Attention Deficit Hyperactivity Disorder (ADHD) is a common and highly heritable neurodevelopmental psychiatric disorder. Here, we critically review four major psychological theories of ADHD – the Executive Dysfunction, the State Regulation, the Delay Aversion and the Dynamic Developmental – on their abilities to explain all the symptoms of ADHD, their testability and their openness to falsification. We conclude that theoreticians should focus, to a greater extent than currently practiced, on developing refutable theories of ADHD. Attention Deficit Hyperactivity Disorder (ADHD) is a common neurodevelopmental psychiatric disorder . Much ofAccording to the scientific philosophy of Popper, a scientific hypothesis must be testable and therefore open to falsification . If a scOther philosophers have argued that in practice, when anomalous (non-predicted) results are reported and accepted as real data, rather than abandoning that theory, scientists are more likely to modify their existing hypotheses to align with the new findings  . AccordiThe strength and health of the field of ADHD behavioural research , is dependent on proposed hypotheses being made testable and falsifiable. ADHD is currently a behavioural construct, defined by behavioural dysfunction rather than by a biological marker . It is aThis review focuses on four key cognitive and behavioural theories of dysfunction associated with ADHD: the Executive Dysfunction, Delay Aversion, State Regulation and the Dynamic Developmental theories. Each of these theories has undergone transformations over the last decade. As Lakatos predicted, theoreticians have added auxiliary hypotheses to explain new data as they have arisen; some of these auxiliary hypotheses are components of the other leading theories.This review does not attempt to be systematic; rather it aims to review each of the theories from a philosophical perspective. After defining each theory, we asked these questions: (1) Does the theory explain all of the symptoms of ADHD? (2) Is the theory testable? (3) Has a falsifiable hypothesis been stated?Executive dysfunction is a term used to explain deficits in "higher-order" cognitive processes, such as planning, sequencing, reasoning, holding attention to a task, working memory, inhibition of inappropriate and selection of appropriate behaviours -15. ThesThe Executive Dysfunction theory of ADHD suggests that the symptoms of ADHD arise wholly as a result of a reduction in executive control, which is caused by abnormalities in the structure, function and biochemical operation of the fronto-parietal and fronto-striatal neural networks . NeuropIn terms of the three broad symptom types of ADHD, the Executive Dysfunction theory explains impulsivity and inattention, but has largely ignored the hyperactivity element of ADHD. Response inhibition, as the neuropsychological marker of impulsivity, has been earmarked by some as the critical deficit in ADHD, leading to secondary impairments in other executive functions, specifically working memory, internalisation of speech, self-regulation of affect-motivation-arousal and novel goal-directed action, resulting in decreased motor control of internally represented information and action . Responset al., meta-analysis concluded that the Executive Dysfunction theory failed to explain the full complexity of symptoms of ADHD [medium effect size. Executive dysfunction is not always found in all children with ADHD. For example, in a recent study, an estimated 35–50% of ADHD combined type cases showed response inhibition deficits [Whilst there is supporting evidence of deficits in performance by participants with ADHD on executive functions such as response inhibition, sustained attention and spatial working memory, the recent Willcutt  of ADHD . The weideficits ; yet we Neuropsychological tests of executive function are often quite complex and involve a number of different executive functions, making it difficult to resolve the exact locus of dysfunction . AdditioMany papers have used the Executive Dysfunction theory to produce testable hypotheses about the role of the frontal cortices and their circuits in the symptomatology of ADHD. One difficulty, however, with the Executive Dysfunction theory is the complexity in defining and then testing an executive function. The behavioural manifestations of ADHD probably represent a combination of cognitive and motor processes. For example, response inhibition, as measured by the Stop Signal task, is the summation of cognitive processes  and control of the primed motor response ,42. IdeaIn its most general form, the Executive Dysfunction hypothesis in ADHD is difficult to falsify, as poor performance on a general executive function task is taken as evidence of an executive function deficit in ADHD, without conditions specified for testing alternative hypotheses. This need not to be the case however and studies have been carried out where the cognitive process under study has been precisely identified (using careful control conditions) and the predictions for an alternative have been explicitly stated and tested (e.g. ). This rThe State Regulation hypothesis states that a non-optimal energetic state could explain performance deficits in children with ADHD. This hypothesis is based on research using the Cognitive Energetic model of Sanders ,46. In tSeveral issues regarding the State Regulation hypothesis remain. Direct supportive evidence for disturbances in the activation pool in ADHD is limited. Only a few studies have tried to directly measure the motor activation pool. Besides the cardiac response studies of Börger and colleagues ,67, mostAlthough most evidence suggest that the state regulation problems in children with ADHD are related to under-activation problems, originally it was suggested that activation and performance take an inverted 'U' function where either increases or decreases in activation from an optimal energetic state lead to performance decrements . In ordeThe state regulation account argues that ADHD symptoms may increase or decrease depending on the child with ADHD's state. For example, symptoms of inattention may appear when tasks are slow or boring. Children may become impulsive or hyperactive in an attempt to increase stimulation (self-stimulation). This may explain findings such as longer RTs, higher intra-subject variability of responding and increased error rates in children with ADHD.With respect to the specificity of a state regulation deficit in ADHD, the following findings should be emphasised. Performance in adults with High Functioning Autism (HFA) was disproportionally impaired by a fast event rate condition, whereas no difference between groups was noted in a slow condition . Poor RTCrucially, the state regulation account suggests that differences between children with ADHD and typically developing children will be minimal when children with ADHD are in an optimal state. Unfortunately, it is difficult in practice to specify the optimal state as this may be task/context dependent and will also differ between children.Falsifiable hypotheses have been stated. The specificity of these hypotheses, however, is not always clear. Most evidence suggests that the state regulation problems in children with ADHD are related to under-activation problems, however originally it was suggested that either increases or decreases in activation from an optimal energetic state lead to performance decrements in ADHD . One wayThe Delay Aversion theory was first described by Sonuga-Barke and colleagues in the early 1990s ,79 and hInattentiveness and hyperactivity are considered to reflect attempts to reduce subjective experience of delay in situations where delay cannot be avoided . The oriMore recently, Sonuga-Barke incorporated the delay aversion hypothesis within a new framework, the Dual Pathway theory ,84. ThisSonuga-Barke (2002)  developeRecently the concept of delay aversion has also been revised such that it no longer is viewed as a competing causal model with impulsivity-as-lack-of-self-control ,88. DelaThe main focus of the theory is on impulsiveness. Inattentiveness and hyperactivity are considered to reflect attempts to reduce subjective experience of delay in situations where delay cannot be avoided .The original delay aversion hypothesis that contrasted the different conditions  includedThe dual hypothesis includes several predictions, including that of the two subtypes, but to our knowledge this is yet to be investigated at the level of individual children. The dual pathway model relies on a correlational pattern of findings, the interpretation of which is somewhat difficult at present. The effects of combining ADHD and control groups in the analyses are unknown. Further development of the model would benefit from a clear description of tasks and variables that measure the proposed constructs. Many of the links proposed in the model are yet to be tested and, overall, replication and extension of findings with independent samples will be important. There are no studies, as yet, describing the neural pathways or role of genetics in delay aversion behaviour in ADHD .Beyond the proposal of the two subtypes, the dual pathway model includes the assumption of poor performance on the Stop Task as reflecting an inhibition deficit. There is ERP evidence to suggest that slower SSRT may be related to early problems with shifting attention to the stop signal, questioning the validity of the SSRT as an inhibition measure . Meta-anThe original delay aversion hypothesis  includedThe Dynamic Developmental Theory (DDT) of ADHD has been developed by Sagvolden and colleagues over the past 20 years and has been the subject of a recent major review process . This coThe DDT has adapted some of the findings from the Executive Dysfunction and Delay Aversion literature, incorporating attentional, behavioural organisation, motor coordination, nondeclarative habit learning deficits and delay aversion, as auxiliary hypotheses, into a larger dopaminergic, fronto-striatal neurological model. Reinforcement and extinction processes are hypothesised to be the core problems in ADHD due to abnormally low levels of dopamine, affecting the functioning of the anterior cingulate, dorsolateral prefrontal and motor circuits and subsequently a variety of behaviours.The DDT holds a theoretical position on hyperactivity, impulsivity and inattention. The shorter delay-of-reinforcement gradient and deficient extinction effect in ADHD is hypothesised to occur within the mesolimbic dopamine branch along the anterior cingulate-fronto-striatal circuit. Hyperactivity, impulsivity and delay aversion are explained through this system. Hyperactivity may be due to a combination of factors including failing extinction resulting in too many responses, a deficit in the pruning of ineffective or inappropriate responses resulting in a relative increase in these, and a short delay-of-reinforcement gradient resulting in poor reinforcement of appropriate behaviour. Impulsiveness may be due to the short delay-of-reinforcement gradient, as the appropriate behaviour is not reinforced well and the significance of the immediate reinforcer is much stronger then the delayed reinforcer, which remains unlinked to the original response. Delay aversion is also explained by the shortened delay-of-reinforcement gradient. Stronger and more salient reinforcers are required to produce the desired behaviour in children with ADHD, compared with control children. The larger but delayed reward loses its saliency .Abnormally low dopamine levels within the mesocortical and nigrostriatal branches are hypothetically linked with poor reinforcement and extinction processes in the dorsolateral prefrontal and motor circuits, respectively. This is hypothesised to lead to deficient attention and poor behavioural organisation  and poor motor coordination, response disinhibition and nondeclarative habit learning (motor circuit) .Behavioural variability may be due to a combination of an increased number of types of responses and a deficient extinction process resulting in a greater number of inappropriate responses. Variation between people with and without ADHD may be due to variation within the biological starting position of the person , the delay gradient and the ongoing neuromodulatory effects of the environment (learning and experience).The DDT is testable. The altered-reinforcement hypothesis makes two explicit predictions . (1) TheResearchers may interpret the results as suggestive of deficits in spatial working memory and indeed Aase and Sagvolden suggest that reduced working memory capacity may be underlying the performance of the children with ADHD . The ratThe DDT provides a well described theoretical framework that has produced falsifiable hypotheses within the confines of the stimulus-response-reinforcer experimental set-up. Theoreticians of the DDT relate the theory to everyday behavioural manifestations of ADHD , for exaIn this review we have considered the strengths and weaknesses of the four major psychological theories of ADHD, especially as they relate to the requirement for scientific hypotheses to be open to falsification . We concReflecting on these four theories of ADHD, it is striking that the researchers coming from different theoretical positions may be describing the same phenomena but utilising different words, concepts and schemas; they may also be defining the same phenomena or process but from a different temporal or anatomical point of view. For example, an arousal deficit in ADHD (lower energetic pool) may affect motivation to complete a task (poor state regulation) or the ability to sustain attention to a task (executive dysfunction), which behaviourally may manifest as an aversion to delay and poor performance on some executive function tasks; this behaviour may be reinforced inappropriately and because of low dopamine tone there may be deficient extinction of the socially inappropriate behaviour.One common assumption of all the hypotheses is that there is a certain degree of homogeneity within the ADHD construct. It is possible that through a direct analysis of the models and an evaluation of how they dissociate and overlap, there will be a subgroup within the broader ADHD group that will show impairments on some other psychological aspect not currently incorporated within these four models. It is hoped that in this event, a new scientific model will be generated. In a similar manner, a comparison of children with ADHD with children with other clinical diagnoses may also inform theorisation.post hoc, to explain many of the phenomena we observe in ADHD; yet this is not where their strength lies and we need to move from post hoc theorisation further towards explicit, a priori  falsifisee also .The authors declare that they have no competing interests.KJ, RW and JK conceived the review idea, prepared, reviewed and approved the final manuscript.


To the Editor:Bartonella are fastidious, aerobic, gram-negative, facultative, intracellular bacteria that infect erythrocytes, erythroblasts, endothelial cells, monocytes, and dendritic cells, and are transmitted by arthropod vectors or by animal scratches or bites  from North Carolina coastal waters. Reptilian erythrocytes are nucleated, and commercial lysis methods clogged filtration columns because of the high DNA content of whole blood. Consequently, DNA was extracted from frozen whole blood by using a modified alkaline lysis method adapted from an avian cell culture DNA extraction method  region  of 37 sea turtle blood samples tested, including samples from 13 free-ranging and 3 rehabilitated turtles. Pap31 PCR was performed for Bartonella ITS-PCR–positive samples. Pap31 amplicons were obtained from 5 samples of which 3 were successfully sequenced. Amplification and sequencing of the 16S-23S ITS region detected 2 Bartonella species: a B. henselae–like organisms and 1 more similar to B. vinsonii subsp. berkhoffii. The 3 Pap31 amplicons successfully sequenced confirmed B. henselae infection. Sequences obtained from 1 sample matched B. henselae strains H1-like, the B. henselae SA2-like strain, and B. vinsonii subsp. berkhoffii genotypes II and IV, which suggests that this turtle was co-infected with multiple Bartonella spp. and strains. Three other samples yielded amplicons 99%–100% identical with B. henselae strain SA2, and 3 yielded sequences most similar to B. vinsonii subspecies berkhoffii genotypes II and IV. Two samples contained an ITS region sequence most similar to B. henselae SA2, but with a 15-bp deletion beginning 617 bases downstream from the 16S rRNA gene. Whether these ITS sequence differences represent distinct strains or nonrandom translocation events is uncertain.The purpose of this study was to determine whether sea turtles are infected with ) region . BartoneB. vinsonii subsp. berkhoffii. However these amplicons were much shorter than expected for B. vinsonii subspecies berkhoffii genotype II and genotype IV sequences in GenBank. Because Pap31 gene amplification was unsuccessful for these samples, it is unclear whether small amplicons represent a species related to B. vinsonii subsp. berkhoffii or a new Bartonella sp.Four sea turtle blood samples contained partial ITS sequences most similar to Bartonella spp. DNA in sea turtle blood represents the first molecular evidence of Bartonella infection in nonmammalian vertebrates. B. henselae infection, now reported in porpoises and sea turtles, may represent an emerging infection of marine animals. According to previous studies, immune status appears to affect disease severity, variation in clinical manifestations, the pattern of histopathologic features, and the relative ease of diagnostic detection of the organism (Bartonella spp. in blood of sea turtles is a function of prior immunosuppression induced by stressors is not known. Such stressors could include mechanical injury, malnutrition, environmental toxins, parasites, or concurrent bacterial or viral infections. Alternatively, sea turtles may be a natural marine reservoir for B. henselae or for a Bartonella sp. genetically related to B. vinsonii subsp. berkhoffii.To our knowledge, detection of B. henselae and an organism genetically similar to B. vinsonii subsp. berkhoffii in the blood of loggerhead sea turtles provides evidence that this genus is not ecologically limited to terrestrial reservoirs. The geographic distribution, prevalence of infection, carrier potential, mode of transmission, and pathogenicity of bloodborne Bartonella spp. in sea turtles await additional studies.In summary, documentation of


The dopamine-derived tetrahydroisoquinolines (TIQ) synthesized endogeneously from aldehydes and catecholamines have shown to modulate neurotransmission, central metabolism and motor activity. Converging evidence has implicated abnormalities of the dopamine metabolism to the pathophysiology of Attention-Deficit/Hyperactivity Disorder (ADHD). Therefore, four TIQ derivatives involved in central dopamine metabolism  have been analyzed for the first time in children and adolescents with ADHD and healthy controls.42 children and adolescents with ADHD and 24 controls from three sites participated in this pilot study. Free and bound amounts of salsolinol, N-methyl-salsolinol, norsalsolinol, N-methyl-norsalsolinol have been analyzed in urine.free, most of the ADHD patients were identified correctly with a sensitivity of 92.5% (specificity 94.4%).In the ADHD group, free and total amounts of the four TIQ derivatives in urine were significantly higher compared to urine levels of healthy controls. For N-methyl-salsolinolUrine levels of salsolinol, N-methyl-salsolinol, norsalsolinol and N-methyl-norsalsolinol are elevated in children and adolescents with ADHD and point to a new perspective on catecholaminergic dysfunction in ADHD. However, replication and extension of this pilot study would progress this innovative and promising field. Attention-Deficit/Hyperactivity Disorder (ADHD) is a common worldwide disorder characterized by inattention, impulsivity, and hyperactivity. Despite a large amount of research its etiology still remains unclear. Actually, ADHD is regarded as a multifactorial disorder caused by many interacting and/or additive risk factors . There iIn this context the dopamine-derived tetrahydroisoquinolines (TIQ) including salsolinol and norsalsolinol derivatives are of high interest  because TIQ are found at low concentrations in postmortem brain , cerebroBecause TIQ occur physiologically not only from their in vivo formation but also from ingestion of various foods ,22, theyBecause there are still different hypotheses on hyper- and hypodopamine deviances in central metabolism in ADHD  and TIQ 42 children and adolescents with ADHD  and 24 healthy controls  from three sites  were enrolled. All patients were referred and fulfilled DSMIV-TR  criteriaControls were recruited from hospital staff, their children and a school class near Goettingen. All controls were screened by an expert child and adolescent psychiatrist for absence of psychiatric disorders and reported no medication before and during study participation.Urine was collected over 12 hours starting at 7 p.m.. Study participants were not allowed to consume food or beverages rich in TIQ derivatives  for 48 hours before urine samples were obtained .The pilot study was approved by the ethics committees of each participating sites. Informed consent was obtained from the children and their parents. This study was conducted in accordance with the Helsinki Declaration.2EDTA. All aliquot samples collected were stored at -40°C and subsequently measured by a two-step chromatography. Urine samples were analyzed at least three times for all conditions, firstly processed by affinity chromatography, and then TIQ derivatives were quantified by high performance liquid chromatography with electrochemical detection (HPLC/ECD) as previously described [bound = TIQtotal-TIQfree.The 12 h urine samples were collected in the presence of 50 mg semicarbazide and 50 mg Naescribed ,27. Undeescribed , dihydroStatistical evaluations were conducted using the Statistical Package for the Social Sciences . Values were expressed in nM ± SEM as indicated . We perfbound was significantly different between the groups.In the ADHD group (n = 42), free and total concentrations of all measured TIQ derivatives were increased in urine samples compared to those of healthy controls (n = 24) , and since the control group itself was heterogeneous in age , we subsequently performed an ANCOVA with the covariate 'age'. Additionally, because of differences in the ADHD group in medication status (24 without versus 18 with) and co-existing psychiatric problems (26 without versus 16 with) two further covariates were included. There were significant effects of the covariates 'medication status'  and 'additional psychiatric diagnoses'  only for N-methyl-salsolinolfree: 92.5/94.4; norsalsolinolfree: 87.8/80.0; N-methyl-norsalsolinolfree: 69.0/93.5; salsolinolfree: 55.5/95.2).To determine the predictive quality (ADHD yes/no) of elevated TIQ levels (elevated yes/no) sensitivity and specificity were calculated. For the definition of an elevated TIQ level an arbitrary limit was set by the mean of the free concentration of the control group for each TIQ derivative plus one SEM. Specificity is the proportion of true negatives (no diagnosis of ADHD and no elevated TIQ level) of all negative cases  in the population; sensitivity is the proportion of true positives (diagnosis of ADHD and elevated TIQ level) of all positive cases  in the population (sensitivity/specificity (%): N-methyl-salsolinolfree , salsolinoltotal , N-methyl-salsolinolfree , N-methyl-salsolinoltotal , norsalsolinolfree , norsalsolinoltotal , norsalsolinolbound  and N-Methyl-norsalsolinolfree . Analogously, the absence of group differences remained for N-methyl-salsolinolbound , N-Methyl-norsalsolinolbound . For salsolinolbound  no group comparison could be performed because in the control group only for one child its concentration could be calculated successfully. For N-Methyl-norsalsolinoltotal the significant difference between both groups including all patients changed to a trend .To exclude possibly confounding effects of age, medication and comorbidity not only statistically by including covariates but also by strict in- and exclusion criteria of both groups, we analyzed in a second step the data of 21 children and adolescents with ADHD compared to 19 healthy controls under the age of 18 years. There were no differences in age  and in the ADHD group there were no patients with medication and comorbidity. The group differences in TIQ levels remained as calculated by the ANOVA in the whole sample for salsolinolfree, N-methyl-salsolinolfree, norsalsolinolfree, and N-methyl-norsalsolinolfree in ADHD patients even when considering three covariates  or when subjects with these confounders were excluded. N-methyl-salsolinolfree showed the highest sensitivity (92.5%) and specificity (94.4%) of the four TIQ derivatives.Comparisons of urine concentrations of TIQ derivatives between children and adolescents with ADHD and healthy controls revealed higher concentrations of salsolinolAlthough Moser et al  demonstrfree and norsalsolinolfree.Interestingly, in the present study there is no hint for an effect of the therapy with the psychostimulant methylphenidate on TIQ levels, although the increase of the endogenously produced synaptic dopamine concentration through inhibition of the dopamine transporter (DAT), which takes up the dopamine into the presynaptic neurons , might hIn any case, concluding an exclusive relationship between increased central dopamine metabolism and elevated urine concentrations of TIQ derivatives might be an oversimplified view because the found elevation of TIQ levels in ADHD could result not only from primary central but also from peripheral synthesis .free derivatives in urine give evidence for endogenous synthesis rather than oral ingestion, because ingested TIQfree derivatives will be rapidly inactivated by gluco- or sulfo-conjugation preventing elevated levels of non-conjugated TIQfree derivatives in urine [Because ingestion of TIQ influences their levels in urine ,22, partin urine . In addiin urine ,24 can nIndependently of considerations of causality, the sensitivity and specificity of TIQ urine levels, especially that for N-methyl-salsolinol, which is much better than that of other neurobiological procedures  needs coIn conclusion, urine levels of salsolinol, N-methyl-salsolinol, norsalsolinol and N-methyl-norsalsolinol are elevated in children and adolescents with ADHD and point to a new perspective on catecholaminergic dysfunction in ADHD. Replication of the findings in a larger sample of children and adolescents with ADHD focusing on subtypes and including a control group of children with other movement related catecholaminergic disorders would progress this innovative and promising field of research.Attention-Deficit/Hyperactivity Disorder (ADHD), Tetrahydroisoquinolines (TIQ), analyses of variance (ANOVA), analyses of covariance (ANCOVA).The author(s) declare that they have no competing interests.VR contributed to the conception and design of the study and was primarily responsible for the interpretation of the data and writing of the manuscript. SW contributed to the study design and data acquisition. FR contributed to the study design and data acquisition. RH contributed to the study design and data acquisition. AR contributed to the design of the study and revision of the final manuscript. MG contributed to the conception and design of the study and the interpretation of the data and writing of the manuscript. AM contributed to the conception and design of the study, to the analyses and the interpretation of the data and writing of the manuscript. All authors have read and accepted the final manuscript.


Attention-Deficit/Hyperactivity Disorder (ADHD) is a heterogeneous disorder from both clinical and pathogenic viewpoints. Executive function deficits are considered among the most important pathogenic pathways leading to ADHD and may index part of the heterogeneity in this disorder.SLC6A3) 3'-UTR VNTR genotypes and executive function in children with ADHD, 196 children diagnosed with ADHD were sequentially recruited, genotyped, and tested using a battery of three neuropsychological tests aimed at assessing the different aspects of executive functioning.To investigate the relationship between the dopamine transporter gene  and on the Wechsler Intelligence Scale for Children, Third Edition (WISC-III) Freedom From Distractibility Index , as well as strong trends on Self Ordered Pointing Task error scores  and WISC-III Digit Span performance . Children with the 9/10 genotype exhibited, on average, a poorer performance on all four measures compared to children with the 10/10 genotype. No effect of genotype on Wisconsin Card Sorting Test measures of performance was detected.SLC6A3 genotype may modulate components of executive function performance in children with ADHD.Results are compatible with the view that  ADHD is a heterogeneous disorder characterized by age-inappropriate inattention, motor activity, and/or impulse control. Its high prevalence in school-aged children 8 to 12%) [2% [1] inThere has been a strong focus on the dopamine transporter in ADHD as psychostimulants, the first line medication for ADHD, have been shown to block this transporter as part of their mechanism of action . FurtherWhile the above-mentioned genetic studies have examined allelic associations between specific polymorphisms and ADHD, genotypic approaches have also yielded support for DAT1 involvement. For instance, Loo et al. have reported that in patients with ADHD, there is an association between 10/10 DAT1 genotype and increased errors on a vigilance task when compared to a compound group composed of children having the 9/9 or 9/10 genotypes . Along sDue to the relatively small frequency of the 9/9 genotype in the general population and to the consequently small sample sizes generally acquired for this genotype, 10/10 genotype groups are usually compared to a combined group of subjects with the 9/9 and 9/10 genotypes (as well as other very rare genotypes). This being said, a few recent studies have compared genotypes separately. For instance, Cornish et al.  have shoOverall, these patterns of results are consistent with a high degree of heterogeneity in genetic effects and with the view that current candidate genes are only indirectly responsible for ADHD as they may instead be associated with specific clinical variants and/or behavioral dimensions of ADHD yet to be precisely identified. If this is the case, selecting candidate genes on the basis of their potential implication in modulating intermediate phenotypes (or endophenotypes) relevant to ADHD could improve the robustness of genetic results. While endophenotypes can exist at a number of levels, most studies to date have focused on the neuropsychological level. One such pathway with considerable empirical support implicates dysregulation of executive function processes . FacWhile traditionally, executive function tasks have been believed to rely mostly on the prefrontal cortex, the involvement of the basal ganglia is now being recognized ,32. For Finding an association between executive function dysregulation and ADHD together with demonstration that dopamine neurotransmission is critical for normal executive functioning would highlight the plausibility of executive function as an endophenotype in ADHD. From a genetic standpoint, the DAT1 gene is known to be preferentially expressed in the basal ganglia and has been reported to influence caudate volume  and aspeThe present study examined executive function performance in children with ADHD in relation to the Variable Number of Tandem Repeats (VNTR) located in the 3' untranslated (UTR) region of the DAT1 gene in order to further characterize the association between genotype and behavior in children with ADHD. It is hypothesized that DAT1 gene genotype has an effect on executive function performance.Children were sequentially recruited from the Disruptive Behavior Disorders Program and the Children Outpatient Clinics of the Douglas Hospital. They were referred to these specialized care facilities by school teachers, community social workers and pediatricians.To be included in this study, children were required to be between 6 and 12 years of age and meet DSM-IV diagnosis criteria for ADHD . Diagnosie 1- response inhibition and execution (includes planning), 2- working memory and updating, 3- set-shifting and task-switching, and 4- interference control), our neuropsychological test battery comprised the Wisconsin Card Sorting Test (WCST) [In order to capture the gist of the four factors of executive functioning reported above (t (WCST) , the Selt (WCST) , the Towt (WCST) , and theThe WCST, the SOPT, and the TOL were administered by trained research personnel as described in Taerk et al. . For botFurthermore, the WISC-III was administered by a trained psychologist to all children not having had it administered in the previous year at school. Where the WISC-III had been administered in the previous year, records of the evaluation were made available by the school authorities. In all cases where records were complete, the Freedom from Distractibility Index (FFDI) was derived from the Arithmetic and Digit Span subtest scores. 17% of children (n = 16) with the 9/10 genotype and 14% of children (n = 18) with the 10/10 genotype were tested at school.In the WCST, the child is asked to sort cards according to three different criteria . Feedback on whether the child achieves a correct or incorrect match is given after each trial. The matching criterion changes after ten consecutive correct matches and the child has to identify the new matching criterion using the feedback (correct/incorrect) provided to him. While the WCST is a task where one has to hold active information in working memory in order to identify the matching criterion, it arguably taps mostly into response inhibition capacity and into set-shifting and task switching ability. Evidence for the high reliability and validity of the WCST for various normal and clinical populations has been reported in many studies . While tie response inhibition and execution), but not with measures of interference control [In the SOPT, series of matrices of 6, 8, 10 and 12 images are presented to the child. The child is asked to chose, by pointing, one different image on each page. Errors occur when the child points to images that he pointed to in previous presentations of the set of images. Each set is presented to the child three times. While performing the SOPT, the demand on keeping information active in working memory is very high. SOPT scores have been shown to highly correlate with measures of working memory, strategy utilization and planning  constituted by the Arithmetic and Digit Span subtests . PerformThe research protocol was approved by the Research Ethics Board of the Douglas Hospital . Parents were explained the study and provided written consent. Children were explained the study and gave their assent to participate.SLC6A3 gene was genotyped using a PCR-based method as previously described [Blood samples were collected from affected children and their parents in order to extract DNA for the purpose of genetic analyses. The VNTR polymorphism in the escribed . The PCRThe VNTR polymorphism has two main alleles: 9- and 10-repeat alleles. Three very rare alleles  were also identified in 13 subjects . Because of the very small sample sizes of these latter genotype groups and because only 21 carriers of the 9/9 genotype were detected, we limited our analyses to the groups of children with the 9/10 or 10/10 genotypes. The total sample size of subjects having been genotyped for the DAT1 gene was 251. Of these, 96 had the 9/10 genotype and 127 had the 10/10 genotype.To test for the effect of genotype on SOPT performance, we implemented a general linear model repeated measures analysis with genotype  as the between subject factor and the 4 SOPT difficulty level error scores as the within-subject repeated measures. A similar procedure was implemented for the 12 difficulty level scores of the TOL. An analysis of variance was implemented for the WCST and WISC-III scores. Age was used as a covariate except when test scores were already age-standardized (eg WISC-III subtests).The mean of the absolute values of the correlations between scores on the different outcome variables  was modest to moderate (mean r = 0.357). Between different tests (eg WCST and WISC subtests), the absolute value of the correlations ranged between a low of 0.042 and a high of 0.379. However, between the different indices of performance on a given test , correlations ranged between 0.495 and 0.874. High correlations between performance values of a given test are expected in cases when performance on one index is in part derived from performance on another index. Taking these correlations into account , a Bonfe2 = 0.043, df = 1, p = 0.84). Gender, ethnic group, age, average household income, and severity of behavioral problems as assessed by the CBCL  were not significantly different between the three genotype groups. Further, no significant differences between genotype groups were found in IQ, in diagnostic subtype of ADHD  or in the number of ADHD items as assessed by the DISC-IV. In keeping with most studies on ADHD, the sample was characterized by a high prevalence of comorbidity including oppositional defiant disorder and conduct disorder. However, there were no significant differences in comorbid disorders between the genotype groups . The genotype distribution did not differ from the Hardy-Weinberg equilibrium  and FFDI performances  as well as a strong trend on SOPT error scores  and Digit Span performance . Children with the 9/10 genotype had poorer performance on all four measures compared to children with the 10/10 genotype . In contrast, the SOPT, TOL, and FFDI subtests all exert a very high demand on working memory whether directly (SOPT and Digit Span FFDI subtest) or through mental manipulations (which is one of the elements of working memory) that have to be made while the child keeps bead positions (TOL) or numbers (Arithmetic FFDI subtest) in active memory. Further work will be needed to confirm this tentative hypothesis.The most important finding of this study is the presence of a significant effect of the While Barkley et al. (see introduction)  found moAs stated in the introduction and in apparent contradiction with the current findings, Cornish et al.  and Loo As the10-repeat allele has frequently been reported as being a risk allele for ADHD, it may appear surprising that those with the 10/10 genotype are not the ones with the worst performance here. However, whether the 10-repeat allele is or not a risk allele for ADHD is not necessarily, by itself, very informative about its potential association with a specific endophenotype 4(e.g. executive functioning) for a subset of subjects with ADHD as a given.SLC6A3 gene and thus does not change the structure of the DAT1 protein. Nonetheless, it has been suggested that it may affect the level of expression of the DAT1 gene, resulting in variable dopamine transporter phenotypes. While this may be the case, the issue still remains controversial. Indeed, it has been reported that DAT binding availability is significantly lower for subjects who are homozygous for the SLC6A3 VNTR 10-repeat allele compared to carriers of at least one 9-repeat allele [While the current study has strengths, including a restricted age range and the assessment of executive function using multiple indices while children were not taking psychostimulant medication, its results should be interpreted with important limitations kept in mind. First, it is noteworthy that some children were tested on the WISC-III by different examiners prior to the initiation of the study. However, if this had an impact on results, it is expected to have manifested itself as statistical noise, making results less likely to appear as significant. Second, the study sample was mainly comprised of children with the combined type of ADHD and not enough subjects had the inattentive or hyperactive/impulsive subtypes in both genotype groups, precluding us from analyzing the relation between DAT1 gene genotype and executive function in each ADHD subtype. Further investigation in larger groups would allow analysis of the aggregation of different clinical aspects stratified by genotype. Third, the polymorphism that we studied in this sample is located in the 3'-untranslated region of the t allele  while tht allele  was repot allele  was founIn conclusion, results from this study show that the DAT1 gene genotype can be a marker of performance of executive functioning (perhaps mostly working memory) in children with ADHD with those having the 9/10 genotype generally exhibiting a relatively poorer performance than those with the 10/10 genotype. As such, and assuming that an endophenotype has a greater potential to target one of the many pathophysiological deficits that in combination may lead to a disorder, present results support the view that it may be possible to characterize with a certain degree of refinement the associations between genotype and semiology. In the wake of frequent false positive findings in behavioral genetics, replication of the current findings is warranted before firm conclusions can be drawn.The authors declare that they have no competing interests.SK analyzed results, conceived, and wrote the manuscript. NG helped design the study, assessed the patients, provided conceptual input on the manuscript content, and provided access to patients. ES-B wrote part of the manuscript and provided conceptual input on the manuscript content. AD provided conceptual input on the manuscript. JB provided conceptual input on the manuscript. VM participated in the cognitive testing of the subjects. AP recruited and cognitively tested the subjects. MT-S recruited subjects and was the coordinator of the project. RDG carried out the molecular genetic studies. JB recruited and cognitively tested the subjects. SS carried out molecular genetic studies and provided conceptual input on the manuscript content. RJ designed the study, assessed the patients, and supervised the conception and writing of the manuscript.The pre-publication history for this paper can be accessed here:


Several studies report that dietary fibre from different sources promotes the feeling of satiety and suppresses hunger. However, results for cereal fibre from rye are essentially lacking. The aim of the present study was to investigate subjective appetite during 8 h after intake of iso-caloric rye bread breakfasts varying in rye dietary fibre composition and content.The study was divided into two parts. The first part (n = 16) compared the satiating effect of iso-caloric bread breakfasts including different milling fractions of rye  and sifted flour). The second part (n = 16) investigated the dose-response effect of rye bran and intermediate rye fraction, each providing 5 or 8 g of dietary fibre per iso-caloric bread breakfast. Both study parts used a wheat bread breakfast as reference and a randomised, within-subject comparison design. Appetite  was rated regularly from just before breakfast at 08:00 until 16:00. Amount, type and timing of food and drink intake were standardised during the study period.The Milling fractions study showed that each of the rye breakfasts resulted in a suppressed appetite during the time period before lunch (08:30-12:00) compared with the wheat reference bread breakfast. At a comparison between the rye bread breakfasts the one with rye bran induced the strongest effect on satiety. In the afternoon the effect from all three rye bread breakfasts could still be seen as a decreased hunger and desire to eat compared to the wheat reference bread breakfast.In the Dose-response study both levels of rye bran and the lower level of intermediate rye fraction resulted in an increased satiety before lunch compared with the wheat reference bread breakfast. Neither the variation in composition between the milling fractions nor the different doses resulted in significant differences in any of the appetite ratings when compared with one another.The results show that rye bread can be used to decrease hunger feelings both before and after lunch when included in a breakfast meal. Rye bran induces a stronger effect on satiety than the other two rye fractions used when served in iso-caloric portions.Trial registration number NCT00876785 Energy-dense foods that require little effort to consume and that are rapidly digested may cause passive over-consumption by failure to provide a feeling of fullness corresponding to the energy content. A diet that is predominantly based on such foods may lead to overweight. It is therefore important to identify properties of foods that facilitate energy balance by creating a high satiety per calorie. A number of studies have confirmed that foods naturally rich in dietary fibre promote the feeling of fullness and reduce hunger in the short term . A recenIn the present study we investigated subjective appetite  during 8 h after consumption of iso-caloric rye bread breakfasts. The study was performed in two parts. The first part (Milling fractions study) compared different milling fractions of rye. The second part (Dose-response study) included different levels of dietary fibre from rye.2; a habit of consuming breakfast, lunch and dinner everyday; fasting plasma glucose 4.0-6.1 mmol/L; haemoglobin (Hb) in men 130-170 g/L, in women 120-150 g/L; alaninaminotransferase (ALT) in men 0.15-1.1 μkat/L, in women 0.15-0.75 μkat/L; thyroid-stimulating hormone (TSH) 0.3-4.0 mlE/L and willingness to comply with the study procedures. Exclusion criteria were the following: intake of medicine likely to affect appetite or food intake; any medical condition involving the gastrointestinal tract; eating disorder; smoking; consumption of more than three cups of coffee per day; change in body weight of more than 10% three months prior to screening; consumption of any restricted diet such as vegan, gluten-free, slimming etc.; and pregnancy, lactation or wish to become pregnant during the study period. Information about the study was first sent out by e-mail, to employees at Lantmännen, Järna, Sweden. Potential participants were screened in a telephone interview and undertook a health control before being recruited. A total of eleven participants is the minimum number to detect a difference of 10 mm in hunger ratings over 4.5 h [The criteria for inclusion were the following: age between 20 and 60 years; body mass index (BMI) 18-27 kg/mA randomised, crossover design was used to compare the effects on subjective appetite 8 h after consumption of iso-caloric breakfast meals. The aim of the study and the content of the test products were not known to the participants. Each participant acted as his/her own control and received each of the bread breakfasts in a randomised order on different occasions, separated by six to eight days. In the time between the test days, the participants kept their ordinary diet. On the day prior to each test day, participants were instructed not to conduct any vigorous physical activity or drink any alcoholic beverages; not to eat or drink after 20:00 and to eat similar type and amount of evening meal. During the test day the participants were asked to fill out a diary, briefly noting their food and beverage consumption and exercise patterns from the previous evening, to ensure that instructions had been followed. Upon arrival on the morning of the test days (08:00) the participants were served one of the breads together with additional breakfast foods, identical on all test occasions in both parts of the study. During intake of the breakfast, the participants were seated in individual booths and instructed not to talk to one another. At 12:00 the participants were given a standardised lunch meal. The breakfast and lunch meals had to be consumed entirely within 30 min. At 14:00 they were allowed to drink coffee or tea. The hot drink, along with a voluntary choice of milk and sugar, was kept identical for each of the participants on all test occasions. Subjective feelings of appetite  were assessed every half hour, starting at 08:00 and continuing until 16:00. The first recording was made in the fasted state immediately before breakfast at 08:00. The data were collected using a specially designed programme  on a palThree rye milling fractions were used table : rye braTo create breads with acceptable palatability and relatively soft texture, the amount of rye that could be used was limited to 60% of the total amount of flour for rye bran and 75% for the intermediate rye fraction. A higher content of rye resulted in compact breads with low ability to rise during fermentation.When baking each type of bread, the amounts of ingredients table  were scaThe different types of breads contained the same amount of energy per portion and were similar in protein, fat and available carbohydrate composition table . The maiFood intake was standardised in terms of type, amount and timing during the test day. The rye breads and the wheat reference bread were served in random order on separate occasions as breakfast meals with identical additional foods: 10 g of margarine (40% fat), 25 g of apricot marmalade, 15 g cheese (26% fat), 200 g of milk (0.5% fat) and one cup of tea or coffee. The participants were allowed to choose between coffee and tea on the first test day and then received the same type of drink for the following test days. The breakfast meal provided in total 1960 kJ . Although dietary fibre is known to contribute energy from short-chain fatty acids following colonic fermentation at around 8.4 kJ/g  , there iRatings for satiety, hunger and desire to eat were analysed using Minitab . The level of significance was set at p < 0.05. ANOVA was performed as paired t-tests and Tukey comparisons using participants as random effect and type of breakfast and time points as fixed effects. Separate analysis was done for the morning (08:30-12:00) and afternoon ratings (12:30-16:00).All participants finished the breakfast and lunch meals completely according to instructions. No adverse events were recorded and no one had problems finishing the test meals well within 30 min.Before breakfast the mean ratings for hunger, satiety or desire to eat were similar between test days. Appetite ratings showed a clear effect of time after breakfast and after lunch, i.e. the VAS ratings visibly demonstrated that the participants responded with lessened hunger and increased satiety directly after the meals and then consecutively rated less satiety and stronger hunger as the time for the next meal approached.The Milling fractions study was designed to compare the satiating capacity of iso-caloric portions of three rye milling fractions included in bread breakfasts. Sifted wheat bread breakfast was used as reference. All of the 16 participants recruited initially (table In the morning 08:30-12:00) the rye bran bread breakfast induced the strongest effect on satiety, stronger than that of the intermediate rye fraction and sifted rye flour bread breakfasts (figure 2:00 the In the afternoon (12:30-16:00), after the standardised lunch, type of breakfast bread did not affect satiety. However, hunger and desire to eat was lower after consumption of each of the three rye bread breakfasts compared with the wheat reference bread breakfast. When the three rye bread breakfasts were compared with each other, no significant differences were seen in any of the appetite measures during the afternoon.The Dose-response study was designed to investigate the satiating capacity of four rye bread breakfasts with rye bran and intermediate rye fraction, each in amounts providing 5 or 8 g of rye dietary fibre/portion. Of the 19 participants recruited initially, 16 completed the study (table On comparisons with the wheat reference bread breakfast the results demonstrated a significantly increased satiety 08:30-12:00) even at the lower levels of rye bran and intermediate rye fraction, as well as for the rye bran bread breakfast, that provided 8 g of rye dietary fibre/portion (figure :00 even In the afternoon (12:30-16:00), after the standardised lunch, type of breakfast bread did not significantly affect appetite. No significant differences were seen in any of the appetite measures, when the four rye bread breakfasts were compared with each other. However, in the late afternoon there was tendency for increased satiety for all the rye bread breakfasts compared to the wheat reference breakfast.The aim of this study was to investigate subjective appetite during 8 h after intake of iso-caloric rye bread breakfasts. Finely milled rye fractions were compared at different levels. When the appetite ratings after intake of the rye bread breakfasts were compared with those after consumption of the wheat reference bread breakfast, significant effects on appetite were apparent in both studies. At comparison between the three milling fractions, a distinct effect on satiety was seen after the breakfast with rye bran, followed by the other two rye bread breakfasts. In the second part no differences between the rye breads were found, despite differences in the amount and composition of rye. This may be explained by some property present in all of the rye breads, for instance relating to bread structure .The levels of rye used in the breads were based on realistic amounts to create palatable, voluminous bread. The bread portion, together with additional breakfast foods, comprised what would be considered a normal breakfast meal. The amount of calories corresponded to recommended breakfast intake. These considerations somewhat limited the amount of rye that could be used in the breakfast meals. Nevertheless, in both parts of the study the rye breads had an affect on appetite compared with the wheat reference bread indicating that the method was sufficiently sensitive.In an earlier study  with simThe mechanisms underlying the satiating effects were not investigated in the present study. However, the satiating properties of dietary fibre have been related to several stages in the physiological processes of short-term appetite regulation . These iIn a broader perspective it is important to consider whether a dietary pattern providing a high satiety per calorie is potent enough to affect energy intake in real life, and in the long run facilitate weight maintenance and/or weight loss. It is evident that satiety signals are being overwhelmed to a certain degree by the potency of external cues to eating . Longer The present study provides support for altering appetite towards an increased satiety for up to 8 h by including rye breads in a breakfast meal.The study was financed by the producer of the test products, Lantmännen. HI and HF are employed by Lantmännen R&D. The authors declare that the data presented in this publication represent a complete, true and faithful representation of the work performed.HI, HF, RA, PÅ and JO participated in designing the study. HI recruited the participants and performed the study. RA and HI analysed the data. HI wrote the manuscript. All authors participated in revising the manuscript and read and approved the final manuscript.


Attention Deficit/Hyperactivity Disorder (ADHD) affects many children, adolescents, and adults and is associated with a number of impairments. Poor academic performance is related to ADHD in clinical samples. However, it is unclear to what extent core ADHD symptoms and scholastic impairment are related in non-referred school-aged children.Data come from three population-based cohorts from Sweden, Denmark, and Finland, which are part of the Nordic Network on ADHD. The combined sample size was 13,087 children who were studied at ages 7–8 or 10–12 years. Teachers rated children on inattention and hyperactivity symptoms and reported children's scholastic performance on basic skills.There was a significant association in all cohorts between core ADHD symptoms and scholastic impairment in reading, writing, and mathematics. Particularly, inattention was related to a two to tenfold increase in scholastic impairment. Prevalence of hyperactivity symptoms was similar across the three cohorts, but inattention was lowest among children from the Finnish cohort, after stratification on living conditions.These results extend previous reports of scholastic impairment among children with clinically diagnosed ADHD to non-referred population samples from three European countries. Surveillance policies should be implemented in school systems to catch children in need of behavioral or scholastic support early. Attention Deficit/Hyperactivity Disorder (ADHD) is the most common neurobehavioral disorder in children and adolescents. ADHD is associated with impairment in all aspects of a child's life, i.e. family, social, and academic ,2. ChildADHD is characterized by inattention and hyperactivity symptoms inappropriate for age or developmental level and diagnosis requires symptoms to be associated with considerable impairment. Scholastic underachievement is associated with clinically diagnosed ADHD in children  and loweOur aim was to investigate the association between core symptoms of ADHD and impairments in basic scholastic skills, i.e. reading, writing, and mathematics in large non-referred samples from three European countries. This approach advances our understanding in several ways. First, we examine the basic academic skills, as opposed to concentrating on reading, which has been previously associated with ADHD. Second, the large sample size enables us to adequately test whether the relations are consistent for girls as well as boys. Third, we have the opportunity to check whether findings replicate in the three participating countries.We focused on teacher-rated restlessness, fidgetiness, and inattention because these symptoms are basic to the diagnostic criteria listed for ADHD in the DSM-IV  and HypeWe studied a large sample of children from three population based birth cohorts from Sweden, Denmark, and Finland. These countries share similar cultural traditions, political structures, and tax-paid healthcare, school systems and social services for children. Despite general similarities, local variations exist and this provides an opportunity to examine whether the associations replicate across a large geographic region. Most previous studies of ADHD have been confined to specific geographic locations. We hypothesized that the core ADHD symptoms and scholastic impairment would be positively related in all cohorts. Further, we expected to find cross-national similarity in the prevalence rates of these symptoms.st Child in the Family, Denmark, The Aarhus Birth Cohort (ABC), and Finland, Northern Finland 1985–86 Birth Cohort (NFBC 1986), which longitudinally investigate medical and psychosocial endpoints. These cohorts are part of the Nordic Network on ADHD, which the Nordic Ministry of Health established and funded to promote a collaborative scientific effort to study various aspects of ADHD. The cohorts share a number of characteristics that make comparison reasonable, although the studies were not originally designed for cross-national comparison. The Ethics Review Board in each of the respective countries approved the studies.Prospective data originate from three birth cohorts from Sweden, 1All cohorts consecutively recruited women in early pregnancy via antenatal health services and achieved high recruitment rates (91% to 99%). Antenatal health care in the three countries is tax-paid. The percentage of pregnant women not receiving this type of antenatal care is less than 0.2% . RoutineAt follow-up participants were traced through the national registries using personal identification numbers in each country, enabling us to trace even those who moved outside the original geographic areas, i.e. anywhere within the national borders. Follow-up data collection occurred when children were approximately 7–8 years in Sweden and Finland and 10–12 years old in Denmark. Permission to contact the child's teacher was obtained from parents in all cohorts. All cohorts collected data on child behavior symptoms and scholastic performance from teachers via postal questionnaire.0 (does not apply), 1 (somewhat true), or 2 (certainly true).Teachers assessed child behavior using the official translations of the Strengths and Difficulties Questionnaire (SDQ)  in SwedeTeachers assessed deficits in reading, writing, and mathematical skills using 7-point and 5-point scales in Sweden and Denmark, respectively, and impaired/unimpaired format in Finland. We dichotomized the scales into below vs. average and above average performance for the purpose of the present study. Previous work shows that teachers using single-item ratings are accurate judges of impairment and their ratings concur with achievements test results ,22. TablEquivalent statistical analyses were conducted separately for each cohort using SAS version 8.2 . The sum of the three core ADHD symptoms  ranged from 0 to 6. We reported the mean sum score by gender across cohorts and examined possible differences using 95% confidence intervals (CI).The cohorts differed on living conditions, maternal education  and on family structure at follow-up . Such indices of disadvantage have been previously shown to relate to ADHD diagnosis . Therefocertainly true because of their clinical relevance in relation to impairment. Core symptoms scores were dichotomized as severe ratings (coded 1) versus lower ratings . We combined the two hyperactivity symptoms and required a severe score on both restlessness and fidgetiness to code 1 and 0 represented any other combination. All classifications were done prior to analyses. Separate models were run for each type of symptom (predictor) and each scholastic skill (outcome).Multivariate logistic regression models were used to assess the association between core ADHD symptoms and scholastic impairment adjusted for maternal education, family structure, and child gender. We examined whether the sum of symptoms, based on a continuous scale, was associated with impairment among boys and girls in each of the cohorts. We also report the strength of the associations using standardized estimates . We then focused on the most severe ratings, i.e. The samples were based on the number of traceable live births in each cohort (rather than on the number of recruited pregnant women) for which parental consent to contact the teacher was obtained and consisted of a total of 13,087 children. Table The unadjusted mean sum score (95% CI) for the three hyperactivity-inattention symptoms for boys in Swedish, Danish, and Finnish cohorts were 1.9 , 1.6 , 1.3 , respectively. The Finnish mean was significantly different from Swedish and Danish means at a probability level of at least .05. Girls' means were 0.7 , 0.6 , 0.5 , respectively and did not differ significantly.certainly true) in comparison to girls. The percentage of boys receiving a high score on any core symptom was lowest in the Finnish cohort. Inattention was most prevalent for children in Denmark.Unadjusted prevalence of high scorers on each of the inattention and hyperactivity core symptoms is presented in table Prevalence of hyperactivity symptoms did not differ significantly across cohorts after stratification by maternal education and family structure (detailed data available on request). Approximately 15–20% of boys from disadvantaged homes  were rated high on hyperactivity symptoms in all cohorts. Well below 10% of girls received such a rating. Less than 10% of boys and much less than 5% of girls in the most advantaged strata  were high on hyperactivity symptoms in all cohorts. However, inattention differed between cohorts. Fewer disadvantaged boys in the Finnish cohort received severe ratings on inattention than in the Danish, 10% vs. 34% respectively. Danish and Swedish cohorts did not differ. Inattention among girls was clearly most prevalent in the Danish cohort in comparison to Finnish cohort, 13% vs. 3%, and there were no differences between the Swedish and Finnish cohorts.Twenty eight percent of children in the Swedish and Danish cohorts and 22% in the Finnish had impaired scholastic performance on at least one skill. Figure In order to determine whether the association between the sum of the core symptoms and impairment applied only to particular skills, we examined the association with each skill separately in each cohort and for each sex. Further, we were also interested in whether the associations held for both boys and girls. Table To assess whether severity in either type of symptom was related to impairment, we examined severe ratings as these may have more clinical relevance. Table In general population based cohorts from three European countries including over 13,000 children, we found a strong and consistent pattern of associations between core ADHD symptoms and scholastic impairment. These results are in line with previous research showing comorbidity between ADHD and learning problems ,27, and Prior work has focused on literacy skills and found an association with ADHD . HoweverGirls had a lower prevalence of severe symptoms in all cohorts, which is in line with the gender disparity in ADHD diagnosis. The larger cohorts showed significantly stronger relative associations between core symptoms and impairment among girls as compared to boys.A major change in the DSM-IV from earlier versions was the addition of impairment requirement. Symptoms must generate impairment in order to be considered a psychiatric disorder. Several reports show that many children are impaired but do not reach the threshold number of symptoms and, therefore, do not meet the diagnostic criteria . ImpairmInitial screening procedures could be conducted in schools as teachers are in a good position to first detect learning problems and notice behavioral deviations because children with ADHD symptoms often disrupt the classroom. Ideally screening for ADHD symptoms could be a part of the school health surveys. Alternatively, teacher ratings on core symptoms could be incorporated into routine progress reports given to parents. Early support in the form of academic assistance, teaching coping strategies to children , and teachers' monitoring behaviors in the classroom would be important first steps. This would also facilitate an alliance between teachers and parents to work on shared goals. This strategy is in keeping with a greater emphasis being placed on support rather than assessment . MultimoPrevalence of core symptoms was related to living conditions in all three cohorts. Our results converge with previous reports showing that disadvantaged children are more likely to receive high ratings on behavioral problems ,43. ThusThe finding that inattention was lowest in Finland (even after stratification on living conditions) merits further research to discern whether etiological factors or other unmeasured factors related to living conditions are at the root of the observed difference. It is unclear to what extent methodological differences between cohorts could have contributed to the differences.There are some limitations to consider. First, methodology differed somewhat between the cohorts e.g., data collection took place 6 years earlier in the Finnish cohort. This difference in time of data collection may explain why Finnish children were rated less inattentive than in the other two cohorts. It may be possible that awareness of ADHD symptoms has been steadily increasing within the last decade and may have affected teachers' willingness to endorse symptoms. However, it seems unlikely that increased awareness would pertain only to inattention and not to hyperactivity symptoms. A Swedish community study , which cSecond, we were limited to teacher-reported core symptoms and scholastic impairment. It may be that teachers tend to rate a child poorly in one area if he or she is rated poorly in another or that behavioral ratings bias perceptions of scholastic performance, e.g. due to a negative halo effect. However, teachers within each cohort rated various behaviors and outcomes pertaining to well-being and development, consequently the connection between the three core symptoms that we study and scholastic impairment was not likely to have been made. The majority of children in our study were first-graders, therefore, official grades or national test scores are not available with which to compare teacher ratings. Nonetheless, our measure has ecological validity as teachers are in the best position to rate academic performance and do so in reality. Teacher ratings of behavior have been found to reflect age-appropriate evaluations . SymptomThird, we related only three core symptoms to scholastic performance rather than clinical diagnosis. Our results show that functional impairment is not only limited to children who fulfill all the clinical criteria for ADHD, but suggest that increases in symptomatology and impairment go hand-in-hand in the general population.Fourth, we used cross-sectional data although we expect a relation between ADHD symptoms and scholastic impairment to develop over time as has been suggested in clinical studies ,48. It iDespite these limitations, the relation between hyperactivity and inattention symptoms and scholastic impairment was replicated in three general population samples of nonreferred children from different countries, which indicates a robust relation. Symptoms were related to all the skills we investigated suggesting overall scholastic difficulties. Scholastic impairment is a powerful predictor of adverse developmental trajectory.The author(s) declare that they have no competing interests.AR participated in the design of the study and drafted the manuscript. KP, JM, and AR performed the statistical analyses. CO provided the Danish, AT and IM the Finnish, and AR the Swedish cohort datasets. JO and TBH, MRJ, and AR supervised the cohort studies in Denmark, Finland, and Sweden respectively. All authors contributed to the manuscript, read and approved the final version.The pre-publication history for this paper can be accessed here:


Purpose. Deep fibromatoses are large, often rapidly growing            but benign soft tissue tumours. Although surgery is the mainstay of treatment, in            unremitting and aggressive cases the use of cytotoxic chemotherapy may produce            objective tumour responses. Fresh tumour samples from four patients with fibromatosis            were investigated as part of a study of drug resistance in soft tissue tumours.                    Methods. Following short-term culture of fibromatosis cells in vitro ,                 chemosensitivity to 4-hydroperoxy-ifosfamide, the active form of ifosfamide and                 doxorubicin was tested. Following 72-h continuous exposure to each drug,                 surviving cell fraction was assessed using the lactate dehydrogenase assay.                    Results. Mean IC50 values for ifosfamide and doxorubicin were                 6.2 and 0.35 µmol/l, respectively. In samples of soft tissue sarcoma (STS) from the                 same study the mean IC50 values for ifosfamide and doxorubicin were 14.8 and                 1.69 µmol. The difference in mean ifosfamide IC50 values for fibromatosis and                 STS samples was statistically significant.                    Discussion. We are not aware of any other report suggesting                 the use of ifosfamide in this condition. These observations suggest that, for patients                 with inoperable or progressive lesions of fibromatosis causing significant morbidity,                 it may be valuable to include ifosfamide in experimental treatment regimens.


Anopheles gambiae and Anopheles arabiensis.Immune responses to parasites, which start with pathogen recognition, play a decisive role in the control of the infection in mosquitoes. Peptidoglycan recognition proteins (PGRPs) are an important family of pattern recognition receptors that are involved in the activation of these immune reactions. Pathogen pressure can exert adaptive changes in host genes that are crucial components of the vector's defence. The aim of this study was to determine the molecular evolution of the three short PGRPs  in the two main African malaria vectors - An. gambiae and An. arabiensis PGRP-S1, PGRP-S2 and PGRP-S3 was investigated in samples collected from Mozambique and Tanzania. PGRP-S1 diversity was lower than for PGRP-S2 and PGRP-S3. PGRP-S1 was the only gene differentiated between the two species. All the comparisons made for PGRP-S1 showed significant P-values for Fst estimates and AMOVA confirming a clear separation between species. For PGRP-S2 and PGRP-S3 genes it was not possible to group populations either by species or by geographic region. Phylogenetic networks reinforced the results obtained by the AMOVA and Fst values. The ratio of nonsynonymous substitutions (Ka)/synonymous substitutions (Ks) for the duplicate pair PGRP-S2 and PGRP-S3 was very similar and lower than 1. The 3D model of the different proteins coded by these genes showed that amino acid substitutions were concentrated at the periphery of the protein rather than at the peptidoglycan recognition site.Genetic diversity of An. gambiae and An. arabiensis regardless of geographic location. This probably relates to its location in the chromosome-X, while PGRP-S2 and PGRP-S3, located in chromosome-2L, showed signs of autosomal introgression. The two short PGRP genes located in the chromosome-2L were under purifying selection, which suggests functional constraints. Different types of selection acting on PGRP-S1 and PGRP-S2 and S3 might be related to their different function and catalytic activity.PGRP-S1 is less diverse and showed higher divergence between  Plasmodium play an important role in the natural control of the infection and are initiated when pathogen-associated molecular patterns (PAMPs) are recognized by pattern recognition receptor (PRR) molecules [Anopheles PGRPs can be divided into two different classes: short and long. In Drosophila short PGRPs are small extracellular proteins about 200 amino acids long and 18-20 kDa that are present in the hemolymph and cuticle. They are constitutively synthesized or induced mainly in the fat-body and, to a lesser extent, also in the epidermal cells, in the gut and in the hemocytes. Long PGRPs, have long transcripts and are either intracellular or membrane-spanning proteins, expressed mainly in the hemocytes [Anopheles gambiae genome [An. gambiae differ in their structure, as PGRP-S2 and S3 have predicted amidase activity while PGRP-S1 does not. The presence of catalytic activity in Drosophila short-PGRPs determines its function. Drosophila has two noncatalytic short-PGRPs, PGRP-SA and PGRP-SD, which are involved in recognition of bacteria and activation of the Toll pathway [Plasmodium infection [Plasmodium.Mosquito immune responses to olecules . Peptido pathway , while P pathway . Transcrnfection  have beenfection ,8 is thaPGRP genes form clusters in the genome suggesting that they may have been originated by gene duplication . This phAn. gambiae and Anopheles arabiensis, the main malaria vectors in sub-Sahara Africa, and considered the type of selective pressure acting on them.Molecular evolution of immune related genes is determined by their interaction with pathogens. In the present study we analysed patterns of evolution in three short PGRPs of the malaria vectors An. gambiae  and 91 from An. arabiensis. Eighty eight sequences were obtained for PGRP-S1 (genbank accession nos FJ821900 - FJ821987), 47 for PGRP-S2 (genbank accession nos FJ821988 - FJ822034) and 102 for PGRP-S3 (genbank accession nos FJ821798 - FJ821899). There were 15 An. gambiae and 18 An. arabiensis for which all three genes were successfully sequenced.A total of 237 sequences were analysed, 146 from An. arabiensis from Mozambique than in the other groups especially in the 5' upstream and 3' downstream non-coding regions (0.013 and 0.023 respectively)  was amplified. For this gene the nucleotide diversity (π) was higher for A total of 1255 bp was amplified in the PGRP-S2 gene, which included the coding region (567 bp). In this gene the π values varied between 0.007 and 0.014 in the coding region and between 0.002 and 0.041 in the non-coding regions . The lowest Fst estimates were obtained in intraspecific comparisons, indicating that differentiation was higher between species than between geographic populations Table . For theTo better understand the relationship between species and geographic regions, a hierarchical analysis of molecular variance (AMOVA) was performed Table . For PGRAn. arabiensis and 83C_GT for An. gambiae. Both were present in the 2 geographic regions and probably correspond to the ancestral haplotype. For An. arabiensis the other 7 haplotypes were unique for each geographic sample. In An. gambiae a lower number of haplotypes was found (5 versus 8) and a second haplotype was shared by the An. gambiae - Mozambique and An. gambiae - Tanzania populations.The median-joining network based on the PGRP-S1 haplotypes showed a clear interspecific separation Figure . Each spversus 13), and the separation between species was not as clear as in the network of the PGRP-S1. There was a single high frequency haplotype, 5-6_AM, that was shared by more than one population and by both species. All the other haplotypes were unique for each population and species, and showed low frequency. All 13 low-frequency haplotypes specific of An. arabiensis were more closely related in the network. For An. gambiae, 9 haplotypes grouped together in the network but two  were closer to An. arabiensis haplotypes.The network based on the PGRP-S2 haplotypes Figure  showed aAn. gambiae. No clear separation was observed either between species or between geographic regions. A network based on pooled PGRP-S2 and PGRP-S3 haplotypes showed interspecific common haplotypes between these two genes   Figure  was madeAn. gambiae-Tanzania population. For the other genes, the results were consistent with neutral evolution  and nonsynonymous (πa) nucleotide diversity was calculated for each gene within each group  [see Additional file There were 14 types of proteins present in our sample [see Additional file The phylogenetic analysis using different methods  show the same structure (data not shown). An isolated branch corresponding to PGRP-S3 type 2 was always present and was the only one with a bootstrap confidence above 70%  in α2 helices and 1 in α3 helices (Thr185Asn). No mutations were present in the β strands or at the recognition sites.An. arabiensis and another to An. gambiae [see Additional file For PGRP-S1 there were 2 protein types, one corresponding to Drosophila and Anopheles [An. gambiae among them PGRP-S1. The nucleotide diversity (π) found for this gene for An. gambiae S form was 0.008; twice as high as the maximum value found in our study. This may relate to differences in geographic location. Although differences were not observed when we compared Mozambique and Tanzania a greater geographical separation could account for differences in genetic diversity between East and West African samples [Nucleotide diversity estimated for the three short PGRP genes was comparable with other studies made on immune related genes of nopheles -19. Cohunopheles  analysed samples .An. gambiae and An. arabiensis for PGRP-S1 gene, which was not observed for the other genes. The phylogenetic networks reinforced the results obtained by the AMOVA and Fst values. The different patterns observed between the three genes may be due to their location on the genome. PGRP-S1 gene is located in the chromosome X (within the Xag inversion) and PGRP-S2 and PGRP-S3 are located in the chromosome 2L (within the 2La inversion). An. gambiae and An. arabiensis display species-specific paracentric inversion arrangements Xag and Xbcd, respectively at the chromosome X. This should contribute to a higher differentiation between the two species at this chromosome due to reduced recombination in the case of rare hybridization between the two species. A recent study has shown that introgressed alleles between An. arabiensis and An. gambiae in the chromosome X were lost in two generations but introgressed alleles were not lost at loci located in the autosomic chromosome 2L [An. gambiae and An. arabiensis. Introgression of genes in autosomal chromosomes through rare hybridisation between these two sibling species has been demonstrated previously, particularly for chromosome 2L [Some differences were found in the three genes studied: PGRP-S2 and PGRP-S3 revealed higher nucleotide diversities when compared to PGRP-S1. With respect to species divergence and population differentiation, once again PGRP-S1 differed from the others. Fst values and AMOVA indicated a clear separation between osome 2L . This maosome 2L ,22.http://www.ensembl.org/Anopheles_gambiae and their coding region have 95% homology. These two genes are both considered functional, since the ratio Ka/Ks presents values much lower than 0.5 [PRGP-S2 and PGRP-S3 are duplicated genes that are physically close (ca 3 kb) at chromosome 2L than 0.5  and seemthan 0.5  suggest Drosophila PGRP-SD interact with GNBP in order to optimally activate the Toll pathway [An. gambiae PGRP-S2 and PGRP-S3 function is not known but transcription profiles of Plasmodium falciparum infected mosquitoes were down regulated in midgut and carcasses when compared with mosquitoes that had a blood meal with a P. falciparum strain unable to produce gametocytes [This agrees with the 3D models of the protein, where substitutions were concentrated at the periphery Figure , while r pathway . Amino aetocytes . Howeveretocytes . Once agAn. gambiae and An. arabiensis split from a common ancestor. The higher complexity of the haplotype network for PGRP-S3, showing more diversity and a higher proportion of shared haplotypes between species suggests that this may be the ancestral gene from which PGRP-S2 may have originated.We cannot determine with certainty when the duplication occurred, but since it is present in both species it is likely that it happened before Anopheles immune related genes, studied so far, are also under purifying selection . Sin. SinAnopon  [Plasmodium by inducing mosquito basal immunity, which is essential to control the infection. Therefore pathogen specificity will indirectly determine the faith of mosquito malaria infection and exert different selection pressure on short PGRP coding genes.The different types of selection observed between PGRP-S1 and PGRP-S2 and S3 might be a consequence of their function as PGRP-S1 does not have predicted catalityc activity and is probably involved in recognition and subsequent activation of an effector pathway, as PGRP-SA and PGRP-SD in osophila . While Posophila  and/or mPGRP-SC2 . FurtherPGRP-SC2 . This dooglycan) . PGRP-S3oglycan) . and micAnopheles short PGRP genes studied are involved in the recognition of pathogens. However, they show different evolutionary pathways. PGRP-S1 gene is located in the chromosome X while PGRP-S2 and PGRP-S3 are located in chromosome 2L. This explains why PGRP-S1 is less genetically diverse and shows higher divergence between An. gambiae and An. arabiensis regardless of geographic location. On the contrary, PGRP-S2 and PGRP-S3 are more diverse and less divergent due to autosomal introgression between An. arabiensis and An. gambiae. Data indicated that PGRP-S2 and PGRP-S3 are likely subject to purifying selection consistent with their role in recognising conserved PAMPs. The 3D model of the proteins showed that no mutations were present at the cleft that forms the peptidoglycan binding groove, once again implying strong evolutionary constrains probably because these proteins need to maintain their PAMP recognition site unaltered, while the periphery that interact with other molecules is more prone to accumulate variation. The lower diversity and apparently higher divergence between species for the PGRP-S2 gene suggests that this is duplication from PGRP-S3. Different types of selection acting on PGRP-S1 and PGRP-S2 and S3 might be a consequence of their different function and catalytic activity.The three An. gambiae (82) and An. arabiensis (46) genomic DNA were analysed from two areas in East Africa: Mozambique and Tanzania. Mozambique samples were collected in Furvela (Inhambane province) in February/April of 2004 using light traps and samples from Tanzania were collected in Ifakara in 2000 using also light traps [Samples of ht traps . MosquitAn. gambiae molecular forms was carried out by PCR as described in Fanello et al. [Genomic DNA was extracted from individual specimens according to the protocol described by Ballinger-Crabtree et al. . Specieso et al. .An. gambiae s.s. genome at Ensembl . Name, sequence and product length of each pair of primers are represented in [see Additional file The primers used to amplify PGRP-S1, PGRP-S2 and PGRP-S3 genes were designed based on the sequences annotated in the complete 2, 200 μl dNTPs, 0.5 μl of each primer and 1.25 U/μl of Taq DNA Polymerase (Fermentas) for all reactions. PGRP-S1 1st nested PCR cycle conditions were: initial denaturation at 95°C for 2 minutes, followed by 35 cycles of 95°C for 45 seconds, 58°C for 60 seconds and 72°C for 2 minutes, with a final extension at 72°C for 5 minutes. PGRP-S1 2nd nested PCR cycle conditions were: initial denaturation at 95°C for 2 minutes, followed by 35 cycles of 95°C for 45 seconds, 58°C for 30 seconds and 72°C for 60 seconds and a final extension of 72°C for 5 minutes.Nested PCRs were performed in a MyCycler™ Thermal cycler (Biorad) with final reagent concentrations of 1× reaction buffer, 1.5 mM of MgClst nested PCR cycle conditions were similar to those used for PGRP-S1 amplification except for the annealing that was performed at 60°C. PGRP-S2 2nd nested PCR cycle conditions were: an initial denaturation step at 94°C for 2 minutes, followed by 35 cycles of 94°C for 45 seconds, 60°C for 30 seconds and 72°C for 60 seconds, with a final step of 72°C for 5 minutes. PGRP-S3 2nd nested PCR cycle conditions were similar to 1st nested but the annealing temperature was 55°C.PGRP-S2 and PGRP-S3 1PCR products were examined on a 2% agarose gel and products of the expected length were sequenced in both directions after purification with the SureClean Kit (Bioline) according to manufacturer's recommendations. Products were commercially sequenced by Macrogen, Korea.n (average number of nucleotide differences between two sequences) whereas in the Fu & Li's D and F tests the difference used is between the ηi  and ηe  or between ηe and πn.Sequence alignments were performed using the BioEdit Sequence Alignment Editor version 7.0.5.2 . Basic pThe tests referred above give little information in the case of young genes . Therefohttp://swissmodel.expasy.org/workspace/index.php. The best fitting 3D model for PGRP-S2 and PGRP-S3 was based on the crystal structure of the human PGRP-Iα (2aphB) and for PGRP-S1 was based on Drosophila PGRP-SA (1sxrB). Nonsynonymous mutations were visualized on the models using the Swiss-PdB viewr v. 4.0.1. [Amino acid sequences were obtained using the BioEdit Sequence Alignment Editor version 7.0.5.2  and alig. 4.0.1. ,13,12 in. 4.0.1. .An. gambiae and An. arabiensis populations was examined with analysis of molecular variance (AMOVA) [F statistics (Fst) according to Hudson et al. [The genetic structure within and among  (AMOVA) . The tesn et al. . Signifin et al. .To better understand phylogenetic relationships between intraspecific data, which normally consist in very similar sequences, we connected haplotypes on a median-joining network  using NECM, RF, MAS and JL performed the experiments; CM, RF, JP and HS analyzed and interpreted the data; JP, JDC and VER contributed with samples. CM, RF, JP and HS wrote the paper. HS conceived and designed the experiments. All authors read and approved the final manuscript.Figure S1. Multiple alignment of deduced amino acid sequences of An. gambiae and An. arabiensis PGRP-S2 and PGRP-S3.Click here for fileTable S1. Frequencies of PGRP-S2 and PGRP-S3 protein types of An. arabiensis and An. gambiae from Mozambique and Tanzania.Click here for fileTable S2. DNA sequences grouped by protein type.Click here for fileFigure S2. Multiple alignment of deduced amino acid sequence of An. gambiae and An. arabiensis PGRP-S1.Click here for fileTable S3. Sequences of primers used to amplify the three Anopheles short PGRP genes.Click here for file