on both social skills and conduct problems (b(SE) = 4.67(1.85), p = 0.018) compared to the other groups . This gain for children scoring low on both social skills and conduct problems indicated that social skills of these children improved approximately half of one standard deviation on the SSRS . No other significant moderating effects were found on social skills.Before assessing the moderating effects on social skills, primary analyses confirmed that that social skills of children in the intervention group significantly improved compared to children in the control group when controlling for baseline social skills , indicating that the effect of the PR program on ADHD symptoms was larger for older children and for children from highly educated families. Results on the SDQ ADHD measure suggest larger treatment gains for children with both high levels of ADHD symptoms and low levels of anxiety, while program effects appeared absent for children with high levels of both ADHD symptoms and anxiety. The effects of the PR program on social functioning was significantly moderated by peer acceptance, indicating that the intervention was more effective for children who were less accepted by peers at baseline, and by the interaction between social skills and conduct problems, indicating superior program effects for children who scored low on social functioning and did not have serious conduct problems.This study isolated what type of children would benefit most from our self-help behavioral teacher program. Results show that our intervention might be more effective for older children, for children from highly educated families, and for children with high levels of ADHD symptoms in the absence of other problem behavior . These findings are in line our hypothesis that this low-intensive behavioral teacher program could be more effective for children from highly educated families, and those without many comorbid psychopathology. For children from low educated families and for those with severe comorbid problem behavior, more intensive therapeutic support might be needed to successfully reduce ADHD symptoms and improve social functioning .Regarding the moderating role of age, our results are in line with  evidence that also revealed larger effects of behavioral programs for older children  althoughThe absence of a moderating effect of gender is in line with the MTA study where gender did not moderate treatment outcomes either , and sugOur finding that the PR program is more effective for children from highly educated parents is in line with several reviews discussing predictors and moderators of treatment outcomes in children with ADHD . In mostThe current results indicate that the PR program is equally effective for all children with ADHD symptoms, regardless of the severity of their ADHD symptoms, which is in line with meta-analytic evidence . HoweverFor children scoring low on social functioning, the PR program appears to be more effective in improving teacher-rated social skills, particularly if conduct problems are absent or not very severe. Possibly, children with limited social skills who do not display oppositional or aggressive behavior  have more room to improve their social functioning thanks to enhanced classroom structure and positive reinforcement provided by the PR program. In contrast, children with both limited social skills and conduct problems, might be more likely to oppose the changes made in the classroom during the implementation of the PR program, which could thus result in smaller benefits for those children.The current findings need to be interpreted in light of the following limitations. First, power was somewhat limited due to the relatively small sample size, urging cautious interpretation of the current findings. Second, our sample mainly comprised boys, limiting the reliability of the finding that the PR program would be equally effective for boys and girls. This problem is common in ADHD research though, where boys are clearly overrepresented in the diagnosed ADHD population . Third, This study investigated the moderating effects of several child and classroom variables on the effectiveness of a low-level behavioral teacher program (PR program) in children with ADHD symptoms. Results indicated that the PR program could be more effective for older children and for children from higher educated families, while comorbid conduct or anxiety problems in combination with high levels of ADHD symptoms appeared to impede treatment gains. Hence, it appears that this self-help teacher program is suitable for children not facing additional challenges, but may not be able to address the needs of children from low educated families and those with comorbid psychopathology. The current findings underline the importance of using the PR program in a preemptive stage before behavioral problems escalate or transfer to multiple domains.BV: data collection, statistical analyses, literature review, and writing entire manuscript. ML and JO: article revisions.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.


The results revealed that: (i) EULP perhaps originated from fatty acid binding protein (FABP) revealed in evolutionary analysis; (ii) Dynamic simulation study shows that EULP is highly stable at below 0.45 Å of root mean square deviation (RMSD); (iii) Docking evaluation shows that EULP has higher binding energy with farnesol and 2-iso-butyl-3-methoxypyrazine (IBMP) than 2-naphthol; and (iv) Competitive binding and quenching assay revealed that purified EULP has good binding interaction with farnesol. Both, In-silico and experimental studies showed that EULP is an efficient binding partner to pheromones. The present study provides impetus to create a point mutation for increasing longevity of EULP to develop pheromone trap for rodent pest management.Transportation of pheromones bound with carrier proteins belonging to lipocalin superfamily is known to prolong chemo-signal communication between individuals belonging to the same species. Members of lipocalin family (MLF) proteins have three structurally conserved motifs for delivery of hydrophobic molecules to the specific recognizer. However, computational analyses are critically required to validate and emphasize the sequence and structural annotation of MLF. This study focused to elucidate the evolution, structural documentation, stability and binding efficiency of estrus urinary lipocalin protein (EULP) with endogenous pheromones adopting  Recognition of social responses and sexual advertisement are greatly mediated by chemosignals, and the chemical information has signaling properties that are communicated among members of the same species through pheromones1368Chemosignals such as thiazole and brevicomin are present in male mouse urine. It has been reported that transport of these chemical messages occurs when they are bound with MUPs and generate broad-ranging effects in the congeners101117The rat MUPs have a lot of structural similarity to the members of lipocalin superfamily1322in-silico and experimental studies.A computational approach is necessary to find the evolutionary clustering pattern of EULP so as to facilitate group categorization and gene ontology (GO) of MLF protein-protein interactions (PPIs) network and pathway association. The networks will be of help in identifying functional annotation of protein interaction pathways based on similarity with the network of another well-characterized speciesin-silico study. Further, it is important to evaluate the binding efficiency of farnesol with EULP by fluorescence binding assay. The central objective is that the elucidation of EULP binding efficiency with pheromones would facilitate development of novel methods for rodent pest management.Therefore, we performed computational analysis of rodent EULP to generate knowledge about sequence and structural annotation and to find the functional backbone. Hence, the present investigation focused on PPIs network, topology, structural superimposition, dynamic changes in structural conformation, and evaluation of pheromone interactions with EULP adopting The computational analysis consisted of sequence analysis and structural annotation followed by stepwise procedure for MD simulation and docking analysis .rd cluster of rat FABP. Therefore, the preliminary sequence of EULP has the largest percentage of alignment with FABP and confirms that it shares conserved sequence motifs to backbone structural fold  values. The evolutionary analysis revealed that the MUP, FABP and retinol binding protein (RBP) are from KL. Also, the distinct more diversified group of OBP belongs to OL. Additionally, the EULP clade occurs as neighbor cluster (NC) in IIIral fold .More than 400 GO entries with p-values were collected and assigned to three major categories viz., biological process (BP), molecular functions (MF) and cellular components (CC). The redundant GO entries were filtered for retrieving a positive dataset. Then GO entries were submitted to reduce visualize gene ontology (REViGO) tool for generating scatter plot of MF  and thenThe PPI network, with 50 nodes and 38 edges, was identified and visualized using Cytoscape (Version.3.2.1). The PPI was characterized and found to have clustering coefficient of 0.3, 1.52 average numbers of neighbors, and the characteristic path length was 1.4. The density of the network was 0.031, heterogeneity 1.3, diameter (∅) 3, and radius (r) 1, without any self loop. The shared neighbors and shortest path length distributions were as shown in The integrated proteins datasets were connected by edges to the nodes of proteins, which indicated the molecular interaction between the proteins. The depiction of P-value significance with log size enrichment as different colored parameter scales are red, blue, green, and yellow stripes for 0x-2x, 2x-4x, 4x-6x, and 6x to a maximum of 7.6x, respectively . The bluThe analysis revealed that the EULP sequence has 16 putative phosphorylation sites viz., Ser2, Ser14, Ser32, Ser56, Ser83, Tyr20, Thr8, Thr12, Thr40, Thr54, Thr61, Thr75, Thr84, Thr86, Thr103, and Thr126, respectively. Further, the EULP showed many phosphorylation kinase sites; among them 10 PKC and 2 PKA were included. Further, PKG, CKI, CKII and INSR has each a phosphorylation site . The kinThe conserved residue analysis of EULP was performed using the ConSurf server. Conservation score was assembled into 1–9 color grade scale. The variable and conserved residue positions present in the EULP were identified. The predicted status of buried (b) and exposed (e) residues showed up in the first row below the sequence. Functionally conserved residues (f) are exposed whereas structurally conserved residues (s) are buried according to neural-network algorithm. The analysis revealed that the predicted functional and structural residues of this protein are highly conserved . There wst β-sheet followed by 310 α-helices in N-terminal (residues 7–15). The topology of H-bond interaction in EULP is shown in The secondary structure-based topology of EULP was obtained from ProFunc server from European Bioinformatics Institute (EBI) and the backbone structure showed significant anti-parallel topology . The secStructural superimposition was performed for prediction of pair-wise structural alignment between the EULP and FABP (PDB ID: 4A60_A) using improved protein block alignment (iPBA) webserver. The structural similarity score was measured using root mean square deviation (RMSD) between both Cα atom of modeled protein and the corresponding template. The results revealed that the EULP model attained identity of 92.3% for 131 aligned residues with a low RMSD of 0.17Å on their backbone atoms. The quality of alignment showed green color in grade scale, which evidently confirmed the high similarity of the model aligned with template and the global similarity of the two proteins as calculated by global distance test total score (GDT_TS) was 92.2. The cartoon representation of structurally superimposed EULP with FABP comprises the topologically equivalent regions represented as I, II, and III square boxes . The ana2) and volume (A3). The potential surface active sites comprise polar atoms in functional region with the highest number of hydrophobic residues. The molecular surface (MS) method was used to measure the area and volume of the protein of interest, which showed 21 binding pockets. Interestingly, the binding pockets 18 and 21 have more hydrophobic residues, in which the phosphorylation site residues such as Ser2, Tyr20, Thr54, Ser56, Thr61, and Thr75, are included (Computed atlas of surface topography of proteins (CASTp) server was used for binding pocket prediction of EULP, which identified and measured as many as of 21 surface active sites with area  interval in the whole protein simulation and the changes are denoted in angstrom units (Å). The EULP obtained lowest RMSD below 0.45 Å and the deviation started from below 0.1 Å  in the phosphorylation site residues such as Ser2, Thr8, Thr54, Ser56, and Thr61. The other phosphorylation site residues such as Try20, Ser32, Thr75, Thr86 and Thr126 have RMSF values in between 0.2 to 0.25 nm  duration with different time scales using GROningen MAchine for Chemical Simulations (GROMACS) Version.4.5.5. During MD simulation, the lowest potential energy of the protein ∆E = −5.3611494e + 05 confirmed that the homology-modeled structure was highly stable and the maximum force was E = 9.9024097e + 02 on 967ow 0.1 Å . The roo 0.25 nm . NotablyProtein-ligand interaction analysis is important to identify the best fit suitable ligand from flexible docking of EULP adopting Discovery Studio. The screening procedure is fully based on physico-chemical properties of pheromone compounds. Seven pheromones and one fluorescent compound (2-naphthol) were screened and considered for visual inspection . ChemicaThe results suggested that significantly two compounds farnesol (CID_445070) and 2-isobutyl-3-methoxypyrazine (IBMP) (CID_32594) have excellent binding affinity and energy to receptor. Compound CID_445070 showed the highest LibDock score of +92.8, and absolute binding energy of +32.7 kcal/mol, with one H-bond connected with EULP; the oxygen atom of Ser2 formed hydrogen bond with the hydrogen atom of farnesol and the distance was . Compound CID_32594 showed higher LibDock score, binding energy of +60.8 and +32.6 kcal/mol, thus forming two H-bonds with EULP, wherein the first H-bond interaction formed between the HG of Ser2 residue and the N-atom present in the benzene ring of IBMP compound , and a second H-bond interaction was formed between the oxygen atom of IBMP and -NH- atom of Leu67 . Also, the 2-naphthol (CID_8663) has a classical H-bond , LibDock score of +59.7 and binding energy of +21.1 kcal/mol with EULP. The residual interactions such as electrostatic and van der Waals forces were represented in pink and green color bubbles . The maxth binding pocket of EULP, which are represented in Interestingly, the first two compounds, CID_445070 and CID_32594 showed, good binding affinity towards EULP and the LibDock score with binding energy of compounds higher than the other compounds, 2-(octylthio) ethanol (CID_19079); 2-sec-butyl-4,5-dihydrothiazole (SBT) (CID_162148); 6-hydroxy-6-methylheptan-3-one (HMH) (CID_129891), 1,2,4 triazone-3,5-diamine (Guanazole) (CID_15078), hydroperoxide (CID_62472), and 2-naphthol (CID_8663), which were subjected to the docking experiments. The absolute energy, LibDock score, H-bonds and bond length along with steric interacting residues of EULP for the selected compounds is shown in The EULP was purified from urine of female rat using gel-filtration chromatography. The column  was packed with Sephadex G-50, and equilibrated with 10 mM Tris-HCl buffer (pH 7.8). After the equilibration, the concentrated urine was poured onto the top of the column in a single motion and eluted with the same buffer at a flow rate of 1 mL/h. The fractions were analyzed for optical absorbance at 280 nm with a UV visible spectrophotometer and the purity was checked once again by 12% SDS-PAGE. The highly concentrated protein was observed at low molecular mass of 14.5 kDa0 at 350 nm and 420 nm in competitive binding experiment (where F is the fluorescence intensity in the presence of farnesol and F0 is absence of farnesol).In fluorescence binding study, the emission and excitation states appeared in different concentrations of protein and endogenous compound. Two important fluorescence experiments viz., competitive binding and florescent quenching, were used to evaluate the binding efficiency of protein and ligand (farnesol) with standard fluorescence probe (2-napthol). In competition and quenching experiment, the fluorescence intensity was measured and the results showed that the increasing concentration of farnesol specifies that 2-naphthol is displaced from the protein binding site by farnesol . The flu0/F measured (The potassium iodide (KI) quenching analysis revealed that the intensity of endogenous farnesol bound more prominently with EULP, which reflects that increased concentration of linear peak was observed for farnesol with EULP compared to 2-naphthol as ratio Fmeasured .It has been shown earlier that mammals in general, and rodents in particular, release many volatile compounds, which are secreted along with body exudates. These volatiles facilitate recognition of conspecifics towards different physiological and behavioral responsesThe odorant binding proteins in general and EULP in particular, belong to lipocalin superfamily. MLF proteins are characterized by their molecular recognition properties which reflect on the capability to bind cell surface receptors and small hydrophobic moleculesThe phylogenetic tree also revealed EULP as distinct from RBP which occurred in the fourth cluster and OL of OBP which aligned with the second cluster. This inference provided lead for further analyzing EULP as an FABP. PPI network provides the valuable framework for perception of the protein functional architecture with interpretation of biological data. Thus, PPI network is useful in deciphering the biological process with which a protein is associatedIt is an established fact that PPI analysis throws light on protein-protein interaction which in turn will greatly depend on the phosphorylation sites in the protein of interest. Therefore, we went for phosphorylation site prediction using Netphos 3.1The Conseq and Consurf are tools that predict the functionally and structurally conserved residues. Particularly, among the buried (B) and exposed (E) states were 23 functionally conserved and 4 structurally conserved residues were identified. More specifically, Gly7, Trp9 and Phe17 are highly conserved. It is to be noted that Gly and Trp are the most reported -GXW- motif regions. This finding threw indication about the residues which are present in the ligands and/or pheromone binding pocketsTopology prediction is an important step to understand the folds and structural motifs. In this analysis, EULP was revealed to contain 3 helices, 10 strands, 8 beta hairpins, 2 beta bulges, 1 helix-helix interaction, and 14 beta turns. Thus in EULP, beta turns supersede the α-helices. This is an important information for us in the sense the preponderance of beta sheets is clear indication that EULP is not a membrane protein but perhaps an extra-cellular functional protein. The topology prediction adopting ProFunc provided for understanding H-bond interaction within residue-residue contacts33st binding site identified from area and volume measurements. Further, the results revealed that the 18th and 21st binding pockets are having more hydrophobic amino acid residues compared to other pockets. The hydrophobic residues are capable of hydrogen bond interaction to the corresponding endogenous ligands. Thus, this part of the result substantiates that the modeled EULP is the best one in the light of having many ligand binding pockets. It further substantiated accuracy and structure of the EULP model.In the next step, we went for binding site analysis adopting CASTp sever, which reveals cavities on protein surface as well as specific amino acid positioning within it to create the physico-chemical properties needed for a protein to perform its function. It also reveals annotated functional information of specific residue on protein structurein-silico analysis led systematically through phylogeny construction, GO and PPI analysis, phosphorylation site analysis, conservation analysis, topology prediction, superimposition, binding site analysis, molecular dynamic simulation and molecular docking. It guided us to conclude that EULP is a binding protein which would potentially bind fatty acid or its derivatives. The conserved residue and topology were revealed and the model was proved to be the best fit. Since functionally the protein would bind fatty acid or its derivatives, further analysis led us to infer that farnesol and IBMP would be the most appropriate ligands that would bind with the protein. Farnesol, in a form bound to α2u-globulin, has been identified in the perpetual gland of house ratWe then subjected the EULP model to analysis of stability and conformational changes at different time scales using MD simulation. The analysis revealed that backbone of the protein has stable structure with lowest RMSD and less RMSF values, which also indicated that the EULP model has the best structural conformation. The MD simulation showed that the energy of the protein was maintained at standard level at the time of simulation and the protein could be used for structure-based drug designing and virtual screening with molecular dockingThe IBMP is an organic liquid state aromatic odorant having green bell pepper odor. It is insoluble in water and a standard compound used in many competitive binding assays of OBP41430/F was measured for 2-naphthol in the presence of EULP at increasing concentrations of farnesol on excitation peak. The binding efficiency obtained by the dissociation constant (Kd) of EULP with 2-naphthol was measured as Kd = 0.42 ± 0.06 μM and it is understood that farnesol might possess the same Kd as that of 2-naphthol. Therefore, the residue, Ser2 reveals similar interaction and optimal binding energy with farnesol and 2-naphthol. This shows that Ser is a dynamic and capable residue for EULP structure modification and for better interaction with pheromones. This preliminary report demonstrated that farnesol has more hydrophobic affinity with binding pockets of EULP. The present results will facilitate our effort to develop a pheromone trap for use in rodent pest management using farnesol and/or EULP, with the expectation that if farnesol in the trap is bound to EULP there will be slower release of the volatile such that the trap will provide for sustained release of the attractant. Based on the predictions, we propose that chemosignal communication in the rat might be facilitated by EULP, and this can act as a carrier protein so as to decode chemical signals of pheromones and odorants to attract the congeners. The results obtained from this study would facilitate generation of a point mutation in EULP adopting protein engineering technologies so as to increase its longevity and thus to develop a novel pheromone trap for rodent pest management (RPM) program.In order to predict the pheromone interactions of intact EULP, competition and quenching analysis were performed using 2-naphthol with KI as quencher. The fluorescence quenching intensity ratio Fhttp://www.ncbi.nlm.nih.gov) and protein data bank (PDB) (http://www.rcsb.org). The retrieval of similar proteins was based on iterated profile search with alignment scoring matrix, which was made by position-specific iterated basic local alignment searching tool (PSI-BLAST)http://www.bioinformatics.org/cd-hit)The MLF protein sequences of rat such as MUP, FABP, OBP, and RBP were retrieved from non-redundant protein (nr) database of National Centre for Biological Information (NCBI) The resulting 34 non-redundant sequences were submitted for analysis of multiple sequence alignment (MSA) executed by GONNET weighted matrix with gap extension in ClustalW2 program  connected to PANTHER classification system. The predicted GO term was used to assign proteins to distinct clusters and generate a scatter plot using REViGO analysis that is specific to molecular function (http://revigo.irb.hr)Identification of GO ID, enrichment, and elucidation of p-values of EULP sequence were carried out through GO consortium (http://www.cbs.dtu.dk/services/NetPhos-3.1/)Phosphorylation sites and kinase-specific phosphorylation sites such as Ser (S), Thr (T) and Tyr (Y) of EULP sequence were predicted using the online web servers NetPhos 3.1 (http://consurf.tau.ac.il/)The EULP model was analyzed for the identification of functional residues and mapping conserved regions in both exposed and buried residues of structure using ConSeq and ConSurf tool (http://www.ebi.ac.uk/thornton-srv/databases/profunc)The prediction of EULP structural topology was carried out using various web-based online servers such as ProFunc (Homology-modeled structure of EULP and its template structure (PDB: 4A60_A) were superimposed using iPBA (http://www.dsimb.inserm.fr/dsimb_tools/ipba/index.php). This algorithm was used for pair-wise comparison of multiple structures and it employs anchor-based dynamic programming for identifying structurally conserved regions as local alignments. The degree of structural similarity between backbone Cα atom pairs of the model and the respective templates was calculated by RMSD. The iPBA was extensively outperformed using a nontrivial benchmark dataset and compared to other well established structural alignment methodshttp://sts.bioe.uic.edu/castp/calculation.php) online web serverThe active site of the modeled EULP was analyzed using CASTp (https://bioinformatics.cineca.it/PMDB/). The database provides information about manually built homology-modeled proteins in a manner simple to deposit or retrieve the model in PMDB. The EULP model was deposited successfully and accepted in PMDB. After submission, a unique identification number (PMDB ID: PM0079851) was provided by PMDB for the EULP model.The 3D coordinates of homology-modeled protein was structurally documented and deposited in Protein Model Database (PMDB) (http://www.gromacs.org/)http://www.ks.uiuc.edu/Research/vmd/) graphics software was used for visualization of the EULP simulation outputs.The MD simulation was carried out for the modeled EULP using GROMACS V.4.5.5 (https://pubchem.ncbi.nlm.nih.gov/) and, then from these, 11 compounds were filtered and eliminated to retrieve the naturally secreted rodent pheromones and odorants in relation to the organisms which produce them. The selection was narrowed down to 7 compounds via Lipinski rule of five (RO5) web server (http://www.scfbio-iitd.res.in/software/drugdesign/lipinski.jsp) and docking experiments. The selected ligands are GuanazoleThe structural coordinates of about 32 endogenous pheromone compounds (ligands) were retrieved from PubChem (2), density, boiling, melting point, octanol/water partition coefficient and Lipinski RO5 were tabulated and presented in Physico-chemical properties of all ligands were identified from various databases such as PubChem, ChemSpider, and DrugBank . In addihttp://accelrys.com/products/discovery-studio/).Interaction of novel endogenous ligands with the EULP model was analyzed by docking module in Discovery Studio. Accelrys Software Inc (V.2.1)  was used for molecular docking studies (Rattus rattus) were trapped at residential areas close to Bharathidasan University, with proper permission from the house owners. The reproductively active female rats (150–200 g) were selected and acclimatized to laboratory conditions for a month. The rats were housed in polypropylene cages (40 × 25 × 15) with 2 cm rice husk lining at the bottom as hygienic bedding material, which was changed once every three days. The regular estrous cycling animals were selected for further experimental study. All animals were maintained in 12:12 L & D cycle throughout the experimental period. All the experimental procedures were followed according to the guidelines of Institutional Animal Ethics Committee (IAEC) and also the experimental protocols were approved by Bharathidasan University, Tiruchirappalli.The female house rats , maintained at metabolic cage for three consecutive estrous cycle and then the urine samples were prepared according to Muthukumar HTX multi-mode microplate reader  and FP-6500 spectrofluorometer. Fluorescence determinations were made using a tungsten halogen lamp with a PMT detector for maximum sensitivity and the data collection, analysis of results, and exporting were done by Gen5 Data Analysis Software.In this study, 2-naphthol was used without further purification and the concentration of EULP protein (24 μM), and 2-naphthol (1 mM stock) were prepared in 100% ice-cold ethanol, which was stored in the dark at 4 °C. The 2-naphthol was further diluted for each experiment to a final concentration of 25 μM and the 10 mM phosphate buffer used at pH 7.2 at room temperature in all experiments. All pH values were measured by a digital pH meter with a combined glass-calomel electrode . Fluorescence intensity and the measurements were carried out using Synergy We were interested in analyzing the binding efficiency of EULP with endogenous pheromone, farnesol -3, 7, 11-trimethyldodeca-2, 6, 10-trien-1-ol) using competition experiments. The farnesol stock solution was 0.7 mM in ice cold ethanol and it was used to displace 2-naphthol from the binding site of EULP. The fresh 2 M solution of KI was used as quencher in fluorescence analysisHow to cite this article: Rajesh, D. et al. Structural elucidation of estrus urinary lipocalin protein (EULP) and evaluating binding affinity with pheromones using molecular docking and fluorescence study. Sci. Rep.6, 35900; doi: 10.1038/srep35900 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


BackgroundRecent studies point to overlap between neuropsychiatric disorders in symptomatology and genetic aetiology.AimsTo systematically investigate genomics overlap between childhood and adult attention-deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD) and major depressive disorder (MDD).Methodn = 93), childhood ADHD (n = 17), MDD (n = 63), ASD (n = 51), childhood dual diagnosis of ADHD–ASD (n = 16) and healthy controls (n = 78).Analysis of whole-genome blood gene expression and genetic risk scores of 318 individuals. Participants included individuals affected with adult ADHD (ResultsWeighted gene co-expression analysis results reveal disorder-specific signatures for childhood ADHD and MDD, and also highlight two immune-related gene co-expression modules correlating inversely with MDD and adult ADHD disease status. We find no significant relationship between polygenic risk scores and gene expression signatures.ConclusionsOur results reveal disorder overlap and specificity at the genetic and gene expression level. They suggest new pathways contributing to distinct pathophysiology in psychiatric disorders and shed light on potential shared genomic risk factors. Worldwide prevalence rates for the five major psychiatric disorders range from 0.2% for attention-deficit hyperactivity disorder (ADHD) to 5.7% for major depressive disorder (MDD), with schizophrenia, autism spectrum disorder (ASD) and bipolar disorder showing intermediate prevalence.7 Large genome-wide studies have now started to reveal risk variants for individual disorders but an outstanding question is whether genetic risk factors or polygenic risk scores are specific to particular disorders, or shared. For instance, certain copy number variations (CNVs) are overrepresented in both patients with schizophrenia and those with ASD.9 Additionally, calcium-channel activity genes have been shown to have shared involvement in ASD, schizophrenia and bipolar disorder, whereas polygenic risk analysis showed strong overlap of genome-wide risk between bipolar disorder, MDD and schizophrenia, and moderate overlap between ASD and schizophrenia.10 In another sample, high sharing was seen between bipolar disorder and schizophrenia, with moderate overlap between MDD and schizophrenia, bipolar disorder and ADHD and low, but significant, overlap between schizophrenia and ASD.11 These results suggest that some shared molecular genetic factors underlie a significant proportion of the risk for development of several psychiatric disorders. To date, however, it is not known whether these genomic overlaps are reflected in overlapping patterns of gene expression in tissues. In this study we therefore explored whole blood gene expression across adult and childhood ADHD, ASD, MDD and healthy controls, using weighted gene co-expression analysis to search for patterns of correlated gene expression between disorders as well as disorder-specific gene expression signatures. In addition, we generate polygenic risk scores to assess overlap between disorders on a genome-wide genetic-risk level. We next investigate whether differences in polygenic risk scores are reflected in disorder-related gene expression profiles and whether they can be used to tease apart genetic and environmental influences on gene expression.Twin study heritability estimates for psychiatric disorders are moderate for MDD (~40%) and high (80–90%) for ADHD, ASD and schizophrenia, indicating a significant genetic component in their aetiology.Participants were from four different projects and included individuals affected with adult ADHD, childhood ADHD, MDD, ASD, childhood dual diagnosis ADHD–ASD and healthy controls . Becausehttps://github.com/snewhouse/BRC_MH_Bioinformatics), excluding sample and probe outliers, after which robust spline normalisation and log2 transformation were applied.12 After excluding samples based on low-quality expression profiles we included only participants with full phenotype data  and RNA concentration per sample). This left 318 participants and 5638 probes for analysis (13 using a linear model. Surrogate variable (SV)1 was not correlated to known covariates, but SV2 and SV3 were highly correlated to cell proportion estimates by the CellMix package:14 SV2 ; SV3 . No correlation of surrogate variables or principal components was found with self-reported ethnicity .Whole-genome gene expression data of a total of 424 individuals were generated using the Illumina HT-12.v4 BeadChips at the SGDP/BRC BioResource Illumina core lab according to the manufacturer's protocol. We rigorously quality controlled and preprocessed the data using a standard pipeline  by the criteria described by Zhang & Horvath.16 Soft-thresholding results in a 5638 × 5638 dimensional weighted adjacency matrix containing pairwise connection strengths. Subsequently, a topological overlap measure is calculated based on the number of shared neighbours. Modules were then defined as branches of a hierarchical clustering tree using a dissimilarity measure . Each module is subsequently assigned a colour. To define a representative module expression profile for each module, we summarised the (standardised) gene expression profiles of the module by their first principal component. This statistic is referred to as the module eigengene: it can be thought of as an average gene expression value for all probes in a module per sample. In order to identify hub genes, we calculated a connectivity measure (‘module membership’) per probe by correlating expression values with the relevant module eigengene. Probes with high module membership are defined as ‘hub genes’ of their module.Corrected gene expression data were analysed using weighted gene co-expression analysis (WGCNA).17 Participants were assigned a main diagnosis of a particular disorder  v. all other participants. We investigated the effect of possible covariates on the association of module eigengenes with our phenotypes of interest. This led us to correct for gender, RIN and RNA concentration. Medication use defined as dichotomous measures of use of antidepressants (n = 73), stimulants (n = 49), antipsychotics (n = 7), benzodiazepines (n = 5) and mood stabilisers (n = 5) were investigated as a possible covariate. Considering the nature of the sample collection of the childhood ADHD–ASD and childhood ADHD samples, analyses were repeated also including age as covariate, allowing us to tease out diagnosis and age-specific effects. We used a Bonferroni threshold for significance .The module eigengenes were correlated to phenotype of interest using a linear model.18 In addition, we performed connectivity mapping on selected modules to investigate overlap with drug-induced gene expression changes through the LINCS/CMap database (http://apps.lincscloud.org).19 We entered the gene content of modules of interest as upregulated, resulting in a list of compounds of which the application to cell lines results in a similar gene expression pattern.Modules of interest were tested for enrichment of blood cell type lists using the userListEnrichment function in WGCNA package with five as minimum number of genes in a pathway. This function compares the number of overlapping genes to the maximal possible overlap and applies Bonferroni correction. Enrichment analyses were performed in WebGestalt using Wikipathways database.For most individuals genotype data were available and after quality control and imputation polygenic risk score analysis and genetic pathway analysis was carried out to investigate the relationship of genome-wide risk to disorder-specific gene expression findings in this study .v. all other participants, covarying for gender, RIN, concentration in one model and also including age in a second model. This results in 7 phenotypes × 6 module eigengenes = 42 tests per model, therefore 84 tests in total when considering both models. We applied a Bonferroni threshold for significance of P = 0.05/84 = 6 × 10−4. Results for the first model are listed in The WGCNA on 318 participants and 5638 probes resulted in seven gene co-expression modules, ranging from 2077 probes in the turquoise module to 80 probes in the red module. The grey module contains 675 probes not belonging to any other module, representing background noise. The network dendrogram is given in online Fig. DS3 and all probes and corresponding module assignments in online Table DS1. The module eigengenes representing a summary of all genes in a given module were related to our traits of interest: participants with a main diagnosis of a particular disorder  P = 0.02) in the green module. The red module, however, is enriched for immune-related pathways: type II interferon signalling , type III interferon signalling .The green (186 probes) and red module (80 probes) eigenvalue estimates per individual correlated negatively with MDD and positively with adult ADHD status , Fig. 1.http://apps.lincscloud.org)19 revealed 200 compounds corresponding to upregulation of genes in the green module, and 3273 to upregulation in the red module. This implies that application of these compounds to cell lines results in a similar gene expression profile as we find for adult ADHD, and opposite to that for MDD. Among the compounds, there were several tricyclic  and other  antidepressants. In addition, there are steroid-related drugs  and other inflammatory drugs . Full connectivity mapping results are given in online Table DS2.Connectivity mapping  and red  modules, indicating that highly connected hub genes generally show the strongest associations with the phenotypes. The six main green hub genes are YY1, AAK1, PAK2, C20ORF94 (now SLX4IP), PAPD5 and SPATA13. The expression of transcription factor YY1 indeed shows one of the highest negative correlations with MDD status, together with TMEM189–UBE2V1, PSMC1, WDR82 and LOC730052 (now UBE2V1P2). M6PR, TPM3, TOB1, ZNF364 (now RNF115) and RAD21 show highest positive correlation with adult ADHD. For the red module, the hub genes are the interferon genes IFIT3, IFI35 and XAF1, PARP14 and two probes for UBE2L6. Of these, PARP14 is highly inversely correlated to MDD and adult ADHD, as are MX1 and EIF2AK2. In addition, two probes for STAT1 show high gene significance for MDD and OAS2 for adult ADHD.P = 2.2 × 10−22), electron transport chain  and oxidative phosphorylation . The correlation between gene significance and connectivity is r = −0.48, P<2.2 × 10−16 for childhood ADHD and r = −0.48, P<2.2 × 10−16 for MDD. Major hub genes in this module are ZRANB2, TMEM126B, RPL15, PCNP and SRP9. The genes with highest MDD gene significance are LOC1001291, ANXA1, RNF13, GMFG and H3F3B. For childhood ADHD these are SUMO3, RPL22, UTP3, POLE3 and CCDC50.The turquoise module (2077 probes) is positively correlated with both MDD and childhood ADHD status . This efP = 8 × 10−4), focal adhesion , prostate cancer  and IL17 signalling pathway . The correlation between childhood ADHD gene significance and connectivity is r = −0.5, P<2.2 × 10−16. Blue hub genes are WAS, MOBKL2A, C15orf39, G6PD and GNAI2. Genes with highest negative correlation to childhood ADHD disease status are TMUB2, STAT5B, DENND3, CA4, FCGR3B.The blue module (1672) is negatively correlated to childhood ADHD status . This efWe did not find enrichment of GWAS signal or association of the module eigengenes with polygenic risk scores for the modules of interest .21 we do not find an immune gene expression signature for these groups, as we do for MDD and adult ADHD.This study of whole-blood gene expression of several psychiatric disorders aimed to reveal overlapping gene expression patterns and disorder-specific signatures. The WGCNA on 318 participants revealed seven gene co-expression modules. Of these, two small modules are inversely related to MDD and adult ADHD, the large turquoise module is associated with both MDD and childhood ADHD and finally, the blue module shows a disorder-specific signature for childhood ADHD. No significant results were found for the ASD, ADHD–ASD and control groups. Even though there is some evidence for increased immune-related comorbidities in ASD23 Previous literature about genetic overlap between MDD and ADHD has yielded conflicting results, reporting a genetic correlation between MDD and ADHD,11 but no significant overlap in polygenic risk scores.10 A reason for these discrepancies could be the lack of distinction between adult ADHD and childhood ADHD in previous studies, which could add to phenotypic heterogeneity.5 The group with childhood ADHD may include individuals whose condition will become less severe with age, whereas the adult group with ADHD contains individuals whose condition is chronic. Another reason why we chose to analyse participants with childhood and adult ADHD in separate groups is because of anticipated biological heterogeneity between children and adults in the context of peripheral gene expression. An explanation for our findings would be gender differences in prevalence of MDD and adult ADHD, however, analyses were corrected for gender and stratified analyses yielded the same results (data not shown). We also performed a correlation with the module eigengenes and an indicator of current state of depression (Beck Depression Inventory24) within participants, which did not show a significant association (data not shown).Two small modules, red and green, are negatively correlated to MDD status, but positively correlated to adult ADHD status. GWAS on both disorders have not yielded genome-wide significant hits to date.YY1, WDR82 and AAK1. YY1 is a transcription factor involved in many processes including transforming growth factor beta (TGF-β) signalling, but has also shown to be active in histone modification.25 In addition WDR82 (WD repeat domain 82) is part of SET1A/SET1B histone H3K4 methyltransferase complexes,26 which genetic pathway analyses has shown to be strongly enriched for association in MDD, bipolar disorder and schizophrenia.27 This implication of epigenetic processes provides a mechanism by which environmental influences can exert their influence on gene expression and thereby contribute to the pathology of neuropsychiatric disorders.29 The green hub gene adaptor-associated kinase 1 (AAK1) is of interest because it is a positive regulator of the Notch pathway.30 This pathway is traditionally implicated in cell-fate determination during development, but has important function in tissue homeostasis and neuronal plasticity later in life. In addition, it has been implicated to play a role in immune functioning.33The green module shows enrichment for a cell signalling category and harbours some interesting highly connected hub genes that also show high gene significance, most notably, 34 Expression differences in cytokines have been shown to differentiate patients with MDD from ones with bipolar disorder and controls35 and studies have shown regulation of the serotonin receptor through cytokines and neurotrophins.37 Importantly, cytokines and polymorphisms in interleukin genes have been shown to predict antidepressant treatment response.39 The majority of patients with MDD (70%) and a few (13%) of the participants with adult ADHD in the current study were on antidepressant medication although medication use did not correlate significantly with any gene expression module. However, connectivity mapping revealed that the upregulation of genes in red and green modules as seen in our adult ADHD group coincides with those seen in response to application of a number of tricyclic antidepressants, indicated for the treatment of depression and ADHD with comorbid depression. Results also contained some anti-inflammatory drugs, converging with the module enrichments for immune system genes. This could support the hypothesis that anti-inflammatory drugs such as non-steroidal anti-inflammatory drugs (NSAIDs) might have a role in the treatment of MDD and, our data suggests, also ADHD. Literature on the effects of NSAIDs in patients with MDD is, however, mixed.41 Our results suggest potential drug repositioning opportunities for NSAIDs for both MDD and ADHD.Even though the immune system has not been implicated in adult ADHD before, there are many studies on its association with MDD although the relationship is still somewhat controversial.42 In order to fully untangle this, we would need childhood diagnoses of ADHD in the MDD cohort, but this was not available.Whereas MDD and adult ADHD showed opposite patterns of gene expression changes (the green and red modules), we found an overlapping gene expression signature between MDD and childhood ADHD, in the form of the turquoise module. One possible interpretation of this finding is that childhood ADHD increases risk for developing MDD in later life.G6PD and WAS. Even though we corrected for gender in our analyses, the childhood ADHD group consisted solely of males, which might explain downregulation of X-chromosome genes, although the blue module is not enriched for genes on sex chromosomes. The significance of the association of childhood ADHD with the turquoise and blue modules disappears when adding age as a covariate in the analyses, but in the absence of a healthy child control group, age and childhood ADHD cohort membership are highly correlated. However, the fact that the childhood dual diagnosis ADHD–ASD group does not show a correlation with these modules suggests that age cannot fully explain these results.Finally, we find a disorder-specific signature of childhood ADHD in the blue module, which is highly enriched for cell signalling genes. There are two hub genes located on the X-chromosome; 43 did not reveal a significant enrichment for the modules of interest (data not shown). Likewise, a sample-handling or collection effect is unlikely because of initial batch correction and the fact that the healthy controls were for the most part from the same project as the participants with MDD and yet do not show an effect for the relevant modules. In addition, the lack of genetic association could also be the result of the initial GWAS results being underpowered to detect variants associated with MDD, ADHD and ASD.We did not find enrichment of GWAS signal or association of the module eigengenes with polygenic risk scores for the modules of interest . This could indicate that the differences in gene expression are driven by environmental rather than genetic factors or, perhaps, that polygenic scores are not yet strong enough in disorders such as MDD and ADHD. One possible known environmental influence on gene expression is smoking. Even though we did not have access to smoking behaviour for all participants, a Fisher's exact test of enrichment of smoking-related genes44Future research could extend this study to include a broader range of psychiatric disorders, such as psychotic disorders (schizophrenia) and anxiety disorders , in order to better understand the genomic correlates of different syndromes. However, our results have several limitations including power to detect effects in relatively small samples and the reliance upon cross-sectional study designs. Our findings in adult ADHD and MDD will require replication and assessment in different study designs to assess potential therapeutic applications. Also, in this study we examined gene expression in blood but it will be important to determine whether the pattern of results holds true for brain tissue. In ASD, for instance, changes in the expression of a number of genes has been reported to be altered in post-mortem studies.In conclusion, in a study of gene expression in peripheral blood of patients with psychiatric disorders and healthy controls, we identified both cross-disorder and disorder-specific signatures for adult ADHD and MDD. With the caveats discussed above, they suggest new pathways contributing to distinct pathophysiology in psychiatric disorders and shed light on potential shared genomic risk factors.