Attention deficit hyperactivity disorder (ADHD) which is characterized by developmentally inappropriate levels of attention, hyperactivity and impulsivity, is considered as the most common neurodevelopmental disorder in childhood. Physical exercise has shown to have several benefits in the improvement of children with ADHD. In this meta-analysis, we aimed to systematically show, with evidence, the impact of physical exercise on children with ADHD.http://www.ClinicalTrials.gov were the searched sources for studies which were based on the impact of physical exercise on children with ADHD. Relevant endpoints were assessed. This evidence based meta-analysis was carried out by the most relevant RevMan 5.3 software. Due to the involvement of continuous data (mean and standard deviation), weight mean difference (WMD) with 95% confidence intervals (CI) were used to represent the final analysis. A significant level of P ≤ .05 was set and a fixed statistical effect model was used throughout the analysis.Web of Science, MEDLINE, EMBASE, Google Scholar, Cochrane Central and P = .00001). Hyperactive/impulsive symptoms  and inattention symptoms  were also improved with physical exercise but the results were not statistically significant. This evidence based analysis showed thought problems ], P = .0007), social problems ], P = .0001), and aggressive behaviors ], P = .02) to have significantly been improved in participants with ADHD who were assigned to physical activity group.Fourteen studies with a total number of 574 participants with ADHD were included in this evidenced based meta-analysis. Two hundred and seventy six (276) participants were assigned to the physical activity group whereas 298 participants were assigned to the control group. Results of this analysis showed that anxiety and depression were significantly improved with physical activity in these children with ADHD ], This current meta-analysis showed with evidence, that physical exercise has a major contribution owing to significant improvement in anxiety and depression, aggressive behaviors, thought and social problems among children suffering from ADHD. Therefore, physical exercise should be incorporated in the daily life of children with ADHD. Further future research should be able to confirm this hypothesis. The diagnosis is mainly based on parents’ and teachers’ reports, with reference to learning disabilities, anxiety and conduction disorders.Attention deficit hyperactivity disorder (ADHD) which is characterized by developmentally inappropriate levels of attention, hyperactivity and impulsivity, is considered as one of the most common psychiatric disorders and as the most common neurodevelopmental disorder in childhood. health, social and academic domains contributing to motivational and learning disabilities and anxiety disorders as well as impulsive behaviors resulting in a negative impact on themselves and therefore contribute to the development of suicidal ideas later during adulthood. A higher risk of academic failure, and poor psychological and occupational outcomes have strictly been related to ADHD. This disorder is also associated with substantial long-term depressive outcomes and mortality.Children with ADHD often have significant impairment in occupational, However, the use of pharmacological drugs have potentially serious adverse events limiting their use in certain cases. Poor adherence, partial or non-response to medications, and uncertainty about the long-term costs of medications, beneficial and side effects are the other limitations of pharmacological therapy for ADHD.To improve the symptoms of ADHD in these children, pharmacological treatment is associated with long-term beneficial effects and has been considered as first line therapy. Several recent studies support the benefits of physical exercise on the cognitive performance and the management of behavioral symptoms in children with ADHD.,17 However, there is limited evidence to show the effects of physical activities on childhood ADHD.Physical exercise has always been beneficial and is considered as a preventive measure for developing or the exacerbation of several chronic medical conditions, and disabilities. would aim to show the effects of physical activity on executive function in children with ADHD. However, it was only a study protocol and there is still no information about when the final study will be published.A systematic review and meta-analysis protocol which was published in the year 2019,In this current meta-analysis, we aimed to systematically show, with evidence, the impact of physical exercise on children with ADHD.22.1http://www.ClinicalTrials.gov were the searched sources for studies which showed the impact of physical exercises on children with ADHD.Web of Science, MEDLINE, EMBASE, Google Scholar, Cochrane Central and The searched terms included:1.Attention deficit hyperactivity disorders;2.Attention deficit hyperactivity disorders and physical exercise;3.Attention deficit hyperactivity disorders and physical intervention;4.Attention deficit hyperactivity disorders and exercise;5.Attention deficit hyperactivity disorders and physical activity;6.ADHD and physical exercise.2.2Criteria for inclusion consisted of studies:(a)Which were randomized trials/observational cohorts (prospective or retrospective studies)/case-control studies which were published before July 2019;(b)Showing the comparison of physical exercise versus a control group in children with ADHD;(c)Reporting relevant endpoints based on the impact of physical activities on health improvement status of children with ADHD such as aggressive behaviors, perseverative errors, internalized and externalized problems, social and thought problems;(d)Published in English language.2.3Criteria for exclusion consisted of studies:(a)Showing the impact of physical exercise in children with ADHD without comparing it to any control group;(b)Relevant endpoints based on health improvement status were not reported;(c)Published in a different language;(d)Literature reviews, systematic reviews and meta-analyses;(e)Repeated studies.2.4Table 2.5The endpoints which were assessed in this meta-analysis included:1.Hyperactive/impulsive symptoms;2.Anxiety and depression;3.Inattention symptoms;4.Oppositional symptoms;5.Thought problems;6.Social problems;7.Aggressive behaviors;8.Strength and agility;9.Internalized problems;10.Externalized problems;11.Perseverative errors;12.Non-perseverative errors;13.Stroop color-word response.2.62), the mean and standard deviation values associated with each endpoints since these values would be required during data analysis, and the types of studies; that is, whether the study involved was a randomized trial, an observational cohort (retrospective or prospective) or a case-control study.The author extracted data including the common endpoints  reported in the original studies consisting of the total number of participants in the physical and non-physical activity groups who suffered hyperactive/impulsive symptoms, anxiety and depression, inattention symptoms, oppositional symptoms, thought problems, social problems, aggressive behaviors, strength and agility, internalized and externalized problems, perseverative and non-perseverative errors, and stroop color-word response; data concerning the methodological quality of the studies, the total number of ADHD children assigned to the physical exercise and the control groups respectively, the baseline characteristics including the percentage of boys and girls with ADHD who were assigned to the physical activity versus the non-physical activity group respectively, the mean age which was reported in years and mean body mass index (BMI) of the participants which was reported in kilogram per meter square .The quality assessment of the randomized trials was carried out with reference to the criteria suggested by the Cochrane CollaborationBased on the assessment, a grade was allotted to each study, grade ‘A’ indicating a low risk of bias, grade ‘B’ indicating a moderate risk and grade ‘C’ indicating a high risk of bias.2.8P ≤ .05 was set and a fixed statistical effect model was used throughout the analysis. In addition to the Q statistic test with a P value ≤ .05 showing the level of significance, heterogeneity was also assessed by the I2 statistic test. The I2 value was reported in percentage. Heterogeneity was determined by an increasing I2 value. The higher the I2 value for a particular subgroup, the greater the heterogeneity for that particular subgroup.This evidence based meta-analysis was carried out by the most relevant RevMan 5.3 software. Due to the involvement of continuous data (mean and standard deviation), weight mean difference (WMD) with 95% confidence intervals (CI) were used to represent the final analysis. A significant level of During data extraction, a mean and standard deviation were provided from the parents’ as well as from the teachers’ perspective view. Therefore, in this analysis, we have calculated an average value of this mean and standard deviation to proceed with the results.Sensitivity analysis was also calculated by an exclusion method. In addition, publication bias was observed through visual assessment of the funnel plot.2.9Ethical or board review approval was not required for this meta-analysis since data were extracted from previously published studies.33.1 An initial elimination of 1386 articles were due to irrelevance. Seventy (70) full text articles were assessed for eligibility based on the criteria for inclusion and exclusion.A total number of 1456 publications were obtained from the above mentioned searched databases (PRISMA guidelines).Full text articles were eliminated since:(a)They did not consist of control group (n = 15);(b)They were literature reviews, systematic reviews and meta-analyses (n = 6);(c)They did not report relevant outcomes (n = 6);(d)They were case studies (n = 8);(e)They were duplicated studies (n = 21).–26,27–34 were included in this evidence based meta-analysis, as shown in Figure Finally, 14 studies3.2A total number of 574 participants with ADHD were included in this evidenced based meta-analysis. Two hundred seventy six 276) participants with ADHD were assigned to the physical activity group whereas 298 participants with ADHD were assigned to the control group, as shown in Table 76 particEight (8) studies were randomized trials, and 6 studies were observational studies.Bias risk assessment of the randomized trials based on the Cochrane Collaboration, and the bias risk of the observational studies based on the NOS resulted in an average grade B score indicating a moderate risk of bias in each of the trial or study.The baseline features including the total number of boys vs girls with ADHD, the mean body mass index of the participants, and the average age were listed in Table 3.3P = .00001) as shown in Figure P = .93) and inattention symptoms  were also improved with physical exercise, but the results were not statistically significant, as shown in Figure This evidence based meta-analysis showed that anxiety and depression were significantly improved with physical activity in these children with ADHD ], P = .0007), social problems ], P = .0001), and aggressive behaviors ], P = .02) to have significantly been improved in participants with ADHD who were assigned to physical activities as shown in Figure P = .21).This evidence based analysis showed thought problems ], P = .01), internalized problems , externalized problems , perseverative errors  and stroop color-word test  as shown in Figure The results for strength and agility (WMD: 5.69; 95% CI: [1.13 – 10.25], A summarized version of the results has been shown in Table Consistent results were obtained throughout. Publication bias was visually observed through a funnel plot which has been demonstrated in Figure 4 In our current meta-analysis, we aimed to show, with evidence, the benefits of physical exercise in children with ADHD.Researchers have found intense exercise or physical activity to have major beneficial effects in children with ADHD.Results of this analysis showed with evidence, that physical exercise significantly improved anxiety and depression, thought and social problems as well as any aggressive behavior. Hyperactive/impulsive as well as inattention symptoms were also improved, but without statistical significance. The authors stated that aerobic exercise had a major impact compared to yoga on these beneficial effects.Similarly, another meta-analysis involving 8 randomized trials based on aerobic and yoga exercises showed yoga and aerobic exercises to effectively improve hyperactivity, anxiety, impulsive disorders and social problems in children with ADHD. A significant improvement in cognitive functions and social competency was noted in these children suggesting a major impact of physical exercises in children with ADHD. Similarly, another study showed physical activity to increase attention and calmness in a 4 year old ADHD boy. Another study has even demonstrated a possibly hypoactive attentional system in children with ADHD that could be induced and enhanced through engagement in physical exercise.In a 6 week prospective trial, including 12 sessions of education and sports therapy whereby the participants were involved in a 90 minutes athletic activity twice a week, the authors demonstrated a strong so-relation of physical sports with improvement of symptoms of attention, cognitive and social skills in children with ADHD.Our current analysis showed that physical activities could reduce and improve anxiety and depression among children with ADHD. Even though this is a major benefit, other studies have shown that the duration of depression might also have an impact on the outcome. To be more clear on this matter, Ghio et al showed that untreated depression reported for a shorter time period was associated with better favorable outcomes and these patients responded well to the treatment given. The authors stated that even if the study designs and the physical activities reported in the included studies varied considerably, all of them lead to similar conclusions indicating the beneficial effects of physical exercise in children with ADHD and led to significant improvements in terms of neuropsychological parameters, strength, motor skills and social behavior.Moreover, another review was also in favor of this current analysis. Parental stress will decrease and parental confidence in their children with ADHD will be increased.Therefore, with this positive impact of physical exercise in children with ADHD, it should be encouraged to improve the condition of children with ADHD in the society. Improvement in the academic field, ADHD children with healthier outcomes, can easily adapt and cope with normal children without ADHD, and would further contribute to the progress of a community, a country, or a continent. A society consisting of a subgroup of patients with ADHD but without depression, without suicidal attempts would further contribute to the well-being and progress of a country. It would be easier to set up better work environment later for these patients with ADHD.,45 Our current analysis showed physical exercise to significantly improve social problems in children with ADHD. Therefore, social training skills would also be improved without the requirement of significant assistance, and implication and patience of parents and teachers would be to a lower extent if these children become socially capable.Social skill training is important in children with ADHD. Therefore, improving depression through physical exercise by these children with ADHD will result in far less use of pharmacological drugs, thus sparing these children from adverse drug events representing another benefit of physical activity on ADHD.Depression is a major problem in children with ADHD. Scientific reports have shown pharmacological drugs which are used for the treatment of ADHD to further exacerbate depression in these subjects. This has been demonstrated in a case study involving a 7-year-old boy with ADHD who was on a minimum dose of methylphenidate. Increasing the dosage of the drug resulted in the development of clinical signs of depression. However, this unwanted condition was resolved when the drug was withdrawn.5The limitations of this analysis were: first of all, only a total number of 574 participants were included. This minor number of participants might not lead to robust analysis. However, data were limited on this particular topic and there was no additional data that could be included. Secondly, the type of physical activity varied in this study. A few studies were based on swimming, basketball, and other physical training. However, since our analysis was based on physical exercise, all these activities were subsets of physical exercises. Thirdly, one study involved adolescents with a mean age of 21 years whereas all the other studies involved children. Another limitation could be the fact that three or four studies were not involved during data analysis since their endpoints were unique and were not reported in other studies for us to be able to make a comparison. Also, a high level of heterogeneity was observed in several of the subgroups. At last, only one researcher was involved in this study, and data extracted by only one researcher could result in bias, further limiting the strength of the results obtained.6This current meta-analysis showed with evidence, that physical exercise has a major contribution owing to significant improvement in anxiety and depression, aggressive behaviors, thought and social problems among children suffering from ADHD. Therefore, physical exercise should be incorporated in the daily life of children with ADHD. Further, future research should be able to confirm this hypothesis.Conceptualization: Yu Zang.Data curation: Yu Zang.Formal analysis: Yu Zang.Funding acquisition: Yu Zang.Investigation: Yu Zang.Methodology: Yu Zang.Project administration: Yu Zang.Resources: Yu Zang.Software: Yu Zang.Supervision: Yu Zang.Validation: Yu Zang.Visualization: Yu Zang.Writing – original draft: Yu Zang.Writing – review & editing: Yu Zang.


Attention deficit hyperactivity disorder (ADHD) is a childhood mental health disorder that can lead to alcohol and other drug (AOD)-related problems if it persists into adolescence and adulthood. Several findings suggest that ADHD contributes to the development of AOD use disorders. ADHD generally precedes alcohol use and is correlated with developmentally inappropriate levels of alcohol use or abuse; conduct problems typically precede the development of alcohol use or abuse. The potential role of ADHD in the development of AOD use problems has important implications for prevention and treatment of such problems. For example, people with ADHD have poor outcomes from AOD abuse treatment. Service providers who work in AOD abuse treatment settings must develop the diagnostic and clinical expertise to address co-occurring ADHD and AOD use disorders. Attention deficit hyperactivity disorder (ADHD) is a childhood mental health disorder that is characterized by inattention, impulsivity, and hyperactivity. It has been discussed in the psychiatric literature for approximately 100 years under various names, including minimal brain dysfunction; hyperkinetic reaction of childhood; attention deficit disorder; and, since 1987, ADHD. The various names reflect an evolving understanding of the disorder and an increasing consensus on its nature. One of the major recent advances in the research on ADHD is the recognition that most people who are diagnosed with it as children continue to suffer problems related to this disorder as adolescents and adults . This reTo provide the appropriate background for understanding the link between ADHD and alcohol use, this article first summarizes the diagnostic criteria for ADHD. It then examines the hypothesis that ADHD is a causal factor in the development of problematic alcohol consumption. Finally, the article reviews some preliminary research suggesting that ADHD and its treatment may have important implications for the prevention and treatment of alcohol-related problems.According to the The symptoms or problems associated with ADHD vary somewhat depending on the patient’s developmental stage . ChildreDiagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision ADHD, predominantly hyperactive/impulsive subtype (ADHD–HI)ADHD, combined inattentive and hyperactive/impulsive subtype (ADHD–C).To be diagnosed with ADHD, a person must:Meet the criteria for one of the three subtypes of ADHD:Predominantly Inattentive type: six symptoms of inattention, as listed belowPredominantly Hyperactive/Impulsive type: six symptoms of hyperactivity/impulsivity, as listed belowCombined type: six symptoms from each of the lists belowHave symptoms that have persisted for at least 6 months and which must be present to a degree that creates problems and is inconsistent with developmental levelHave at least some symptoms that caused impairment before the age of 7Have some symptoms that are present in at least two different settings , and that cause clinically significant impairment in social or academic functioningHave symptoms that are not associated with another disorder .A person with Predominantly Inattentive ADHD often:Fails to give close attention to details or makes careless mistakesHas difficulty sustaining attention in tasks or play activitiesDoes not seem to listen when spoken to directlyDoes not follow through on instructions and fails to finish tasks Has difficulties organizing tasks and activitiesAvoids or dislikes tasks that require sustained mental effortLoses things necessary for tasks or activities Is easily distractedIs forgetful in daily activities.A person with Predominantly Hyperactive/Impulsive ADHD often:Fidgets with hands or feet or squirms in seatLeaves seat in classroom or in other situations in which remaining seated is expectedRuns or climbs excessively when it is inappropriateHas difficulty playing or engaging in leisure activities quietlyIs always “on the go” or acts as if “driven by a motor”Talks excessivelyBlurts out answers before questions have been completedHas difficulty awaiting turnInterrupts or intrudes on others .Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision . Furthermore, these symptoms must cause impairment in two or more settings and contribute to problems before age 7, and must not be attributable to another psychiatric or medical disorder . As withClinical Workbook, may reduce this time demand. However, such tests are useful only when the number of positive responses required to indicate a diagnosis is reasonably low in order to avoid “false negatives”—cases in which a client has a disorder but does not show up as positive for the disorder on the test. The ability of screening tests for ADHD to avoid false negatives is particularly important because adolescents and adults typically underreport symptoms of ADHD  must precede the effect .ADHD and alcohol use, abuse, or related problems must be correlated, and the correlation should be large enough to mandate consideration from a theoretical or applied standpoint.ADHD must be a unique cause of alcohol-related problems, independent of other variables that might plausibly cause those problems.A fourth consideration that is helpful, but not required, is to have a theory of the mechanisms that mediate the relationship between ADHD and alcohol use. The following sections discuss data that pertain to the three necessary conditions for a causal effect. Potential mechanisms that might mediate or influence such a causal relationship are discussed in the section “Mediators Between ADHD and Alcohol Use and Abuse.”Given that a formal diagnosis of ADHD requires that problems are evident before age 7 and that 7-year-olds almost never exhibit any deviant behavior related to alcohol, the requirement that cause precede effect  is easily satisfied. It is noteworthy that the typical length of time between recognition of ADHD and the onset of drinking provides ample opportunity for interventions to prevent alcohol-related problems. Some non-pharmacological interventions for children and adolescents with ADHD and their families, such as training in effective parenting, hold promise for reducing rates of alcohol-related problems among people with ADHD e.g., . The rolThe second condition for inferring that ADHD contributes to alcohol use and abuse requires that ADHD and alcohol use be correlated. It is important to note, however, that correlation does not prove causation, but causation implies correlation. Therefore, correlation is one of the three minimum conditions for inferring a causal link between ADHD and alcohol use.At first glance, the evidence for a correlation between ADHD and alcohol use is mixed. However, careful examination of data regarding alcohol use in early adolescence and alcohol abuse and dependence in early adulthood suggests that there is a meaningful correlation between ADHD and alcohol use.In one study in which children diagnosed with ADHD and control children were followed for 8 years , investigators found that at a mean age of 14.9 years, 40 percent of the ADHD children, but only 22 percent of the control children, had used alcohol . This fiuse during adolescence and young adulthood do not necessarily imply that people with ADHD experience more problems, elevated rates of alcohol use disorders  are clear signs of a link between ADHD and AOD-related problems. Therefore, researchers have investigated the rates of alcohol use disorders in people with and without ADHD. One study of adolescents with ADHD (mean age 14.4 years) found no statistically significant differences in the rate of alcohol use disorder diagnoses  compared with a matched control group   . In a stpercent) . Becausepercent) . Althougpercent)  and thisThe fact that alcohol use disorder rates do not differ between adolescents with and without ADHD is possibly an indication that the diagnostic criteria for alcohol use disorders may not be developmentally appropriate for adolescents . It mighTo summarize, it appears that a correlation exists between ADHD and AOD-related diagnoses but that this phenomenon is mostly evident during adulthood. Also, there appears to be a correlation between ADHD and alcohol use, but this is mostly evident during early adolescence. Further study using measures that are more age-appropriate or more sensitive to differences in populations, such as measures of heavy drinking, may be necessary to document a clear link between ADHD and alcohol use during mid- to late adolescence.The third condition required for ADHD to be considered a cause of alcohol-related problems is that the presence of ADHD independently  predicts those problems. The comorbid disorder that is most often implicated as a confound to the link between ADHD and alcohol is conduct disorder (CD), or more broadly, the antisocial spectrum of behavior that includes aggressivity and oppositional defiant disorder in childhood, CD in adolescence, and antisocial personality disorder (ASPD) in adulthood. In clinical samples of children with ADHD followed into adolescence, elevated rates of AODD were found only among those who had developed CD e.g., . This fiIn a comprehensive review of the literature on comorbidity between ADHD and AODD in adults, Given the strong link between ADHD, CD, and AODD, it may be worthwhile to comment further on the relationship between CD and ADHD. CD is characterized by gross violations of social norms, such as irresponsibility, lying, criminality, and aggression.  Approximately 25 percent of children and 50 percent of adolescents with ADHD also meet the diagnostic criteria for CD . FurtherIt appears, then, that the link between ADHD and AODD is most pronounced in people with severe behavior problems. As a cautionary note, however, most of the research on this topic is based on studies with potentially significant selection biases. For example, any association between a psychiatric condition and AODD in clinical populations should be viewed cautiously because referral patterns may result in a selection bias for the more severe cases . TherefoAssuming that a causal link exists between ADHD and alcohol use, an important issue related to selecting the most appropriate treatment for patients with ADHD and an AODD is that various factors may underlie, or mediate, the relationship between ADHD and AODD. Several such mediating factors have been suggested, including certain brain chemicals (described below), relative susceptibility to developing alcohol-related problems, and levels of sociopathy.Clinical neuroscience has begun to identify some possible brain-based links between ADHD and alcohol use and abuse. Of particular interest is the dopamine hypothesis of ADHD  and the The dopamine system also has been implicated in mediating some of alcohol’s effects on the brain and therefore may play a role in the development of alcohol use disorders. Accordingly, disturbances in the dopamine system may underlie both ADHD and alcohol use disorders and may therefore contribute to the association between the two disorders.Another possible mediating mechanism between ADHD and AODDs is that people with ADHD may have lower thresholds for AOD-related problems. For example, people with ADHD typically have low baseline levels of impulse control. As a result, they would experience alcohol’s detrimental effects  after consuming less alcohol  compared with people without ADHD. Similarly, greater levels of inattention associated with ADHD could augment alcohol’s detrimental cognitive effects.It also is possible that people with ADHD use AODs in an effort to self-medicate distress related to ADHD or co-occurring conditions . Thus, iFinally, children with ADHD commonly experience social deficits and academic problems, and these problems may play important roles in the mediational chain linking ADHD and alcohol problems. Appropriate intervention with parents and teachers to provide better social structure and academic support might prevent many problems related to ADHD, including the potential for developing an AODD .Some researchers have argued that in people with co-occurring CD and ASPD there may be two mechanisms linking ADHD and alcohol use . Both ADAlternatively, low behavioral control related to extreme impulsivity in people with ADHD and comorbid CD may account for elevated AODD problems. Thus, people with ADHD and comorbid CD might be regarded as “super impulsive.” To better understand these competing, but not mutually exclusive, mediating theories, research that measures and models sociopathy and impulsivity as separate dimensions is needed.Understanding the unique treatment implications related to inattention, impulsivity, and callous/unemotional states may be one of the keys to unlocking the link between ADHD and alcohol use. Unfortunately, the current diagnostic criteria for CD muddle the distinction between impulsivity and callous/unemotional traits.Most of the available data suggest that ADHD has detrimental effects on the prevention and treatment of alcohol-related problems. For example, one of the most commonly used approaches in the prevention of AODD is cognitive therapy designed to improve self-control and appropriate problem solving. Unfortunately, these cognitive techniques do not work with children with ADHD , their eIf prevention fails, people with ADHD who develop an AODD fare poorly in AODD treatment compared with peers who do not have ADHD. In one study, people with ADHD took more than twice as long to recover from AODDs than did people without ADHD . SimilarAnother possible explanation for poor outcomes of patients with ADHD in AODD treatment is that, because of their core deficits of inattention and impulsivity, these patients may respond poorly to standard treatments based on cognitive therapy or semistructured group therapy. Effective treatment of the core symptoms of ADHD with stimulant medication may make patients more amenable to standard AODD treatment; however, stimulants work for approximately 70 percent of people with ADHD, leaving a substantial number of clients who may not respond to AODD treatment . MoreoveIn addition to treating the core symptoms of ADHD, another consideration is the high stress level observed in families of individuals with ADHD and the contribution of poor family functioning to poor AODD outcomes . There cIn summary, professionals working with clients with ADHD must be prepared for intra- and interpersonal dysfunction resulting from one or more common problematic symptom clusters, including high levels of inattention, impulsivity, or—among those with comorbid CD or ASPD—callous disregard for others. In addition, the specific problems that must be addressed as well as the appropriate therapeutic approaches most likely will differ among clients. Thus, people with the ADHD–IA subtype may have different history, prognosis, and treatment needs than do people with the ADHD–HI subtype. If comorbid CD or ASPD adds callous/unemotional traits to the clinical picture, these features also must be considered in the treatment plan. Research has not yet determined whether patients with different ADHD subtypes respond differently to treatment. It is clear, however, that ADHD patients with comorbid CD or ASPD have poorer outcomes .Clinical researchers and AODD service providers need to be prepared for co-occurring ADHD and AODD. The rate of ADHD is at least 25 percent among patients receiving treatment for AODDs, and 20 to 50 percent of adults diagnosed with ADHD meet criteria for an AODD. Furthermore, the rate of ADHD may be as high as 50 percent in high-risk populations


CLOCK, considered the master gene in the circadian rhythm, has been explored by few studies. Understanding the relationship between ADHD and CLOCK may provide additional information to understand the correlation between ADHD and sleep problems. In this study, we aimed to explore the association between ADHD and CLOCK, using several genetic markers to comprehensively cover the gene extension. A total of 259 ADHD children and their parents from a Brazilian clinical sample were genotyped for eight single nucleotide polymorphisms (SNPs) in the CLOCK locus. We tested the individual markers and the haplotype effects using binary logistic regression. Binary logistic and linear regressions considering ADHD symptoms among ADHD cases were conducted as secondary analysis. As main result, the analysis showed a risk effect of the G-A-T-G-G-C-G-A  haplotype on ADHD. A suggestive association between ADHD and rs534654 was observed. The results suggest that the genetic susceptibility to circadian rhythm attributed to the CLOCK gene may play an important role on ADHD.Circadian and sleep disorders, short sleep duration, and evening chronotype are often present in attention-deficit/hyperactivity disorder (ADHD).  Attention-deficit/hyperactivity disorder (ADHD) is one of the most common neurodevelopment disorders during childhood and adolescence, affecting ~5% of people at this age worldwide . ADHD coCLOCK is one of the most important genes of the endogenous master clock system. The main function of this gene relies on the transcription activation of downstream core clock genes and on the promotion of rhythmic chromatin opening, also regulating DNA accessibility of other transcription factors [CLOCK gene has already been associated with the evening chronotype as well as with some circadian and sleep disorders, such as delayed sleep phase syndrome [The circadian rhythm comprises a regulation underlying a 24 hour-physiological cycle, including metabolism, body temperature, hormone secretion, and sleep/wake patterns in mammals, and it is especially important for sleep behavior ,10. It i factors . In humasyndrome .CLOCK gene provide putative mechanistic links between circadian/sleep disorder and ADHD pathophysiology [CLOCK in neuronal function, mainly in dopamine output regulation, has been demonstrated [CLOCK as well as in other circadian genes’ expression, providing additional evidence linking circadian system regulation and ADHD [Animal experiments concerning the ysiology ,13,14,15nstrated . Furtherand ADHD ,14,15. CLOCK gene has been the most investigated [CLOCK gene and ADHD focused only on one genetic variant: The 3’UTR rs1801260 SNP. Only one study has evaluated several variants and observed a haplotype effect [The nature of the association between sleep and circadian problems and ADHD is unclear . An undestigated ,17,18,19e effect ,17,18,19CLOCK and ADHD described above used European and/or Asian samples. Population genetic structure, allele frequency, and heritability could vary across populations around the world [CLOCK gene is more explored, ancestry seems to be an important factor in understanding the mixed results. The association direction and magnitude seem to be highly impacted considering ancestry [CLOCK gene and ADHD has never been explored in admixed populations, especially from Latin America. Therefore, we aimed to explore the association between the CLOCK gene and ADHD, using several genetic markers to comprehensively cover the gene extension in Brazilian patients with ADHD. The association studies between he world , stressiancestry . To the A sample of 259 Brazilian probands with ADHD and their parents were enrolled in this study. The probands were recruited at the ADHD Outpatient Program (ProDAH) from Hospital de Clínicas de Porto Alegre (HCPA). ADHD was diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria . The asshttps://github.com/Nealelab/ricopili), which covers both individual and markers quality control (QC). The filters used before imputation were Hardy–Weinberg equilibrium (HWE)-p-value < 1 × 10−6 calculated from founders and exclusion of invariant variants. Blood samples were collected from probands and their parents. DNA was extracted from lymphocytes by standard procedures . DNA froThe Brazilian sample comes from a population of essentially European ancestry with little evidence of African or Native American admixture. The Principal Component Analysis considering genotyped markers shows an overlap between the Brazilian samples and the European population from 1000 Genomes Project . Additiop-value higher than 1 × 10−6.For the secondary analysis considering symptoms’ severity  were included.In order to examine the 2 test, implemented in PLINK 1.9 software [For the 259 trio data, we applied a case-pseudo control design, which is an alternative procedure to the genotype transmission/disequilibrium test in which the pseudo control is created with the non-transmitted alleles from the parents . Allele software ,29. p-value. These analyses were performed using PLINK 1.9 [p-value for multiple tests was estimated by Type 1 Error Calculator GEC [p-value thresholds to declare significant SNPs for association in a set of dependent p-values. For our set of markers, a total of six effective markers was detected. According to GEC, the p-value threshold for significance was set at 0.0081 and, suggestive, at 0.0162. Since pseudo controls are perfectly matched to each case, no covariates were used in the association analysis.In order to estimate the effect of individual markers, binary logistic regression was performed using an additive genetic model. The estimates are given through the odds ratio (OR), 95% confidence interval, and LINK 1.9 . The p-vator GEC , a Java-p-value is the probability of observing a likelihood ratio statistic at least as large as this one, if the null hypothesis was true. Then, the estimated haplotype effects against the reference are reported by OR and their 95% confidence interval. For the haplotype analyses only, haplotypes with a frequency higher that 0.01 were considered.Linkage disequilibrium and visualization were done by Haploview . HaplotyAs a secondary analysis, we explored the symptomatology of hyperactivity/impulsivity, inattention and total ADHD in patients (obtained from KSADS-PL). Symptom data was available for 221 subjects. To analyze this data, two regression models were considered: Binary logistic and linear regression. First, the symptoms were categorized using the percentile 50%. Since we identified that from this percentile, the symptom number showed a greater difference, we were able to split the sample into higher and lower scores . The cutH. sapiens genome. It attributes a score for functionality, which varies from 1 to 6 and the lower the value, the higher the probability of impact in a regulatory site. VEP determines the effect of variants  on genes, transcripts, and protein sequences, as well as regulatory regions. The CADD v1.4 provides a measure of variant deleteriousness namely the normalized score, PHRED-scaled, with higher values indicating that a variant is more likely to have deleterious effects [In order to provide further information about the putative functionality of each marker included in this study, we used the regulatory and functional information provided by Regulome DB, HaploReg v4.1 , Varian  effects .This study was approved by the Ethics Committee of Hospital de Clínicas de Porto Alegre (HCPA) (Project identification code 45210715.1.0000.5327) and was conducted in accordance with the Declaration of Helsinki. All subjects gave their informed consent for inclusion before they participated in the study. The parents provided written informed consent and probands provided verbal assent to participate.The results for the association analysis considering the eight markers included in the study are shown in CLOCK locus. High pairwise D’ values were observed, and all SNPs were in a single block. The global association test suggested a significant effect for at least one haplotype (p = 0.027). The individual haplotype effect showed that G-A-T-G-G-C-G-A was considered as a risk genetic factor for ADHD   . The logistic regression model for symptomatology showed that three SNPs were nominally associated with inattention symptoms: rs11931061 , rs3817444 , and rs726967  . The lasCLOCK variants to better explore our results. According to HaploReg and RegulomeDB data, rs4864548 and rs1801260 are placed in regulatory sites . rs1801260 is a 3’ UTR variant placed in a site of interaction with Transcriptional repressor CTCF (CTCF), Basic leucine zipper transcriptional factor ATF-like (BATF), and Signal transducer and activator of transcription 3 (STAT3) proteins. It is also an eQTL for CLOCK.In silico analyses were performed to predict the functionality of the ry sites . rs48645Considering the regulatory regions specific in brain tissue, both markers are placed in active domains of chromatin . The actAccording to CADD, the highest scores are observed for the coding variants , was associated with an increased ADHD risk. According to in sillico functionality analysis, many variants in this haplotype present a putative functional role, mainly rs4864548, rs1801260, rs6855837, and rs34897046. None of the eight variants was significantly associated with ADHD when analyzed separately. The only study in the literature exploring haplotypes in ADHD observed that a single haplotype block, covering 73 kb (rs3805148 (A/C), rs12504300 (C/G), rs4864542 (C/G), and rs12649507 (A/G)), was associated with inattention symptoms [We covered markers in a block of 131kb in the CLOCK and other clock genes and ADHD subtypes are needed. Although the functional molecular role in vivo and in vitro presents compelling findings linking the gene with ADHD etiology, the mechanism through which sleep/circadian rhythms problems affect ADHD still needs to be elucidated. The most consolidated theory about the association between circadian rhythm and ADHD is that sleep deprivation and circadian disorders would lead to a deficit in pre-frontal cortex executive functions, which at the pathological level, would represent ADHD symptoms ,39,40. OBHLHB2 gene and rs1534891 in the CSNK1E gene in association with bipolar disorder. The SNP rs534654 showed the strongest individual association signal in our study. However, this SNP did not survive to multiple test correction. This variant has never been tested in ADHD. It was already reported as nominally significantly associated with bipolar disorder in a genome-wide association study  and in aCLOCK gene, we were not able to find any study exploring the SNP rs4864548 in ADHD susceptibility. On the other hand, the studies concerning ADHD were mostly conducted with rs1801260. It is of note that we were not able to replicate the previous findings, which observed an association with rs1801260 (3111T/C). This polymorphism is located in an interaction site with miRNA-182 [Considering the markers with potential regulatory effects in the iRNA-182 ,44, an i1801260 31T/C. ThiiRNA-182 ,47. FurtiRNA-182 ,47,48,49Kissling et al. (2008) and Xu et al. (2010) found a risk effect of the T allele of rs1801260 on ADHD in a clinical European adult sample and a clinical European and Asian child sample, respectively. Jeong et al.(2014) also observed that rs1801260  was associated with ADHD scores in an Korean adult sample . These tCLOCK gene and ADHD are comparable to ours [Our results should be interpreted considering its limitations. The first is related to sample size. We studied a small clinical sample and our potential to find positive association was limited due to low statistical power. However, it is important to note that the sample sizes of other studies involving the  to ours ,19. Seco to ours . HoweverCLOCK gene was neither associated with ADHD nor with chronotype, sleep duration, or sleep disorders in the most comprehensive and recent genome-wide association studies (GWAS) [CLOCK gene may be a peripheric gene of small effect on ADHD, involved in the regulation of core genes, as proposed by the omnigenic model of complex traits [CLOCK, the literature is scarce and studies aiming to explore it are needed. Additionally, it is possible that the CLOCK gene may be more related to a specific ADHD subtype. Then, the use of global ADHD phenotypes would mask the CLOCK gene effect in this disorder.It is important to highlight that the s (GWAS) ,51,52,53s (GWAS) . Second,x traits . Then, ex traits ,55. For CLOCK gene polymorphisms and ADHD phenotypes in a non-European and non-Asian sample. Brazil is a country of continental proportions and unique populational admixture characteristics. The population has originated from a tri-ethnic parental populations , which resulted in one of the most admixed and heterogeneous populations in the world [CLOCK gene [CLOCK being important for these phenotypes in other population ancestries. To the best of our knowledge, this is the first study to explore the relationship between several he world . Within he world ,58,59. Ahe world , which hOCK gene . FurtherOCK gene  ThereforCLOCK and circadian rhythm/sleep problems in ADHD. It is plausible to expect that the relation between sleep/circadian rhythm phenotypes and ADHD may arise from a polygenic complex mechanism involving several clock genes as well as different SNPs, besides the regulation through the CLOCK gene. Further studies, especially based on genome-wide approaches, are needed to clarify the role of CLOCK, other clock genes, and sleep phenotypes in ADHD clinical subtypes in different populations.In conclusion, we provided additional evidence suggesting a putative role of


Methylphenidate (MPH) is a first line drug for attention-deficit/hyperactivity disorder (ADHD), yet the neuronal mechanisms underlying the condition and the treatment are still not fully understood. Previous EEG studies on the effect of MPH in ADHD found changes in evoked response potential (ERP) components that were inconsistent between studies. These inconsistencies highlight the need for a well-designed study which includes multiple baseline sessions and controls for possible fatigue, learning effects and between-days variability. To this end, we employ a double-blind placebo-controlled cross-over study and explore the effect of MPH on the ERP response of subjects with ADHD during a Go/No-Go cognitive task. Our ERP analysis revealed significant differences in ADHD subjects between the placebo and MPH conditions in the frontal-parietal region at 250ms-400ms post stimulus (P3). Additionally, a decrease in the late 650ms-800ms ERP component (LC) is observed in frontal electrodes of ADHD subjects compared to controls. The standard deviation of response time of ADHD subjects was significantly smaller in the MPH condition compared to placebo and correlated with the increased P3 ERP response in the frontoparietal electrodes. We suggest that mental fatigue plays a role in the decrease of the P3 response in the placebo condition compared to pre-placebo, a phenomenon that is significant in ADHD subjects but not in controls, and which is interestingly rectified by MPH. Attention-deficit/hyperactivity (ADHD) is a developmental psychiatric disorder involving problems with attention and/or hyperactivity and impulsivity. ADHD is often associated with anxiety, depression, difficulties with social interactions, learning disabilities and behavior disorders ,2. WhileElectroencephalography (EEG), an inexpensive and non-invasive method, enables the study of neurotransmission and is a means to understand neurobiological dysregulation . Thus, iAccordingly, extensive investigation of ERP differences between subjects with and without ADHD during various cognitive tasks, such as the Go/No-Go, oddball, Stroop, auditory and working-memory tasks, has been performed over the past years. This has identified differences mostly in the earlier components, namely N1, P2, N2 and P3 –16. SomeThe P3 component is often reported to be abnormally smaller in children with ADHD compared to controls during attention-related tasks, such as the continuous performance task (CPT) ,19 and tFurthermore, studies involving CPT found reduced N1 in boys with ADHD , yet larIn addition to inconsistencies between studies, a gap exists in the analysis of previous work, since most of it has ignored the later components (>500ms post stimulus), focusing instead on the earlier components. However, late components do provide important information, for example, De France et al., 1996  suggesteMethylphenidate (MPH) is the most prescribed medication for ADHD  and is kFor example, several studies involving CPT (X version) reported mixed-gender children with ADHD having abnormally small P3 amplitudes compared to controls, which were normalized after administration of MPH –30. HoweIn addition to the reasons mentioned above that may lead to variability between studies, a main reason for inconsistencies across these MPH studies has been attributed to the varied experimental designs . Due to To overcome some of these limitations, and building upon Sunohara et al., 1999 , we condAs the SART task has been found to affect the P3 amplitude ,40 and lTwenty children diagnosed with ADHD and twenty-one gender- and age- matched controls were recruited for the study. The children with ADHD were recruited from the child and adolescent psychiatry unit in Sheba Medical Center in Tel Hashomer, Israel. Healthy controls were recruited through ads sent to the workers of the Weizmann Institute or posted on bulletin boards in the Interdisciplinary Center (IDC) in Herzliya, Israel.One child with ADHD and two controls were excluded due to excessive noise in their EEG recordings. One control participant was excluded from the study due to technical errors during his EEG recordings. We therefore report results from a group of 19 ADHD participants , and a comparison control group of 18 controls .Subjects with ADHD were evaluated by a trained psychiatrist during the first baseline visit, and were included in the study only if they were diagnosed with ADHD according to the Diagnostic and Statistical Manual of Mental Disorders (DSM5) criteria. Thirteen of the patients were naive to ADHD medications, and six were regular users of MPH. All participants with ADHD were diagnosed with at least a moderate severity of ADHD symptoms . All controls were screened by a child psychiatrist using the child strength and difficulties questionnaire and were not included in the study if they had a history of a psychiatric disorder or neurological cognitive impairment .Criteria for exclusion from the ADHD study group were: age lower than 9 or above 18 years; history of seizure, head trauma, substance abuse or medication other than MPH; or an inability to swallow pills. Criteria for exclusion from the control group were: age lower than 9 or above 18 years; history of seizure, head trauma, substance abuse; current medication use; or current or lifetime psychiatric disorders.The study was approved by the institutional review board at Sheba Hospital and was conducted according to the principles of the declaration of Helsinki. Since participants were children and adolescents, both parents signed an informed consent form and assent to participate in the study was obtained from all children. Subjects' parents were reimbursed for travel and loss of time.All participants (ADHD and controls) completed a general demographic questionnaire and a standardized intelligence test based on Raven's Progressive Matrices scale . SubjectFor all participants, each experimental session consisted of the following steps:A baseline EEG recording of 5 minutes, during which subjects were instructed to sit comfortably with their eyes open.EEG recordings during which participants completed the computerized SART task . FollowiThe number of sessions differed between the ADHD and control groups, as detailed below.For the ADHD group, the study was performed based on a double-blind placebo-controlled cross-over design . The subThe controls performed two sessions on one test day and did not receive any medication. They were similarly given an hour break between the two sessions, during which the EEG cap remained on their head.Single digits (‘1’ through ‘9’) were presented in black centered on a white background. Subjects were asked to respond by pressing a designated button (Go condition), with the exception of the digit ‘3’, for which participants were asked to refrain from pressing (No-Go condition) . A totalAt the beginning of the test day, a BioSemi head-cap of suitable size  was placed on the participants’ head. Continuous EEG recordings were collected from 64 Ag-AgCl active electrodes  at a sampling rate of 1024Hz.For 9 ADHD subjects and 3 controls, some electrodes were removed due to excessive noise . Out of the removed electrodes, only two  are included in the reported results. For these electrodes, the data were reported from 18 subjects instead of 19.For the ADHD group, the experiment took place at Sheba Hospital, Ramat Gan, Israel. Cognitive stimuli were presented to the subjects using the E-Prime software . Triggers that marked the time of the stimuli presentation were sent from the E-prime software to the BioSemi system via a USB relay . For the control group, 9 subjects were also tested at Sheba Hospital with the same experimental system as the ADHD subjects. The other 9 subjects were tested at the Interdisciplinary Center (IDC), Herzliya, Israel. In this case, the E-Prime software sent triggers to the BioSemi system via a standard parallel port. Due to differences in the internal processing time between the systems in IDC and Sheba hospital, the experimental system at Sheba Hospital was 25ms faster than the experimental system at IDC and the data taken at IDC were corrected accordingly.rd order linear Finite Impulse Response (FIR) band-pass filter , low transition bandwidth 0.2Hz, high transition band width 10Hz) and a notch-filter of 50Hz , transition bandwidth 8Hz). Go and No-Go data were segmented into epochs from -1000ms to 2000ms around the stimuli presentation, and an offset  was removed for each epoch. Epochs which were excessively corrupted by movement artifacts were manually removed  and the data were then referenced by subtracting the average of all electrodes [The continuous EEG recordings were filtered in EEGLAB using a hamming-windowed 3ectrodes . This dip≤0.05, and we considered significance only if it occurred in three or more consecutive windows.The statistical analysis was performed in Matlab . For each subject and group of electrodes, the MRW was divided into sliding windows of 150ms, which is the typical width of the ERP components. The windows were bounded between 0ms-1000ms post stimulus (with an overlap of 125ms between two consecutive windows) and the mean amplitude of the signal, averaged over time for the duration of the window, was calculated. Repeated measures ANOVA was performed for each window and group of electrodes. The statistical significance level α for all analyses was set by For the ADHD group, three binary criteria describe each EEG recording: whether the recording preceded or followed the treatment (labeled “pre” or “post”); whether placebo or MPH was administrated (labeled “PLB” or “MPH”); and whether MPH was given on the first or second day (labeled “first” or “second”). We grouped the data into 4 conditions, “pre-PLB”,”pre-MPH”,”post-PLB” and “post-MPH”, and tested for differences between the groups using Repeated Measures ANOVA with Time (pre or post) and Intervention (PLB or MPH) as within-subject factors, and Order (first or second) as between-subjects factors. The subjects were classified as children (< = 12) or adolescence (>12) , and theTo investigate the differences between ADHD and controls, we grouped the data of the ADHD subjects according to session number, and considered their two treatment-free sessions: the first session of the first day (labelled “S1”) and the first session of the second day, which was the third session overall (labelled “S3”). The control group performed two sessions on the same day and did not receive any treatment, and their sessions were simply labelled H1 (first session) and H2 (second session). Differences between the groups were tested using Repeated Measures ANOVA with Session (“first” or “second”) as a within-subject factor and Diagnosis (“control” or “ADHD”) as a between-subjects factor. Post-hoc analysis with unpaired-sample t-tests was conducted for electrodes’ groups in which a significant effect of “Diagnosis” was found.To examine within-day changes, difference waves were calculated for each subject and group of electrodes. We subtracted the ERPs of the pre-placebo condition from the post-placebo condition (labelled as “ΔPlacebo”); the ERPs of the pre-MPH condition from the post-MPH condition (labelled as “ΔMPH”); and the ERPs of the H2 condition from the H1 condition (labelled as “ΔControl”). Then we could examine differences between the Δ conditions using paired t-tests.Correlations between behavioral variables  and ERP amplitudes were calculated using the Pearson correlation coefficient. We considered those behavioral variables for which significant difference between groups were found. Furthermore, in order to avoid multiple comparisons errors during the correlation analyses, we focused only on time windows and electrodes’ groups in which significant differences in amplitude were already evident. For the relevant electrodes’ groups, the correlation analysis was performed per-electrode.F1,34 = 5.5, p = 0.02, Cohen’s d = 0.89; Middle frontal: F1,34 = 8.2, p = 0.007, Cohen’s d = 0.94) (Differences between the groups were tested using Repeated Measures ANOVA with Session (“first” or “second”) as a within-subject factor and Diagnosis (“control” or “ADHD”) as a between-subjects factor. Significant “Diagnosis” effects (p < .05) were found in the left and middle frontal regions in 5 consecutive time windows, ranging from 550ms to 800ms post-stimulus. Post-hoc analysis revealed significant differences between the first session of the healthy controls (H1) and the first session of the ADHD subjects (S1). The statistical results are reported for the window 650-800ms, in which the effect-sizes (Cohen’s d) were most dominant  . No “SesDifferences between the groups were tested using Repeated Measures ANOVA with Time (pre or post) and Intervention (PLB or MPH) as within-subject factors, and Order (first or second) as a between-subjects factor. Significant “Time x Intervention” effect (p < .05) was found in 4 consecutive windows ranging from 200ms to 500ms in the right/middle/left frontal regions and in the left/right parietal regions. No “Order” interaction was found, nor any age or gender effects. Post-hoc analysis revealed significant differences between the post-placebo and the post-MPH conditions, as well as between the pre-placebo and post-placebo conditions. The statistical results are reported for the third window, 250-400ms, in which the effect-sizes (Cohen’s d) were most dominant , Fig 2. For the frontal and parietal regions that were found in Figs F1,18 = 10.35, p = 0.005, Cohen’s d = 0.72; Middle frontal: F1,18 = 13.02, p = 0.002, Cohen’s d = 0.83; Right frontal: F1,18 = 18.26, p = 0.0004, Cohen’s d = 0.98). Furthermore, a significant decrease in the amplitude of the ΔPlacebo condition was found in the P3 window , while no change was found in the ΔMPH condition. No significant change was evident in these regions for ΔControl. Finally, one can see that the waveform pattern of the ΔMPH condition follows the ΔControl waveform pattern along the full post-stimulus time window, suggesting a normalizing effect of the MPH towards the control’s brain activity (A significant difference in the P3 amplitude (250-400ms) was evident in the right/middle/left frontal regions when comparing ΔPlacebo and ΔMPH , while no difference was found post-placebo compared to pre-placebo. In addition, the ADHD subjects made significantly more omission errors post-placebo compared to pre-placebo (p = 0.01), but not for post-MPH compared to pre-MPH. No differences were found between the H1 and H2 conditions.Task performance results  showed tp = 0.001, see p<0.05) to the P3 amplitudes (250-400ms) of the post-placebo condition in the parietal and frontal electrodes  (The standard deviation of the response time (RTSD) significantly differed across the conditions of subjects with ADHD  .Well-designed experiments are particularly important when inconsistency is reported in the literature, as is the case of ERP measured in ADHD with relation to cognitive tasks and to the effects of MPH. Less than a handful of studies have used placebo-controlled cross-over designs ,28,32,34In this study we used a unique design, which involved two test days for the ADHD group  and one test day for the controls group (two treatment-free cognitive sessions separated by an hour break). This design enabled us to investigate the differences in ERPs for ADHD vs. Controls, MPH vs. placebo, and treatment- vs. treatment-free sessions. Furthermore, the design allowed us to account for possible learning and fatigue effects. We also examined the data over a wide time window (0-1000ms post stimulus), in search of possible differences in later components.When investigating differences between the first, pre-treatment session of ADHD (S1) and the first session of the controls (H1), significant differences were found in frontal regions only in the LC (650-800ms), which was lower for ADHD compared to controls . Late ERTo assess the involvement of learning effects, we tested the treatment-free sessions of the ADHD subjects for “Session” effects. As we hypothesized, our results indicate that no learning effects were evident across the two test days. In addition, no effect of “Order” (whether MPH was administrated in the first or second day) was evident. This confirms our ability to disregard the day label and group the conditions such that they reflect only the type of treatment .and correct responses), and differences in the P3 amplitude were still evident. We therefore hypothesize that the inconsistency in the results, despite relative similarity in the design, could be attributed to their different conditions for placebo administration. In our design, the placebo was given only in the afternoon, after the first cognitive task was performed. In Sunohara et al., 1999 [In agreement with our hypothesis, studying the effects of MPH on ERPs revealed higher P3 amplitude following MPH compared to placebo in parietal and frontal electrodes in the ADHD group . Howeverl., 1999  placebo While several studies ,28,32,34lower post-placebo P3 amplitude indicates lower levels of attention capabilities. This conclusion is consistent with our findings that MPH intake resulted in lower RTSD (hence in higher level of attention), as well as higher P3 amplitude compared to placebo.Since the P3 component differed between placebo vs. MPH conditions, we examined whether the P3 amplitude correlated to behavioral indices. We focused on the RTSD (standard deviation of the response time) of the ADHD subjects, which was found to be significantly lower after MPH compared to placebo, in consistency with the literature ,61. InteTo account for within-subject variations that might be caused by time-on-task effects and by mental fatigue, our design included two pre-treatment sessions, prior to both placebo and MPH administration. Interestingly, we found differences in the P3 amplitude when comparing between the pre-placebo and the post-placebo conditions, but not between the pre-MPH and post-MPH conditions. The lack of differences between pre- and post-MPH sessions could result from an inhibiting effect of the MPH, as suggested by Nigg, 2001 . It has Although we used a more optimal design than most previous studies, our study has limitations that need to be considered. Due to the limited number of participants, we did not use a narrow age band, and therefore could not account for age variation. However, our design was more focused on examining the repeatability within-subjects, and therefore age was less of a concern. For the same reason, we could not account for the different ADHD sub-types. In order to account for these limitations in retrospect, we included the age and sub-type as covariates in the statistics and found no significant relations. While this limitation does not appear as a substantial one in our case, future improvement of our design should take these factors into account.In conclusion, this study tested differences between adolescents with ADHD and controls during attentional effort and investigated the normalizing effects of MPH while accounting for effects of placebo, learning and fatigue. We found MPH to have a normalizing effect on the P3 component, which decreased following placebo. This effect was evident due to our unique design, which included a treatment-free baseline session before each placebo or MPH treatment. In accordance to our hypothesis, we did not find between-days learning effects, yet we identified effects between consecutive sessions which could be attributed to task-on-time/fatigue. This result emphasizes the need for well-designed within-subject studies which account for between-days variability, especially in the context of the literature inconsistencies regarding ADHD. The differences we found between ADHD and controls in the late component suggest that the attention difficulties associated with the disorder are also manifested during the preparation for the next task. This highlights the importance of broader outlook on the ERP waveform to later times than usually considered in the context of ADHD.S1 FigThe electrodes are placed according to the 10/20 International system. PFC = Prefrontal Cortex; SM = Sensory-motor; Par = Parietal; Occ = Occipital; Temp = Temporal.(DOCX)Click here for additional data file.


Moderate to high intensity exercise can improve cognitive function and behavior in children including those with attention-deficit/hyperactivity disorder (ADHD). However, exercise with long periods of the same activity, or inactivity can fail to engage or maintain their attention. This study examined the effect of exercise sessions developed to engage children with ADHD. Twelve children (10–11 years), six with a diagnosis of ADHD and six with no diagnosis, undertook 40-min sessions of short-duration, mixed activities bi-weekly for eleven weeks. ADHD symptoms and exercise enjoyment were recorded before six and eleven weeks of intervention. Teacher-reported data showed ADHD symptoms were significantly decreased in the children with ADHD, with a moderate to large effect size. There were no changes in the control group. All children indicated equal enjoyment of the exercise sessions. Specially designed exercise sessions stimulate and maintain engagement by children with ADHD and may reduce ADHD symptom levels in the school environment. The method that supports inclusive practice in physical education (PE) was successfully transferred to the study school and led by the usual class teacher. Children evaluated the exercises as acceptable and enjoyable for those with and without ADHD. This inclusive exercise method might help children manage ADHD symptoms. Attention-deficit/hyperactivity disorder (ADHD) is a syndrome of inattention, impulsivity, and over-activity in variable proportions, sufficient to cause impairment in functioning. Systematic review and meta-analysis data estimate the prevalence to be between 5 and 7% ,2.Children with ADHD may struggle to maintain age appropriate behavior and show poor social competence or emotional dysregulation ,4,5. ThiRecognizing that children with ADHD need a different approach to teaching and learning, DuPaul et al. , developStudies involving children with ADHD have been undertaken to examine the effects of exercise at moderate to high levels of intensity under experimental conditions. Improvements in motor skills, cognitive function, and behavior have been found after acute and long-term exercise ,10,11. HInterest in exercise as a possible adjunct to treatment for ADHD has been stimulated by concern over side effects from medication and difficulty implementing behavioral interventions . HoweverThe evidence indicates that exercise might benefit those with ADHD more than those with few or no symptoms of ADHD. In addition, no studies reported adverse effects of exercise ,16. TherIt is also interesting that progressive teaching methods have addressed the need for children with ADHD to have some physical movement during seated classroom activities . ChildrePreviously, this research group developed a series of 40-min, moderate to high intensity mixed-activity exercise sessions for children with ADHD, suitable for normal school facilities . These iThe exercise sessions were developed with children diagnosed with ADHD. However, these sessions were not tested by children who do not have ADHD. Current developments in education policy encourage inclusive strategies for children with special educational needs including ADHD . TherefoThis feasibility study followed a repeat-measures design to examine the effect of an exercise program developed for children with ADHD. An ADHD study group and an equally sized, age-matched control group were included. Ethical approval for the study was granted by the University of West London, FREC04. Children aged 10–11 years (school years five and six) attending a school in the UK were invited with their parents or guardians to take part. Exclusion criteria were the diagnosis of a cardiovascular condition, a respiratory problem, or a current infection . InclusiAfternoon exercise sessions were held twice weekly over one 12-week term. Baseline measures of ADHD symptoms for children in the study and control groups were recorded by both parents and teachers before the exercise intervention began. Activity sessions included different combinations of activities to stimulate interest. Each session started with a session-specific warm-up for 5–10 min with children taking turns to lead. This was followed by two different gym-based and outdoor blocks of mixed activity, each lasting 10 min with a mini-break (20–30 s) between. The session then finished with a 5–10-min cool-down. All activities had previously been chosen by a sports scientist and sports psychologist to be engaging for the children and to enable a moderate to intense work-rate. The researcher used existing facilities and equipment belonging to the school. In addition, the exercise sessions can be adapted to utilize the existing equipment of each school to avoid extra cost. The activities had been tested and confirmed as acceptable by children with ADHD . The exeChild-friendly hand signals were used during exercise sessions to monitor progress and enjoyment during each activity, but these signals are not a validated rating scale and the responses were not enumerated. The children’s own special learning assistants (SLAs) were available to assist should any child need to withdraw from a session. The total ADHD symptoms were recorded by the class teachers and parents at three time-points: baseline before the intervention started, then on weeks six and eleven of the exercise intervention.The home and school versions of the ADHD Rating Scale-IV were used to measure the symptoms of ADHD . This psThe study group was derived from the limited number of children with ADHD in years 5 and 6 of one primary school. The small sample did not meet the criteria for normal distribution. Test–retest reliability was examined using Kendall’s tau due to the non-normal distribution, small sample size, and large number of tied ranks. The Wilcoxon signed ranks test was used to compare the ADHD scores for the study group and for the control group pre to post intervention. Univariate ANOVA, with Bonferroni correction, was then used to examine ADHD scores for the study group over the 12-week period of the study. Planned contrasts were used for within participant comparisons between time-points.Twelve children took part and completed the first six weeks of the study. The hand signals during the exercise sessions and the verbal responses from the control group children confirmed their enjoyment of the exercises matched that of the children with ADHD. Activities chosen as favorites by the children in the previous work were confirmed as favorites by all the children in the current study giving the thumbs up signal.p < 0.001, indicating good test–retest reliability.The test–retest reliability, of the teacher-rated ADHD symptoms scores, was examined by a Kendall’s tau calculated for teacher-rated symptoms scores at six weeks compared with baseline. There was a positive correlation between the two variables τ = 0.881, n = 12, p = 0.028, r = −0.64  completed the first six weeks of the study. After week six, five of the control group were unable to complete all the exercise sessions. There were no differences in the teacher-rated or parent-rated symptom scores, which ranged from 0–2 at the three timepoints for the children in the control group.All of the control group  = 14.81, p = 0.004, ω2 = 0.34. The multivariate tests also showed that the ADHD symptom level was affected by the number of weeks of exercise undertaken. There was a significant effect of exercise on the ADHD symptom level reported by teachers, V = 0.82, F = 9.33, p = 0.031. Planned contrasts were undertaken. The first contrast compared the teacher-rated ADHD scores at baseline with those after six weeks of the exercise sessions. This showed a significant reduction in ADHD scores from baseline to week 6 of exercise, F, = 7.5, p = 0.041, with a large effect size r = 0.78. The second showed a significant reduction in teacher-rated ADHD scores from week six to week eleven of the exercise sessions, F, = 14.76, p = 0.012, with a large effect size r = 0.86.A one-way repeated-measures analysis of variance (ANOVA) was conducted to evaluate the null hypothesis that there would be no change in the ADHD symptom level of the participants of the study group when measured before, during, and after the exercise sessions. Mauchly’s test showed the data met the assumption of sphericity. However, the Greenhouse–Geisser estimate of the departure from sphericity was In this study the use of exercise sessions designed for children with ADHD in PE lessons was associated with a significant reduction in ADHD symptoms observed by the teachers in school. In addition, the teachers informed the researchers that they had observed a general increase in engagement in learning activities in the classroom by the children with ADHD. The observations were spontaneous and qualitative, because no quantitative measure of engagement in sedentary classroom activities was included in this study. Together, the results and observations suggest these exercise sessions may be a practical way to reduce ADHD symptoms through regular school PE. The program applied in this study is distinguished from other exercise programs used in previous research by its original elements including multiple different activities with short periods of time spent on each. While this differs from other programs, the use of moderate to high intensity exercise is similar to previous work. In addition, the observed change in behavior associated with the exercise intervention supports previous findings ,12,13,14Following the pattern of the teacher-rated ADHD symptom reduction, the median parent-rated symptom scores  showed aAll the children in the study, both with and without ADHD, engaged fully with the physical exercise sessions. Their feedback through the regular monitoring by the exercise leaders indicated that their evaluation and enjoyment of the exercise sessions matched that of the children with ADHD who had contributed to the original selection of the activities. The exercise leaders in this study were not the same as the exercise leaders in the previous school study, indicating that this program can be successfully led by other appropriately qualified people, for example class teachers who lead PE lessons as well as subject specialist PE teachers. In addition, the school in the current study was not the same as the one in the development study, so none of the class teachers or children had previously been involved. This transferability suggests that it is the design and content of the exercise sessions that is important in engaging and maintaining the attention of the children.Research indicates that many school practitioners including teachers, special education needs coordinators, and specialists in pupil referral units use a range of strategies aimed to be generally inclusive rather than specifically designed interventions for ADHD children alone . This maThis feasibility study took place in only one primary school, following the development of the method in a separate single primary school. Therefore, there were a limited number of children of the correct age with ADHD available to take part. So, the study design could not include a randomized allocation of children with ADHD into two groups, one undertaking the intervention plus normal PE lessons and the other just the normal PE lessons. An unforeseen limitation arose when places for a different activity for children were provided after week six, which eventually attracted five children and their parents from the control group. Future research involving more than one primary school could address both limitations. In addition, to mitigate against a possible Hawthorne effect, a future study should be designed to enable all the children of the school classes to take part if they want to, so the physical activity is not perceived as a special privilege for some children.The rating scale used in this study only measures symptoms of ADHD so any other changes in the control group would not be detected. In addition, the drop-out rate of the control group was high, due to an unforeseen school timetable change at half-term. However, the control group had reported equal enjoyment through the six weeks of their participation. Therefore, further research is warranted, first, to determine whether the effect on symptoms in children with ADHD can be replicated in a larger sample over a longer period; second, to discover whether other measurable benefits  can be detected in a non-ADHD control group; and finally, to test whether the general acceptability of the exercise sessions would enable their use as an inclusive activity in regular PE lessons, rather than an ADHD-specific intervention.The results from this feasibility study indicate that specifically designed exercise sessions that stimulate engagement by the children with ADHD may be useful for symptom reduction. This study has shown that the exercise sessions and methods can be transferred to other schools and led by the general class teachers. This study has also provided the first evidence that these physical activity sessions are acceptable and enjoyable for other children. Therefore, they are potentially useful in the PE curriculum. While these positive results suggest the sessions could be used to increase engagement by children with ADHD in regular school PE classes and reduce symptoms in the classroom, a randomized controlled trial involving children with ADHD in both a study and a control group should be undertaken to test this over the full school year.


Background: Heavy metals are known to be harmful for neurodevelopment and they may correlate to attention deficit/hyperactivity disorder (ADHD). In this study, we aim to explore the relationships between multiple heavy metals , neurocognitive function, and ADHD symptoms. Methods: We recruited 29 patients with ADHD inattentive type (ADHD-I), 47 patients with ADHD hyperactivity/impulsivity type (ADHD-H/I), and 46 healthy control children. Urine samples were obtained to measure the levels of the aforementioned heavy metals in each child. Participants’ cognitive function and clinical symptoms were assessed, respectively. Results: We found ADHD-H/I patients demonstrated the highest antimony levels (p = 0.028), and ADHD-I patients demonstrated the highest cadmium levels (p = 0.034). Antimony levels were positively correlated with the severity of ADHD symptoms that were rated by teachers, and cadmium levels were negatively correlated with the Full Scale Intelligence Quotient. Lead levels were negatively correlated with most indices of the Wechsler Intelligence Scale for Children–Fourth Edition (WISC-IV), but positively correlated with inattention and hyperactivity/impulsivity symptoms (p < 0.05). Conclusion: Lead, cadmium and antimony were associated with susceptibility to ADHD and symptom severity in school-age children. Eliminating exposure to heavy metals may help to prevent neurodevelopmental disorders in children. Attention deficit/hyperactivity disorder (ADHD), which is characterized by inattention, impulsivity, and hyperactivity, is among the most common psychiatric disorders occurring in children ,2. The APrevious studies have reported that environmental toxicants, especially lead (Pb) and mercury (Hg), contribute to the risk of ADHD . A meta-Hjortenkrans et al. reported that brake linings was an important source of zinc (Zn), cooper (Cu), and antimony (Sb) emissions and tires are one of the main sources of Zn and cadmium (Cd) emissions . Cd is bSb is frequently used as a catalyst in the polycondensation reaction to produce polyethylene terephthalate (PET) and it may leach from PET and recycled bottles into drinking water. Humans can be exposed to Sb by breathing air, drinking water, and eating foods that contain it, as well as by skin contact with substances that contain it. The effect of Sb on human heath has not yet been fully established. Only one study has reported no significant association between prenatal exposure to Sb and cognitive function at the age of four years . BismuthBased on clinical characteristics, the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR) classified ADHD into three subtypes  . PreviouOur research protocol was approved by the Institutional Review Board at Chang Gung Hospital in Taiwan. We recruited patients with ADHD who were treated in the outpatient Department of Child Psychiatry at Chang Gung Children’s Hospital in Taiwan for this cross-sectional study. We obtained written informed consent from all of the participants or their guardians. The inclusion criteria consisted of the following: (a) clinical diagnosis of ADHD by a senior child psychiatrist based on the DSM-IV-TR through structured interviews using the Chinese version of the Schedule for Affective Disorders and Schizophrenia for School-Age Children, epidemiologic version (K-SADS-E) ; (b) betThe control subjects (children without ADHD) consisted of healthy children from communities surrounding Kaohsiung Chang Gung Memorial Hospital or children that were suffering from upper respiratory tract infection (URI) whose symptoms were currently in remission. We excluded any patients with major psychiatric disorders  or major physical illnesses . In total, 46 healthy control children, 48 patients with ADHD inattentive type (ADHD-I), and 65 patients with ADHD hyperactivity/impulsivity type (ADHD-H/I) were recruited for this study. Because maturation processes and education effects could be diverse with age, we selected patents <10 years old into analyses.A urine sample (10 mL) was collected from each child and sent to the Chang Gung Memorial Hospital laboratory. We measured metal concentrations, including Mn, Pb, Cd, Hg, Sb, and Bi, using inductively coupled plasma mass spectrometry (ICP/MASS) and mass spectrometry in order to simultaneously measure the concentration of various heavy metals. ICP/MASS uses argon to generate high temperature plasma (6000K) in order to atomize and then ionize the heavy metal compounds. The mass spectrometer then directed the metal ions and analyzed each metal’s content based on its mass.Each ADHD patient was interviewed by a senior psychiatrist using the K-SADS-E diagnostic tool. An experienced child psychologist conducted the Wechsler Intelligence Scale for Children–Fourth Edition (WISC-IV). Furthermore, the Swanson, Nolan, and Pelham Version IV Scale (SNAP-IV) parent form and SNAP-IV teacher form were completed by each patient’s parents and a teacher, respectively.The K-SADS-E is a semi-structured diagnostic interview that is designed to evaluate current and past episodes of psychopathology in children and adolescents, according to the criteria of the DSM-III-R and DSM-IV . The K-SThe Chinese version of the WISC-IV is an individually administered and norm-referenced instrument developed to measure the intelligence of children aged from 6 to 16 years old . The WISThe SNAP-IV is a 26-item questionnaire for evaluating ADHD symptoms and severity, and it needs to be completed by parents or teachers . The 26 p-values of <0.05 were considered statistically significant.Data were analyzed using the statistical software package SPSS, version 16.0 . Variables are presented as either the mean (standard deviation) or frequency. Two-tailed Categorical variables among the healthy control group, ADHD-I group, and ADHD-H/I group were compared using the chi-square test. We adopted one-way analysis of variance (ANOVA) to compare the continuous variables among groups, followed by a post-hoc Least Significant Difference (LSD) test. Levels of heavy metals in the urine samples demonstrated a significant level of positive skewness. Therefore, the Kruskal-Wallis test was adopted to compare continuous variables among the three groups. Spearman’s correlation was performed to analyze the relationships between heavy metal levels, neuropsychological functions, and ADHD clinical symptoms.Our study sample consisted of 46 healthy control children (mean age 8.1 years), 29 ADHD-I children (mean age 8.0 years), and 47 ADHD-H/I children (mean age 7.7 years). Among these groups , ADHD-H/p = 0.034) levels. Compared to the healthy controls and ADHD-I group, children with ADHD-H/I demonstrated the highest Sb levels (p = 0.028). However, we observed no significant differences among the groups for Mn (p = .485), Pb (p = 0.125), Hg (p = 0.132), or Bi (p = 0.434).n = 122). We found that Pb levels negatively correlated with FSIQ, VCI, WMI, and PSI of the WISC-IV and positively correlated with the inattention, hyperactivity/impulsivity, and oppositional scores rated by parents and teachers, respectively. Cd levels were negatively correlated with FSIQ scores, while Sb levels were positively correlated with inattention, hyperactivity/impulsivity, and oppositional scores that were rated by teachers. Levels of Hg were positively correlated to hyperactivity/impulsivity scores rated by parents. Levels of Mn and Bi were observed to not have a significant association with ADHD clinical symptoms or cognitive functions.In the present study, we investigated the exposure of such metals as Mn, Pb, Cd, Hg, Sb, and Bi and its relationship to the specificity of ADHD symptomatology, measuring severity and cognitive function with WISC-IV. We found that Pb levels were positively correlated with ADHD symptomatology, including inattention, hyperactivity, and impulsivity. From previous studies, we know that Pb interrupts the dopamine pathway, which is one of the major neurotransmitter pathways that is involved in ADHD, resulting in dopaminergic neuron damage and the disruption of the homeostasis of calcium-dependent neurotransmitters . PreviouOur study also suggested that Pb levels were negatively correlated with FSIQ, VCI, WMI, and PSI of the WISC-IV. Such cognitive function impairment may result from Pb-induced directed cell death and the interruption of intracellular biological activities . Our finAnother interesting finding in our study is that the highest Sb levels presented in the ADHD-H/I group and were lower in both the ADHD-I group and the healthy control group. Furthermore, Sb levels were positively correlated with the inattention, hyperactivity/impulsivity, and oppositional scores rated by teachers. Other experimental studies and case reports have suggested that Sb may cause peripheral neurotoxicity , cerebelWhen compared to the healthy controls and the ADHD-H/I group, children with ADHD-I demonstrated the highest Cd levels. We also found in our study that levels of Cd were negatively correlated with FSIQ scores. A prospective study of 1305 subjects identified significantly inverse associations between urinary Cd levels and VIQ and FSIQ . AnotherIn the present study, we also found that the levels of Hg were positively correlated to hyperactivity/impulsivity scores rated by parents. Hg exerts the detrimental effect on enzymes, cellular membrane function, and neurotransmitter levels . A meta-This study has certain limitations. First, the cross-sectional nature of the study design and the single measurement of Pb and the other environmental chemicals were not sufficient to establish a definite causal relationship. In addition, the range of the half-life of these heavy metals was widely dispersed. For example, Sb had the shortest half-life with 95 hours after exposure, but Cd had the longest half-life for decades. The single measurement of urine sample may reflect recent exposure. However, it is difficult to clarify the effect of heavy metals during the critical period of neurodevelopment. Second, using urine samples to measure chemicals may be related to more individual variations, especially in Pb. Furthermore, urinary Pb levels are less sensitive in the low range of exposures  . As for Our study’s strength came from measuring various metals, as well as demonstrating the effect in subjects with a formal ADHD diagnosis that is based on DSM-IV-TR criteria. Our findings indicate metals’ relationship to susceptibility to ADHD, especially for Pb, Cd, and Sb. Children’s neurocognitive function is particularly correlated to Pb levels. These findings suggest that children should avoid heavy metal exposure and prevent the adverse effect of metals on children’s neurodevelopment. However, Taiwan is a cramped and densely populated country. The demarcation of the cultivated field, industrial area, and the residential area was often not clear. Therefore, it is crucial for legislating public health policy for reduction of the environmental heavy metals exposure.


The preliminary results from antibacterial assay which were tested against Gram-positive bacteria such as Bacillus subtilis, Staphylococcus epidermidis, Staphylococcus aureus and Gram-negative bacteria such as Escherichia coli, Salmonella typhimurium are also described.This paper provided comprehensive data on spectroscopic and antibacterial activities of thioureido compounds which are relevant with research article entitled “Synthesis, Spectroscopic Studies and Antibacterial Activity of New Lauroyl Thiourea Amino Acid Derivatives” [1]. Based on the reported study, four new thioureido derivatives, namely 3-(3-dodecanoyl-thioureido)propionic acid (R1), 2-(3-dodecanoyl-thioureido)-3-methyl butyric acid (R2), (3-dodecanoyl-thioureido)acetic acid (R3) and 2-(3-dodecanoyl-thioureido)-3-phenyl propionic acid (R4) were characterized by elemental analysis, Fourier Transform Infrared (FTIR),  This re, 2- and ammonium thiocyanate  were dissolved in acetone (25 mL) and stirred for an hour to give white precipitate. Then, β-alanine  was added dropwise and the mixture was heated at reflux until reaction was completed being filtered into ice. This was monitored by thin layer chromatography. The resulting solid was collected by filtration, washed with acetone and dried under vacuum to give 3-(3-dodecanoyl-thioureido)-propionic acid (R1). The synthesis of 2-(3-dodecanoyl-thioureido)-3-methyl-butyric acid (R2), (3-dodecanoyl-thioureido)-acetic acid (R3) and 2-(3-dodecanoyl-thioureido)-3-phenyl-propionic acid (R4) were prepared in similar manner as described for R1, employing Bacillus subtilis ATCC 11774, Staphylococcus epidermidis ATCC 13518 and Staphylococcus aureus ATCC 25923) and Gram-negative  strains using common well diffusion method. Mueller-Hinton media were seeded with bacterial inoculum using cotton swab. Wells of 6.0 mm diameter were bored into the media using sterile cork borer and 90 μL of the diluted compounds at a dose range of 10–0.01 mg/mL were added in each well. Streptomycin  was used as the positive control while methanol served as negative control. All plates were incubated overnight at 37 °C. The antibacterial activity was evaluated by measuring the zone of inhibition (mm) and minimum inhibitory concentrations (MIC). Ligand that have high specific surface area structure gave higher antibacterial activity [The antibacterial activity of compounds R1-R4 were screened against test strains of Gram-positive (