Wnt signalling is a key regulatory factor in animal development and homeostasis and plays an important role in the establishment and progression of cancer. Wnt signals are predominantly transduced via the Frizzled family of serpentine receptors to two distinct pathways, the canonical ß-catenin pathway and a non-canonical pathway controlling planar cell polarity and convergent extension. Interference between these pathways is an important determinant of cellular and phenotypic responses, but is poorly understood. Here we show that TNIK (Traf2 and Nck-interacting kinase) and MINK (Misshapen/NIKs-related kinase) MAP4K signalling kinases are integral components of both canonical and non-canonical pathways in Xenopus. xTNIK and xMINK interact and are proteolytically cleaved in vivo to generate Kinase domain fragments that are active in signal transduction, and Citron-NIK-Homology (CNH) Domain fragments that are suppressive. The catalytic activity of the Kinase domain fragments of both xTNIK and xMINK mediate non-canonical signalling. However, while the Kinase domain fragments of xTNIK also mediate canonical signalling, the analogous fragments derived from xMINK strongly antagonize this signalling. Our data suggest that the proteolytic cleavage of xTNIK and xMINK determines their respective activities and is an important factor in controlling the balance between canonical and non-canonical Wnt signalling in vivo. The Wnt signalling pathway is a key player in embryonic development, in cancer and in the maintenance of stem cell lineages In Xenopus, the canonical Wnt pathway initially defines the dorsal-ventral axis of the embryo and subsequently directs differentiation along the anterior-posterior (A/P) axis The PCP pathway passes via the cell surface receptor Frizzled to JNK and to the cytoskeleton, and implicates a number of genes whose function in PCP is conserved from worm to man, the so-called “core” PCP factors Drosophila Msn C. elegans ortholog Mig-15 −/− mice fail to develop posterior mesodermal structures and die postgastrulation − and JNK2−- and probably also in JNK1,2,3− mice Xenopus, a signal from the Wnt receptor Frizzled7 (Fz7) through Dsh to JNK regulates CE during gastrulation and neurulation Msn belongs to the HPK/GCK family kinases, a family that encompasses eight subfamilies. The GCK-IV subfamily, or Msn subfamily, includes NIK/HGK (Nck-interacting kinase/HPK/GCK-like kinase) Vertebrates express a large number of Wnt ligands and Frizzled serpentine receptors, and these were initially classified as either canonical or non-canonical Xenopus xTNIK and xMINK cDNAs were found to encode proteins closely related to their human orthologs, and, contrary to recent claims Knockdown of either xTNIK or xMINK was achieved by injection of specific antisense Morpholino™ (Mo) into the two dorsal blastomeres of four cell embryos, see . AlternaThe knockdown data suggested that xTNIK and xMINK were both required for CE suggesting that they may function in the same pathway. In contrast, re-expression of either xTNIK or xMINK significantly rescued the phenotypes induced by knockdown of either kinase , suggestPCP signalling is first apparent in the extension of the primitive ectoderm and the involution of the primitive mesoderm during gastrula. As expected for effects on this pathway, knockdown of xMINK or xTNIK delayed the onset of gastrulation and the closure of the blastopore . Dorsal Dorsal knockdown of either kinase also inhibited formation of the neural folds and neural tube closure, while ventral injection had little effect, neural tube closure mid-trunk being only slightly delayed . At lateConsistent with an early requirement for xTNIK and xMINK in CE, the notochord was also severely foreshortened in knockdown embryos. Analysis of the early mesodermal marker Xbra in embryos depleted dorsally of either xMINK or xTNIK revealed that its induction in the notochord was delayed, but could be significantly rescued by the injection of xMINK mRNA . Again . xMINK oInitiation of Xbra expression in the equatorial zone e.g. . This efOur data suggested that xTNIK and xMINK functions were to some degree interdependent, knockdown of either giving similar effects  is an essential component of the PCP signalling pathway downstream of Wnt and the membrane receptor Frizzled. However, ectopic expression of both Xdsh and certain Xdsh mutants such as XdshD2, a PDZ domain deletion mutant, act as dominant negative mutations of PCP signalling, preventing CE and neural tube closure To better understand the mechanism of xMINK and xTNIK action downstream of the Xdsh adapter, we asked whether either kinase interacted directly and co-localized with Xdsh. Both xMINK and xTNIK were found to co-immunoprecipitated with Xdsh, strongly suggesting that they formed an integral part of the PCP pathway . In the Our data showed that xTNIK and xMINK were required for CE and hence functional PCP signalling, and that they interact genetically in this pathway as well as interacting physically. However, knockdown of either xTNIK or xMINK also suppressed the induction of Xbra , suggestCo-expression of either full-length xTNIK or full-length xMINK also very effectively suppressed secondary axis induction by ß-catenin. At first sight, this appeared somewhat to contradict the activities of the truncation mutants. However, as we have shown the full-length kinases are very probably auto-inhibited most probably by interaction between Kinase and CNH domains and/or dimerization. In this auto-inhibited state both kinases quite possibly compete for pathway components such as Xdsh, explaining their abilities to suppress both the PCP and canonical signals when expressed ectopically.Specific antibodies raised against the unique central domains of xTNIK or xMINK , induced axis truncation and an open back very much like the N-terminal deletion mutants ΔNT and ΔNM . This CEWe further investigated the effects of the isolated Kinase and CNH domains on the ß-catenin pathway using the secondary axis induction assay . The CNHAntagonism between the canonical and non-canonical Wnt pathways is an important factor in determining the phenotypic and physiological effects of signalling during development, differentiation, tissue regeneration and disease. Several possible explanations for this antagonism have been put forward including the integration of multiple Wnt and non-Wnt signalling inputs xTNIK and xMINK contain N-terminal Kinase and C-terminal CNH domains, separated by long and poorly conserved Central domains. We found that xTNIK and xMINK form both homologous and heterologous interactions and are essential components of the non-canonical PCP pathway in Xenopus. The full-length kinases appear to be autoinhibited by interactions between Kinase and CNH domains and this inhibition can occur via either homologous or heterologous interactions, suggesting that inhibition occurs via the formation of dimers. Consistent with this, deletion of the Kinase domain of either kinase acts as a dominant negative mutation to inhibit convergent extension. Kinase domain deletion mutants can be rescued by corresponding CNH domain deletion mutants, but rescue is dependent on an active Kinase domain. Several lines of evidence also suggest that both kinases function cooperatively downstream of Dishevelled (Xdsh). They co-localize with Xdsh within the cytoplasm, and along with Xdsh both can be recruited to the plasma membrane in the presence of Frizzled (Fz). Further, inhibition of PCP signalling by ectopic Xdsh, or the PCP specific XdshD2 mutant, is rescued by the catalytically active, CNH domain deleted xMINK, but not by the equivalent kinase-dead mutant. We could not test the CNH domain deleted xTNIK in this assay since it caused early loss of cell adhesion. However, since xMINK and xTNIK are in greater part functionally redundant  and deleUnexpectedly, both xTNIK and xMINK were also found to regulate canonical ßcatenin-dependent signalling. Consistent with recent data Unexpectedly, we further found that both xTNIK and xMINK are subject to extensive proteolytic cleavage in vivo. This cleavage predominantly releases the Kinase domains of each protein from the CNH domains and leads to their functional activation. CNH-domain polypeptides inhibit both the canonical and non-canonical Wnt pathways, causing CE defects and suppressing secondary axis formation via ß-catenin. The Kinase domain fragments of xMINK are able to rescue CE defects downstream of Xdsh and hence to activate the non-canonical pathway. The equivalent Kinase domain fragments of xTNIK appear to have a similar activity, however their tendency to cause loss of blastomere adhesion limited their study. Surprisingly, Kinase domain fragments of xMINK suppressed the canonical pathway downstream of ß-catenin while the corresponding catalytically inactive forms did not. In contrast, Kinase domain fragments of xTNIK suppressed the canonical pathway when catalytically inactive. The data therefore strongly suggest that xTNIK and xMINK are regulated by proteolytic cleavage and that their catalytic activities cooperate in non-canonical PCP signalling, but antagonize each other in canonical ß-catenin dependent signalling. Yet another level of differential regulation of xTNIK and xMINK activity is suggested by the subcellular localization of the cleavage products of either kinase. For example, the CNH domain proteolytic products of either kinase accumulate in the nucleus and suppress both canonical and non-canonical signalling, while N-terminal fragments can prevent this nuclear accumulation and rescue signalling. The subcellular distribution of xTNIK and xMINK cleavage products also appears to vary throughout the embryo, providing a possible explanation for the disparate responses of different embryonic zones to Wnt signalling .We were not able to determine the relative roles of the two other members of the Msn-related kinase family (NIK/HPK and NRK/NESK) that exist in mammals. TNIK, MINK and NIK share around 50% identity, but show only about 20% homology with NRK. The latest database data reveal that NIK and NRK genes exist in Xenopus. However, while xTNIK and xMINK are expressed relatively robustly and at similar levels throughout early development , microarray analysis indicates that xNRK expression is at least 10 times lower than xTNIK Our data identifies xTNIK and xMINK as key components of both the non-canonical and canonical Wnt pathways. As such, the data suggest a new paradigm for the interdependence of these pathways. Wnt ligands may signal simultaneously to both pathways, but the pleiotropic effects of this signalling may depend on the local cellular balance of xTNIK and xMINK and their cleavage products. This balance may differ throughout the embryo, leading to differential responses in different embryonic regions. This of course begs the question of what determines the status of xTNIK and xMINK and hence the outcome of any particular signalling event. The data do, however, suggest that the answer may come when we understand the regulation of xTNIK and xMINK proteolysis.The studies described in this manuscript do not involve human participants. Animal studies were performed exclusively using Xenopus leavis under protocol #2010-026 conforming to the standards approved by the Canadian Council for the Protection of Animals and reviewed by the Animal Protection Committee of the Centre Hospitalier Universitaire de Québec (CHUQ).xMINK cDNA was obtained from ATCC as an EST clone (GenBank ID: BU901472) and xTNIK cDNAs were cloned from stage 32 mRNA. N-terminal HA, Myc and Flag tags as well as GFP, YFP and RFP sequences were inserted and mutations generated by standard methods. Constructs for Xdsh, Xfz7 and ß-catenin were provided respectively by R. Harland, H. Steinbeisser and R. Moon. For mRNA generation constructs were linearized and mRNA synthesized using the appropriate mMessage mMachine Kit (Ambion) as recommended by the manufacturer.4, 2 mM CaCl2, 5 mM HEPES, 0.1 mM EDTA, pH 7.8), in vitro fertilized, injected in 0.3× MMR, 5% Ficoll 400 (Amersham Biosciences) and then cultured in 0.1× MMR at 14 to 18°C. Embryos were staged according to Eggs were recovered in 1× MMR ;MoT#1 (CATCTTTCGTTT-3′5′-GGCAGTGGCTCCC);cMoT#1 (5′-GTACACTGCTCCCCGTTCTTCCCAC -3′);MoT#2 (CAT-3′5′-TCCGAGCTGGTGGGTCTGATGC);MoM (GAT-3′5′-TCGGACCTGGTCGGTCTCATGC).cMoM  to a final concentration of 12.5 ng/cell.in situ hybridization was carried out on Xenopus laevis embryos as described Whole-mount 5′-CCTAAGAAAGCCTTGGACTA-3′, xMINK (reverse) 5′-AACGCAGTCTCCTTCACGCT-3′. Primers against TNIK: xTNIK1192rtpcr.FOR 5′-CAGAAGGAACAAAGGAGGAG-3′ and xTNIK1552rtpcr.REV 5′-TGATAGAGTGGCTTTTTCTC-3′.Primers against xMINK were designed as follows: xMINK (forward) HEK293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Wisent) and transfections performed by calcium phosphate precipitation as described Xenopus embryos were analyzed on a 6% or 10% SDS-PAGE using rabbit polyclonal antibodies raised against bacterially expressed proteins containing aa476-540 of xTNIK (αT2), aa981-1061 of xTNIK (αT3), aa641-803 of xMINK (αM5) and aa798-946 of xMINK (αM7).HEK293T cells were processed for coimmunoprecipitation and Western blotting essentially as 4, 0.7 mM CaCl2, 5 mM Hepes, 2.5 mM NaHCO3, pH 7.8) until stage 17.For DMZ explants the appropriate mRNAs were injected radially into the two dorsal blastomeres of four-cell embryos. Explants were isolated at stage 10+ and then cultured in 1× MBS solution . For indirect immunofluorescence, animal caps were fixed in MEMFA for 1 hr and permeabilized overnight at 4°C in Dent's fixative. After blocking, primary and secondary antibody incubations were performed overnight at 4°C. Caps were finally cleared and mounted in Murray's medium , and viewed on a Leica SP5 II scanning confocal microscope in standard scanning mode.Figure S1A) Sequence alignment of Xenopus TNIK (xTNIK-full) with its human orthologue (hTNIK) (Ac. No. AAF03782) and with the truncated sequence used by Satow et al. (Ac. No. BC108456) (PDF)Click here for additional data file.Figure S2A) posterior views of stage 13 knockdown embryos. B) Anterior and dorsal views of stage 19 dorsal and ventral knockdown embryos. C) Dorsal and ventral knockdown embryos were also allowed to develop to stage 32. The indicated Morpholinos were injected into the two dorsal or ventral blastomeres of 4 cell embryos together with fluorescein dextran as lineage marker. D) Stage 12.5 dorsal, ventral and lateral knockdown embryos were subjected to in situ hybridization for Xbra mRNA.(PDF)Click here for additional data file.Figure S3A) Interaction between epitope tagged xMINK and xTNIK and their respective deletion mutants was determine by co-immunoprecipitation from co-transfected HEK293T cells. In this case immunoprecipitation was performed via the HA epitope of the xMINK constructs. B) and C) Coimmunoprecipitation analyses of self interactions of xMINK or xTNIK and their respective deletion mutants. The indicated constructs were co-transfected into HEK293T cells. See (PDF)Click here for additional data file.Figure S4A) Xbra in situ hybridization of stage 10.5 embryos expressing dominant negative xTNIK and xMINK mutants in comparison with those expressing Xdsh or the Xdsh mutant D2. B) and C) Rescue of CE in embryos expressing ectopic Xdsh or the Xdsh mutant D2 by co-expression of the catalytically active C-terminally deleted xMINK mutant MΔC, but not the inactive M(K54R)ΔC or the N-terminally deleted mutant ΔNM. Embryos are shown at the equivalent of stage 39–40. The fractions of embryos displaying the indicated phenotype are given below the panels. In A, B and C the dorsal blastomeres of four cell embryos were injected with the indicated amounts of the RNAs. D) Coimmunoprecipitation analyses of interactions of xMINK or xTNIK with Xdsh. The indicated constructs were co-transfected into HEK293T cells. See (PDF)Click here for additional data file.Figure S5A) and B) show Western blots of the endogenous xTNIK and xMINK products throughout early development. Proteins were detected using the anti-xTNIK and anti-xMINK antibodies αT2 and αM7, see (PDF)Click here for additional data file.Figure S6A) Four cell embryos were injected in the two dorsal blastomeres with the indicated RNAs and allowed to develop to stage 39–40. The numbers of embryos displaying each phenotypes is indicated. B) Summary of embryo scoring for the constructs tested in the ß-catenin secondary axis assay shown in (PDF)Click here for additional data file.


Children with attention deficit hyperactivity disorder (ADHD) are hyperactive and impulsive, cannot maintain attention, and have difficulties with social interactions. Medical treatment may alleviate symptoms of ADHD, but seldom solves difficulties with social interactions. Social-skills training may benefit ADHD children in their social interactions. We want to examine the effects of social-skills training on difficulties related to the children's ADHD symptoms and social interactions.rd Edition subscale for 'hyperactivity-impulsivity' between the intervention group and the control group. The outcomes will be assessed 3 and 6 months after randomisation. The primary outcome measure is ADHD symptoms. The secondary outcome is social skills. Tertiary outcomes include the relationship between social skills and symptoms of ADHD, the ability to form attachment, and parents' ADHD symptoms.The design is randomised two-armed, parallel group, assessor-blinded trial. Children aged 8-12 years with a diagnosis of ADHD are randomised to social-skills training and parental training plus standard treatment versus standard treatment alone. A sample size calculation estimated that at least 52 children must be included to show a 4-point difference in the primary outcome on the Conners 3We hope that the results from this trial will show that the social-skills training together with medication may have a greater general effect on ADHD symptoms and social and emotional competencies than medication alone.NCT00937469ClinicalTrials (NCT):  Attention deficit hyperactivity disorder (ADHD) affects 3% to 5% of all children . The maiSocial-skills training aims to develop, improve, and maintain the individual's social skills. This is achieved by teaching how to regulate verbal and nonverbal behaviours involved in social interactions and in compliance with social norms ,28. The A child's ability to form attachments is developed in early childhood through interaction with primary caregivers. Different forms of attachment are secure, insecure dismissing, insecure preoccupied, and disorganised. It is assumed that these different forms of attachment will influence the outcome of social-skills training. A connection between early disorganised attachment and later ADHD has been demonstrated by Punto et al. , who folThe primary aim of the SOSTRA trial is to examine the effect of the combination of social-skills training and parental training plus standard treatment versus standard treatment alone in children with ADHD and their families on the outcome measures of ADHD core symptoms, social skills, and the attachment between the child and the parents.Children aged 8-12 years with a diagnosis of ADHD and their parents are randomised to the combination of social-skills training and parental training plus standard treatment versus standard treatment alone. The trial flowchart is shown in Figure Children and parents randomised to the experimental intervention are included in 1 of 4 identical 8-week social-skills treatment programs with 12 to 16 participants per program. Here, the children are offered weekly social-skills training sessions of 90 minutes in duration. During that time, the parents attend parental training. Each group has two therapists who have been trained in management of social-skills training at Langager School in Aarhus . The expSocial skills are based on the broad area of cognition and emotions. The iceberg model  emphasizes the importance of considering each child's problems and resources distinctly and incorporating these in the training programme. For instance, some children may need more sessions with visualization techniques to better learn the management of aggression. When they get better at managing their aggression, they will also be able to improve their social skills. The social-skills experimental intervention will focus on strengthening the ability of the children to control themselves and start a self-help process. For the parents, efforts will be directed at helping them develop strategies to assist their children in controlling their impulses. It is important that this training equips the children and parents with the skill to cope better and to reverse the bad circles. The efficacy of the experimental intervention will be assessed by improvements in ADHD symptoms and social skills per se or by assessing psychological functioning on a broader aspect, including the quality of peer relationships and emotional competencies.Efforts must be made to create a safe environment in the group. The children should feel safe enough to play and experiment with exploring their own and other people's understanding of them along with understanding the other participants and the different topics in focus. The assignments must be clear and simple. The educational style in the groups will take into account the children's special cognitive difficulties; accordingly, the structure in each session will be predictable. This is secured by regular items on the agenda, which are written on the blackboard every time: Opening round--what has happened since the last time? — revision of the previous session; homework from previous session; presentation/education; role play/creative activities; new homework; closing round.The therapists must be clear and direct but not confrontational or critical. Weight is attached to empathy, positive reinforcement, and a curious 'non-knowing' mentalizing attitude. A relaxed atmosphere with room for humour is the aim.The therapists who are responsible for the children's group in the experimental intervention arm are also responsible for the parents' group in the standard treatment arm. This risk of bias will be limited by writing the detailed manual that will describe the content in each group. Content forms are to be completed by the therapists after each group sessions and will function as a control of the content of the sessions given.In the parental groups, the themes from the children's groups will be presented and discussed. Likewise, the children's homework will be discussed and parents are encouraged to discuss their specific problems with their children and there will be an exchange of experiences among the participants.The standard treatment offered to both the experimental group and the control group encompasses the normal practice regarding ADHD patients at the Child Psychiatric Daytime Clinic in Holbaek. The overall objective is to secure compliance with the treatment, which means that the team attaches importance to building an alliance with the family, creating safety, and ensuring that the family receives sufficient counselling, psychological education, and support to enable them to be more confident and autonomous regarding the problems and challenges of having a child with an ADHD. After assessment and confirmation of the ADHD diagnosis, the family is offered medical treatment for the child. The medical protocol is shown in Figure The children are those referred to the Child Psychiatric Clinics in Holbaek and Roskilde with an ADHD diagnosis. They are screened according to the inclusion and exclusion criteria Table . The parWe estimate that 20 children will be assessed per treatment program . This is the number that we believe is needed to produce approximately 12 to 16 participants for randomisation.All outcomes will be assessed before the interventions starts and at 3 months and 6 months after randomisation. The primary outcome measure is an assessment of ADHD core symptoms. The secondary outcome measures are an assessment of the children's social skills. The tertiary outcomes are an assessment of the attachment between the child and the parents and an assessment of the parents own ADHD symptoms Table .CAI [secure attachment, insecure, disorganized/secure, or disorganized/insecure attachment. This test can be used for children aged 6 to 14 years. (Danish version).• CAI . The CAIK-SADS clinical diagnostic interview [• nterview . This teConners CBRS Teacher [•  Teacher . This quConners 3rd Edition Teacher [•  Teacher . This quASRS Symptom Checklist [• hecklist . This quSCQ [• SCQ . This isSDQ [• SDQ . This isWisc 3 [• Wisc 3 . This isCentral randomisation is performed by the Copenhagen Trial Unit (CTU) with computer generated, permuted randomisation sequence with unknown block size for the investigators. A research secretary will call the CTU and providing a personal pin code, patient number, and the stratification variables of sex  and comorbidity (yes/no). Then the randomisation will be announced.The interventions are not blinded to participants, parents, treating physicians, or personnel in the clinic. However, the outcome assessor of the primary and secondary outcomes is the teacher, who is kept blinded of the child's allocated intervention. The involved parties (the parents and children) are instructed not to inform the teacher of the allocation. To secure integrity of trial data, the principal investigator will collect the questionnaires blind to the intervention. Blinded data will be handed over to the CTU, which will be in charge of data entry and statistical analyses blinded to intervention. Standardised procedures will be assured.rd Edition Rating Scale 'hyperactivity-impulsivity' sub index (primary outcome) and an assumed standard deviation of 5 on the same scale [The sample size is calculated on the basis of a type I error (α) of 5% and a type II error (β) of 20%, thus a power of 80%, and an allocation ratio of 1:1. With a clinically relevant difference of a score of 4 between the experimental intervention group and the control group on the Conners 3me scale ,46, a saThe statistical analysis of the outcomes will be based on the 'intention-to-treat' principle, i.e. all randomised participants will be included in the analysis in the intervention group to which they were randomized, irrespective of how much of the intervention they have received. Per-protocol analyses will be conducted secondarily for the participants who have completed 50% or more of their randomised intervention.According to the ICH Harmonised Tripartite Guideline, Statistical Principles for Clinical Trials E9 Analysis of Drug Trials , the anapost hoc explorative subgroup analyses prompted by the result may be conducted.The analysis of each outcome measure investigates if the outcome measure changes (increases or decreases) significantly over time in one intervention group as compared with the other one. The group coding is concealed for the statistician. Provided the outcome measure is a primary or secondary one, it will be investigated to determine if the effect depends on each of the tertiary outcome measures as measured at entry. If this is the case, To deal with the multiplicity problem, the hypotheses will be ordered into families of hypotheses, and these families will in turn be ordered into a hierarchy of families. The general multistage gate keeping procedure of Dmitrienko et al. (2008) will then be applied . The two1. Hypotheses related to the effect on the primary outcome measure.2. Hypotheses related to the effect on the secondary outcome measures.3. Hypotheses related to the effect on the tertiary outcome measures.post hoc analyses for hypothesis generating purposes.Once none of the null hypotheses in a family are rejected, the procedure stops and the rest of the null hypotheses are accepted. However, the raw P-values of the remaining tests will be calculated and presented as the results of The complete analysis of each outcome measure is the same for the primary and secondary outcome measures. The outcome measures is set to be M (continuous variable), the indicator of intervention to be I , time to be t ), and the tertiary outcome at baseline to be O3-baseline . The possibility that the value of M increases between month 3 and 6 is a real one. Therefore, the full model may not necessarily be a linear model in t. Consequently, we will include the quadratic component t2 in the full model. However, the final choice of model may be tempered by the impression obtained from the inspection of the marginal mean values.The mixed model repeated measures method will be used. The model statement without the O3-baseline included will be specified as follows:where a thru e are coefficients in the model. This model will test if the mean level as well as the linear and the quadratic effect of time differ significantly between the two interventions. A sequential hypothesis testing is used, which is appropriate for polynomial models. Initially, four types of covariance matrices will be tested: compound symmetric, AR(1), AR(1) with heterogeneous variances, and unstructured. Using the Akaike and the Schwartz Bayesian criteria, the best covariance structure will be chosen. In the analysis of the continuous tertiary outcome measures (measurements at entry and after 6 months), compound symmetric and unstructured covariance structures will be compared. When the O3-baseline is included, the full model will be augmented by the main effect of O3-baseline and interactions between O3-baseline and previously included components containing I, the intervention indicator.Each analysis will be repeated twice for comparison . Missing observations imputed by the multiple imputations (MI) method will be included the first time, and only 'complete cases' will be included the second time. However, the main analysis will be a mixed model analysis including all original values (without any imputed ones included).The second tertiary outcome measure is an ordinal variable with four possible categories. The proportional odds model with the same type of model statement as explained above will be used. If the assumption of this model is not fulfilled, various types of ordinal regression (SPSS version 17) will be attempted and, if this fails, a multinomial model will be used. If more than 5% of the values are missing, two analyses will be made, one only including 'complete cases' and the other including MI values.The teachers will be personally informed of the questionnaires by the therapist, who will assess the behaviour of the children in school. This therapist will inform the teacher of the importance that every question is answered in the questionnaires. The teachers will receive the questionnaire, along with a letter, at entry and at 3 and 6 months after treatment. In this letter, the necessity to answer all of the questions will be emphasized, and the teachers will be encouraged to call the principal investigator in case of any queries. The principal investigator and research secretary will play active roles in contacting the teachers of the children with ADHD and ensuring data collection in case the teachers fail to return the questionnaires. After receiving the questionnaires, the principal investigator and research secretary will assess all of the responses. If they find any questions unanswered, they will contact the teachers within 1 week of receiving the questionnaires, ensure that the missing data is completed, and find out why those questions were left unanswered. A short 1-day course will be arranged after the final follow-up and after receiving the final questionnaires for all of the teachers whose students participated in the trial. The teachers will be informed about this course once they receive the questionnaires at the baseline. All children and their parents will be contacted by the ADHD team for a long period of time after the end of this treatment, often as long as several years; therefore, all children and their parents will be bound to ambulatory settings and this study.Table Outcome measure results may also be missing because the corresponding form was not completed due to drop out or other causes . In this case, it is not safe to presume that the pattern of missing values is not related to the unobserved data. The potential impact of type 2 missing values is explored using a worst-case analysis (see below).One approach for dealing with missing values is to use a mixed model for repeated measures (MMRM) in the statistical analysis . This model prevents bias only if the missing at random (MAR) assumption is fulfilled . The results of the study are those obtained by this method when it is applicable for the analysis of the data .For comparison, the method of MMRM is supplemented by that of MI using the model variables and additional variables significantly related to the variables with missing values and/or the absence of these variables. The method used is the fully conditional specification method of SPSS (version 17.0). This is an iterative Markov chain Monte Carlo method that can be used when the pattern of missing data is arbitrary (monotone or non-monotone). The default number of iterations is used initially and then increased if the Markov chain has not converged. Prior to the MI, the distributions of the continuous variables are inspected to see if serious deviations from the normal distribution that need transformations are present. Constraints are set to restrict the range of imputed values of continuous variables so that they are plausible. In total, 10 imputed data sets are produced.Two sensitivity analyses will be performed.1. Parameter estimates obtained by complete case analysis, MMRM, and MI followed by MMCM on the imputed data sets will be compared .2. A worst-case analysis of the effect of type 2 missing values will be conducted as follows. The group coded Effect group will be designated the group with a significant and beneficial effect on the outcome measure (say it decreases over time) as compared to the group coded No Effect group. Missing values will now be imputed as follows: A). Value(s) missing in the Effect Group: A single value missing is imputed with an average of the 2 observed values. Two values missing are both imputed by the third observed value. Three values missing are all imputed by the grand mean of the No Effect group. B). Value(s) missing in the No Effect-group: A missing 6-month value is imputed by the smallest observed 6-month value in the data. A missing baseline value is imputed by the largest observed baseline value in the data. A missing 3-month value is imputed by the average of 0 and 6-month values whether observed or imputed as explained above. This should minimize the response to intervention based on imputed values in patients from the Effect group and give a maximal negative linear response based on imputed values in patients from the No Effect group given the constraint that values more extreme than those observed must never be used.MI will only be used if the missing values exceed 5%.To establish if participant characteristics at trial entry are relatively similar in the 2 intervention groups (thereby a low risk of selection bias and confounding), demographic data (sex and age) and other factors that can be expected to influence the primary outcomes will be presented in a table of the entry characteristics.Participants will be informed of the trial in writing and orally; written informed consent will be obtained from the participant's principal caregivers. There are no apparent ethical problems since all participants are offered standard medical treatment, and standard control treatment in this population, further, there are no known disadvantages of social-skills training. Nevertheless, any adverse events of the intervention will be reported. The trial has obtained approval by the Regional Ethics Committee of Zealand (SJ-85) and is registered with the Danish Data Protection Agency (J. nr.2008-41-2613) and at ClinicalTrials.gov (NCT00937469).This trial compares the effects of social-skills training groups supported by parental training plus standard treatment versus standard treatment alone on the outcome of core ADHD symptoms of hyperactivity and impulsivity. A secondary objective is to examine differences in the effect of the treatment in relation to the children's different abilities in forming attachments: secure, insecure, disorganized/secure, or disorganized/insecure attachment. The last objective is to examine differences in the effect of the treatment in relation to the degree of parents' symptoms of ADHD.The results from this trial can greatly benefit children with ADHD because social-skills training may have a greater general effect on social and emotional competencies than medication alone. Additionally, the connection between the ability to form different attachments and the effect of the social-skills training can influence both the understanding and the treatment of the disease. One of the trial's strengths is that it is related to very important matters in the development of comprehensive treatment programs for ADHD children. Many children with ADHD have serious problems with peers because of their lack of emotional and social abilities. ADHD children are often lonely, and their social problems often lead to a vicious circle, which is difficult to break ,17.Other strengths of this trial are the measurement of attachment styles in children with ADHD and the comparison between the effect of the social-skills training and the attachment competencies. We do not know of other studies in which children with an ADHD diagnosis are tested with the CAI test. Punto et al. stated that there is a significant connection between disorganised attachment in early childhood and ADHD symptoms in the school-age period . This shThe experimental intervention in this trial is relatively short (8 weeks) and is therefore not very costly. This will allow other child psychiatric units to incorporate social-skills training for children with ADHD. A limitation of this trial is the relatively small sample size, which could make it difficult to draw firm conclusions about the research questions. However, if successful, it will be indicative of further directions for research on this topic.ADHD: Attention Deficit Hyperactivity Disorder; ASRS: Adult Self-Report Scale; CAI: Children Attachment Interview; DSM-IV: Diagnostic and Statistical Manual of Mental Disorders; ICD-10: International Classification of Diseases; IQ: Intelligence quotient; K-SADS-PL: Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime version; MTA: Multimodal Treatment Study of ADHD; SCQ: Social Communication Questionnaire; SDQ: Strength and Difficulties Questionnaire; SOSTRA: Social Skills Training and Attachment; WISC-III: Wechsler Intelligence Scale for Children; Conners CBRS: Conners Comprehensive Behaviour Rating Scales.Per Hove Thomsen has received a fee for lecturing on behalf of NOVARTIS and UCB as well as lecturing and consulting on behalf of Eli-Lilly. He also serves on the advisory board of Eli-Lilly.All the other authors declare that they have no competing interests.All authors contributed to the design of the trial. OJS drafted the manuscript. All authors contributed to the further review of the manuscript. All authors read and approved the final manuscript.


Brain dopamine dysfunction in attention deficit/hyperactivity disorder (ADHD) could explain why stimulant medications, which increase dopamine signaling, are therapeutically beneficial. However while the acute increases in dopamine induced by stimulant medications have been associated with symptom improvement in ADHD the chronic effects have not been investigated.11C]cocaine (dopamine transporter radioligand) to measure dopamine transporter availability in the brains of 18 never-medicated adult ADHD subjects prior to and after 12 months of treatment with methylphenidate and in 11 controls who were also scanned twice at 12 months interval but without stimulant medication. Dopamine transporter availability was quantified as non-displaceable binding potential using a kinetic model for reversible ligands.We used positron emission tomography and [Twelve months of methylphenidate treatment increased striatal dopamine transporter availability in ADHD ; whereas there were no changes in control subjects retested at 12-month interval. Comparisons between controls and ADHD participants revealed no significant difference in dopamine transporter availability prior to treatment but showed higher dopamine transporter availability in ADHD participants than control after long-term treatment .Upregulation of dopamine transporter availability during long-term treatment with methylphenidate may decrease treatment efficacy and exacerbate symptoms while not under the effects of the medication. Our findings also suggest that the discrepancies in the literature regarding dopamine transporter availability in ADHD participants  may reflect, in part, differences in treatment histories. Attention deficit/hyperactivity disorder (ADHD) is considered to be the most prevalent psychiatric disorder of childhood and its increasingly being recognized in adults too. The disorder is often chronic, with prominent symptoms  and impairment frequently continuing into adulthood. Deficits in dopamine (DA) neurotransmission have been associated with the disorder 11C]cocaine in 18 stimulant-naïve adult patients with ADHD whom we scanned before and after 1 year of clinical treatment with MPH. The retest PET scan was conducted 24 hours after the last clinical MPH dose to ensure that the acute occupancy of DAT would have dissipated. In parallel we also measured DAT availability in 11 healthy controls at baseline and 12 months later with no intervening medication treatment. We hypothesized that long-term MPH treatment would upregulate DAT availability in the brain in response to stimulant-induced elevations in synaptic DA whereas there would be no changes in DAT availability in controls tested at a 12-month interval.To test this hypothesis, we conducted a Positron Emission Tomography (PET) study using cocaine evaluation of DAT availability in these 18 patients as well as 11 of the controls (without medication). The mean total daily dose used of MPH was in the vicinity of 1 mg/kg or its equivalent in long acting dose form. Progress of treatment was followed sequentially using the CAARS and the Swanson Nolan and Pelham ADHD scale For the 18 ADHD patients, we provided clinical treatment with MPH for 1 year, and then conducted another PET study with [11C]cocaine (specific activity 0.5–1.5 Ci/µM at end of bombardment). Dynamic scans were obtained for a total of 60 minutes as previously described 11C]cocaine in plasma for quantification of DAT availability PET studies were conducted with a Siemens HR+ tomograph  in 3D mode. Dynamic scans were started immediately after injection of 4–10 mCi of [11C]cocaine) 1) in striatum and cerebellum using a graphical analyses technique for reversible systems (Logan Plots) ND), obtained as the ratio of the distribution volume in striatum to that in cerebellum minus 1, was used to quantify the DAT availability, that is the number of transporters that are free to bind to the radiotracer. These measurements are insensitive to changes in body weight.Regions of interest analysis in striatum  and in cerebellum were drawn directly on an averaged emission image (summation of images obtained between 10 to 60 minutes for [ND) for each striatal regions  were evaluated using a repeated (baseline vs. follow-up scan) ANOVA for the comparison between repeated scans within the control or the ADHD group. One between subject factor (group) and one between subject factor (baseline vs. follow-up scan) ANOVA was used for the group comparison (ADHD vs. controls) on changes in DAT (baseline vs. follow-up scan). Differences between the baseline and the follow-up scan are expressed as percent change in BPND from baseline. The significance level was set at p<0.017 after Bonferroni correction (3 regions).Differences in DAT availability (BPThe ADHD subjects and control subjects are similar in age and socioeconomic background . Prior tp<0.01) but the difference was not significant in controls (controls .p<0.007) and the putamen (p<0.005) of the ADHD participants than in the controls. There were no differences at follow up on DAT availability between the groups in the ventral striatum.The comparison of DAT availability between groups (ADHD vs. Controls) for the baseline scan did not differ . It was This study shows that long-term treatment with MPH up-regulated DAT availability in the ventral striatum, providing the first evidence of DAT neuroplasticity after long-term treatment with a clinically relevant dose of MPH in the human brain. DAT is responsible for recycling DA from the extracellular space into the pre-synaptic terminal Similarly, subchronic MPH treatment results in an attenuation of DA release in rodents, which was ascribed to either an upregulation of DAT or enhanced autoreceptor sensitivity Few studies have investigated the behavioral consequences of long-term exposure to MPH and the extent to which chronic exposure results in tolerance is still a matter of debate. Indeed, studies on the chronic effects of MPH have reported conflicting results with some documenting sensitization to the locomotor effects of MPH We hypothesize that the increased DAT availability is a compensation for the pharmacologic occupancy of DAT (estimated to be greater than 50%) 1. This study was designed as an open MPH treatment protocol. Ideally it would have been desirable to have a have a “double-blind, drug-placebo randomized” design, which would have allowed us to assess the changes in DAT measures in ADHD subjects who are not receiving medication. 2. It would have also been ideal to have a control group that also received 12 months of medication to assess if adaptation changes differ between the controls and the ADHD subjects. However, these other designs raise ethical issues, which make them inappropriate. The current design was constructed to address if there are changes in DAT with long-term treatment but did not evaluate the duration of the DAT increases once medications was discontinued. 3. Several participants were female and in them the DAT measures may be confounded by the time of the menstrual cycle at which the studies were performed, which we did not controls nor did we measure gonadal hormones. 4. Since we did not obtain clinical measures for the follow up scans while subjects were not under the acute effects of MPH it is not possible for us to test whether the increases in DAT observed with chronic treatment were associated with worsening of inattention.Here we report an upregulation of DAT secondary to long-term treatment with stimulant medication, which could result in further decreases in dopaminergic signaling when the individual with ADHD is not medicated (i.e. over weekend holidays). To the extent that reduced DA release in ADHD is associated with inattention


In response to Brondum’s comments we would like to reiterate that the main purpose of our review  was to eIn our review, we did not seek to identify individual behavioral tests or functional domains that could serve as surrogates for ADHD diagnosis. Nor did we make the case that developmental lead and PCB exposure are responsible for the rise in ADHD diagnoses in recent years. To the contrary, in our section on other environmental contaminants we specifically highlighted the fact that PCB and lead exposures are declining, whereas exposures to other chemicals—including brominated flame retardants, bisphenol A, phthalates, polyfluoroalkylated compounds and certain pesticides—are increasing. We note that studies of the potential role of these emerging contaminants in the etiology of ADHD are equally, if not more, important than further studies of lead and PCBs. In addition, there is a clear difference between exploring contaminants as potential contributors to ADHD risk as opposed to causing ADHD. Examining our Table 6 , which sNo one is debating whether parental psychopathology should be considered as a possible confounding factor in studies that examine the association of contaminant exposure with specific neurobehavioral diagnoses, including ADHD. We hope that this reply clarifies the goals of our review for those faced with the challenge of assessing the neurobehavioral effects of emerging contaminants and their possible contribution to the phenotypic expression of ADHD or other neurodevelopmental disorders.


Bartonella spp. in bats of 13 insectivorous and frugivorous species collected from various locations across Kenya. Bartonella isolates were obtained from 23 Eidolon helvum, 22 Rousettus aegyptiacus, 4 Coleura afra, 7 Triaenops persicus, 1 Hipposideros commersoni, and 49 Miniopterus spp. bats. Sequence analysis of the citrate synthase gene from the obtained isolates showed a wide assortment of Bartonella strains. Phylogenetically, isolates clustered in specific host bat species. All isolates from R. aegyptiacus, C. afra, and T. persicus bats clustered in separate monophyletic groups. In contrast, E. helvum and Miniopterus spp. bats harbored strains that clustered in several groups. Further investigation is needed to determine whether these agents are responsible for human illnesses in the region.We report the presence and diversity of  DNA in the blood of 5 of 60 tested bats , a mammalian reservoir has not been determined despite a wide range of tested animals collected in these countries  to isolate and identify detected Bartonella spp. and create a reference collection of Bartonella isolates from East Africa with further characterization and diagnostic investigation; 3) to evaluate genetic heterogeneity of circulating Bartonella strains by using the partial sequence variability of the citrate synthase gene (gltA), proven to be an excellent genetic marker for analysis of Bartonella strains  potentially to identify new species of Bartonella.This study was conducted within the framework of the Centers for Disease Control and Prevention (CDC) Global Disease Detection program, which is designed to estimate the health and financial effects and transmission patterns of emerging infectious pathogens associated with humans and animals  in Kenya and other locations. This study had 5 objectives: 1) to estimate prevalence of More than 500 bats were collected from 25 locations across Kenya . Samplinwww.barcodinglife.org). Because the taxonomy of African bats is under development and reference sequences for several species were unavailable in the BOLD database, the examined sequences were identified to the genus level only.Species were identified phenotypically by using available field keys. Additionally, representative tissues of each collected species were submitted for confirmation to Guelph University , where partial sequences of the cytochrome oxidase gene were generated and compared with those available from the database of the Barcode of Life Data Systems  or in an Eppendorf Mastercycler Gradient . PCR products were separated by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining.A heavy suspension of the microorganisms was heated for 10 min at 95°C followed by 1-min centrifugation at 8,000 rpm to precipitate the lysed cells. The supernant containing the genomic DNA was then moved to a clean centrifuge tube to be examined. PCR amplifications were performed in a 25-μL reaction mixture containing 5 μL 5× Green GoTaq PCR buffer , 0.4 μmol of each primer, 200 μM each dNTP, 1 U Taq DNA polymerase (Promega), and ≈20 ng of template DNA. Two oligonucleotides, BhCS781.p (5′-GGGGACCAGCTCATGGTGG-3′) and BhCS1137.n (5′-AATGCAAAAAGAACAGTAAACA-3′) were used as PCR primers to generate a 379-bp amplicon of the gltA gene. The Clustal V program within MegAlign (DNASTAR) was used to align and compare homologous Bartonella spp. gltA sequences obtained from bat samples and from the GenBank database.PCR products of correct size were purified with the QIAquick PCR Purification Kit  according to manufacturer’s instructions and sequenced in both directions by using an Applied Biosystems Model 3130 Genetic Analyzer . Sequencing reactions were performed in a PTC 200 Peltier Thermal cycler by using the same primers as the initial PCR with a concentration of 1.6 µM. Sequences were analyzed by using Lasergene  sequence analysis software to determine consensus of sequences for the amplified region of the Bartonella spp. cultures have been conclusively evaluated in samples from 331 bats of 13 species from 9 genera. Further estimation of the infection rate was determined exclusively on the basis of the specimens from these 331 animals. The time required for growth of Bartonella spp. colonies on agar greatly varied from 3 days, observed for several samples from Triaenops persicus bats to 28 days in one of the samples from Rousettus aegyptiacus bats.Culturing from bat blood pellets, especially from small-sized bats, presented a challenge because of the limited sample volume and the potential for contamination with other bacteria and fungi, problems hard to avoid during field sampling. Consequently, of >500 processed blood samples, the presence or absence of Bartonella isolates were cultured from the blood of 30.2% (106/331) of the bats tested. All isolates were confirmed genetically by amplification and sequence of the gltA. Prevalence of bats positive for Bartonella spp. by culture was as follows: Eidolon helvum (straw-colored fruit bat), 23/88 (26.1%); R. aegyptiacus (Egyptian fruit bat), 22/105 (21.0%); Coleura afra (African sheath-tailed bat), 4/9 (44.4%); T. persicus (Persian trident bat), 7/8 (87.5%); Hipposideros commersoni (giant leaf-nosed bat), 1/4 (25.0%); and Miniopterus spp. (long-fingered bats), 49/87 (56.3%)  for being infected with Bartonella spp. when each was compared with all other bats, and R. aegyptiacus and Epomophorus spp. bats were at significantly lower risk for infection (p<0.01 for both) ( (56.3%) . Minioptor both) .Bartonella isolates obtained from bats revealed 58 gltA genotypes (unique sequence variants with ≥1 nucleotide difference) that represented 11 genogroups with a sequence identity value of >96% between genotypes within each group, as proposed by La Scola et al.  and distant from all sequences found in other bat species (<91% identity) and previously described Bartonella species (<84% identity). Six unique genetic variants identified in R. aegyptiacus bats were clustered in a monophyletic genogroup, marked as R in All 15 gltA sequence identity between 16 Bartonella genotypes identified in E. helvum bats varied greatly, ranging from 78.6%–100%. Eight genotypes (sequence variants) were clustered around 4 clades (genogroups), which are marked as E1, E2, E3, and EW in Bartonella genus .All 4 Miniopterus were compared by using a similarity between the gltA sequences. In total, 51 Bartonella spp. gltA sequences, obtained from 3 identified Miniopterus spp.  and from Miniopterus spp. bats that have not been identified at the species level, were analyzed. Among the 51 bats, 24 unique gltA genotypes represented at least 3 genogroups with a level of divergence ranging from 6.6%–18.5%, higher than has been recommended previously for differentiation of Bartonella spp , but was relatively close (identity 94.2%) to the genotype identified in a whiskered bat (Myotis mystacinus) from the United Kingdom  and long-fingered bats (Miniopterus spp.) harbored strains clustered around 3 and 4 different groups of Bartonella spp., respectively, based on their gltA identity. Nevertheless, all strains of Bartonella spp. recovered from E. helvum bats were typical for this species of bats only. This pattern of cospeciation resembles the Bartonella spp.–host relations observed in cotton rats , and Duvenhage virus, which circulates in insectivorous bats . In addition, Shimoni bat virus was identified recently in an insectivorous bat Hipposideros commersoni  can be helpful in the search for potential reservoirs. The reagents prepared from bat isolates of Bartonella spp. can be used for serologic surveys conducted in high-risk areas of the world. Application of antigens produced from bat-derived strains is especially relevant to serologic investigations of cases of infectious disease of unknown origin among persons occupationally exposed to bats. In addition, cocirculation of Bartonella spp. with other pathogens in a bat population can affect the bat’s ecology and pathobiology. These aspects need further investigation.Characterization of the isolates obtained from bats and comparison with those obtained from human cases associated with


Objective: The purpose of this study was to examine whether persisting hyperactivity into adulthood was associated with impaired family, friendship, and partner relationships or poor coping skills in everyday life. Method: A 20-year community-based follow-up of 6- to 7-year-old boys showing pervasive hyperactivity (n = 40) and unaffected controls (n = 25) was conducted. At age 27 years, participants were assessed with detailed interview techniques as well as self-report ratings. Results: ADHD in adulthood was associated with problems in intimate relationships and negotiation skills. Antisocial behavior did not influence the association, but remitting childhood hyperactivity was not associated with social relationship difficulties in adulthood. Conclusion: In an untreated, community-based sample of hyperactive children, the risk for unsatisfactory social relationships is largely confined to those patients who still show ADHD in adulthood. The majority of patients who experience childhood hyperactivity have positive social relationships in adulthood. The persistence of ADHD into adulthood has become the focus of widespread research attention, and ADHD is currently considered a life span condition . It has DSM criteria; retain at least 50% or more of their original samples into adulthood (above age 18 years); give details on participants lost to follow–up; and have a low attrition rate.There are several long-term prospective longitudinal studies on the persistence of ADHD into adulthood in the literature. As apparent from One purpose of epidemiology is the completion of the clinical picture. Hyperactive behavior is more common in childhood than the diagnosis of ADHD . To deveThe nature of any deficits in adult life also needs fuller understanding. Distinguished research on the adaptive functioning of adults with ADHD has stressed that their problems extend to poor academic grades, low engagement with further education, involvement with the juvenile justice system, and risky behavior, especially in driving . Some deSocial relationships are an important determinant of the quality of life, and need to be understood as part of the natural history of disorder. It has been shown that clinic-referred adults with ADHD have poorer marital adjustment and family functioning , as wellIn a previous article from this prospective epidemiological study , it was Taking the previously mentioned findings into account, we decided to examine social relationships in a never-medicated sample of adults, 20 years after they were ascertained as showing high levels of hyperactive behavior, and hypothesized that childhood hyperactivity would lead to disturbed social relationships in adulthood.The following were the specific aims of the study:To document the extent to which adults who had been hyperactive in middle childhood had significantly different levels of satisfaction in family, friendship, and intimate relationships, as well as impaired ability to negotiate, by comparison with those not hyperactive in their childhoodTo examine whether continuing presence of hyperactivity in adulthood influences the level of satisfaction in family, friendship, and partner relationships, and the ability to negotiateTo examine whether antisocial behavior influences the possible effect of hyperactivity on relationships in adulthoodThe original survey, from which all the participants were taken, has been previously described . As a brThe Rutter B(2) questionnaire was completed for 99% of the children by their class teachers, and the A(2) questionnaire for 80% by the parents. In total, 2,462 had both screening questionnaires completed. Hyperactivity was defined as present if the teacher and parent questionnaire gave a score of 3 or more on the Hyperactivity subscale. Conduct problems were defined as present if the score on the teacher scale was 9 or greater, or that on the parent scale was 13 or greater, and the score on the Conduct Disorder subscale was greater than that for “emotional disorder.” These cutoffs had been validated in previous surveys and on the Isle of Wight studies see .mixed; constituting 5.3% of the study population), those who met criteria for pervasive hyperactivity but not for conduct problems , and those not meeting criteria for either condition (control). The cases were stratified by behavioral group and then randomly sampled from the resulting groups, to give approximately equal numbers in each group for detailed study. Participants were excluded if they had scores of 5 or greater on the Emotional Disorder subscale of either the parent or the teacher scale. With respect to their hyperactive symptoms, the children with mixed hyperactivity and emotional symptoms were similar to those with hyperactivity only , and 33 control children with no detected behavioral problems. Those who only met criteria for conduct problems were not included in the present analyses.For the present study, 121 participants  were initially selected because on the basis of their childhood behavior, they fell into either the hyperactive or control groups, and had been included in the detailed second wave of study. Of these, the follow-up of the children of first-generation immigrant families  will be reported separately. The marked differences in the way they were identified by parents and teachers by comparison with children of native British families, made it inapproprite to use the same criteria of identification . The samOf the 96 adults, 10 could not be traced, and 12 had refused permission for future contact when approached in a previous 10-year follow-up . Of the A first follow-up was carried out 9 years after the second stage of the original survey, when the participants were aged 16 to 18 years . The secTracing the participants was undertaken by a variety of means, including previously recorded addresses, electoral records, and personal contacts. The reliance on multiple methods was intended to reduce the bias that might be introduced by those who fail to be contacted by any one technique. When the young men and their families were contacted, permission was sought for interviews and a test session. A small sum of money was paid to them in recognition of the expenses involved. Informed consent was obtained before interviewing the participants.Adult Functioning Interview is an investigator-based standardized interview schedule, administered to the participant by a trained interviewer. It is a modification of the Adult Personality Functioning Assessment APFA; . The APFReliability checks were carried out throughout the investigation by another researcher listening to and rating randomly selected tapes. The interrater agreements in terms of kappas were .74, .81, .79, and .67 for overall satisfaction with friendships, negotiation skills, partner relationships, and family relationships, respectively.2. Psychiatric outcome was measured by the Schedule for Affective Disorders and Schizophrenia (SADS) interview, and the cognitive function assessed by Cambridge Neuropsychological Test Automated Battery . Algorithmic diagnoses were generated from the SADS. Telephone or postal enquiry was made where possible from the men’s parents.Diagnostic and Statistical Manual of Mental Disorders , which was blind to the person’s childhood hyperactivity levels. All information available from the participants and informants concerning their interviewer and self-rated adjustment over the year prior to the assessment was systematically presented for Mean differences between the hyperactive and nonhyperactive groups as defined in childhood—and, within the previously hyperactive, between those who shared ADHD at outcome and those who did not—were compared by analysis of variance and by analysis of covariance with antisocial behavior as a covariate. Where a scale was dichotomized at the level of those showing “good” function, chi-square comparisons were made.SD = 1.2) and had 0.5 children (SD = 0.8); 83.4% were in paid employment, whereas 13.3% were either unable to work, in full-time education/training, or caring for others (3.3% missing data). The average age of leaving school was 16 years (SD = 1.1), leaving home 20.5 years (SD = 3.6), and starting employment 16.9 years (SD = 2.4).On average, the participants were 27.6 years old  were only slightly older than the controls, M = 27.4, SD = 1.5; t(63) = 3.76, p > .5, and had left home at a younger age  than did the controls, M = 21.3, SD = 3.6; t(45) = −1.21, p > .5. There was no significant difference regarding the employment status, χ2(3) = 1.5, p > .5. The key measure for social relationships is the overall level of satisfaction with friendships. To arrive at this rating, several specific aspects were inquired about: Several aspects of social relationships were measured, such as joint activities and levels of discord with friends. In the sample as a whole, 55% reported having four or more friends with whom they engaged in joint activities and 52% had two or three friends they could confide in. The overall perceived level of satisfaction with friendships was significantly correlated with the number of friends with whom the participant engaged in joint activities , had a confiding relationship , and had received practical help from , as well as with the participant’s own level of sociability . For each friendship, the interviewer inquires further about the quality of relation to arrive at the judgment of overall satisfaction. There was no significant effect of persisting hyperactivity with regard to the overall level of satisfaction with friendships  = 1.7, p = .19.When the participants identified as hyperactive in childhood and the controls were compared for age, employment status, number of children, age leaving school, and starting employment, no statistically significant differences in any of these aspects were found. By age, those designated hyperactive  and those with a “good” level of support were as common in the previously hyperactive as in the controls, χ2(1) = 5.2, p = .27.When the degree of support the participants received from and gave to other members of their family was examined, it was found that a majority (81.2%) reported their families giving them practical and emotional support. A third stated that they were supported by both of their parents, as well as siblings; 28.1% had received support only from their mothers and siblings, whereas the rest either received support only from by their mothers or did not receive support from anyone in the family. As shown in F = 2.44; df = 1, 63; p = .12; 2(1) = 0.25, p = .62.Another aspect examined was the participants’ ability to handle negotiations, defined as the ability to secure rights and obtain their goals using discussion, assertiveness, or even humor, instead of aggression and discord. Again, those identified as hyperactive in childhood did not differ significantly from controls in their level of ability to negotiate (SD = 2.4), and the first steady relationship was at 17.8 years (SD = 2.8), which on average lasted for 27.8 months (SD = 31.2). At the time of the assessment, 41% had no current relationship, 21.7% were in a steady relationship, 35% had been in a cohabiting relationship/marriage for more than 6 months and the rest were in shorter lasting cohabitations. Among those 56.7% with cohabitations lasting more than 6 months, 20% had cohabited with more than one partner, 37% with one partner only, and 10% had never had a steady relationship. Concerning the level of satisfaction in partner and intimate relationships, as shown in F = 0.63; df = 1, 62; p = .43), and those identified as hyperactive in childhood were no less likely to show a “good” level of satisfaction, χ2(1) = 0.41, p = .84.Regarding partner and other intimate relationships in the sample as a whole, the average age the participants began dating was 15.2 years  and less satisfaction in partner and other intimate relationships , compared with the participants without hyperactivity. In contrast, both groups did not differ with regard to the level of satisfaction with friendships or support received from the family, and both groups were similar to the controls.According to the F = 3.50; df = 1, 38; p = .07), satisfaction with relationships  or family support . These results might suggest that persistence of ADHD could be linked to adverse social outcomes, but the number of participants is too small to base conclusions.Comparisons between those hyperactive only in childhood and those with hyperactivity in childhood and ADHD in adulthood were also carried out. Those identified as hyperactive in childhood with ADHD in adulthood did not differ significantly from those hyperactive in childhood only and from controls in their level of ability to negotiate  = 0.43; negotiation skills, F = 0.60; family support, F = 1.4; or friendships, F = 0.72, and the predictiveness of hyperactivity was unaltered.Finally, to examine whether antisocial behavior had an influence on the associations, a global measure of antisocial behavior was created. The number of offenses found in criminal records, self-reported aggressions, and fights and use of weapons were added to create this measure. Using an ANCOVA, with hyperactivity levels in adulthood as predictor, antisocial behavior as covariate, and relationship satisfaction levels as dependent variables, antisocial behavior was not found to be significantly related to intimate partner relationships, This study shows that adult ADHD is associated with difficulties in social relationships, especially intimate partner relationships, and with poor negotiation skills. These findings generally corroborate and extend those of previous studies suggesting that children with ADHD are at an increased risk for social dysfunction in later life . Adults Another interesting finding from this study was that the participants who had been hyperactive in childhood, but did not have ADHD in adulthood, were not obviously less satisfied with their social relationships than those who had never been hyperactive. The rather negative picture of the social outcome of children referred to specialist clinics, and diagnosed with ADHD, should not necessarily be generalized to the broader range of hyperactive children in the community. Most hyperactive children may have a different adult outcome from that reported in clinic-referred ADHD children .It is possible that the method of self-report may have underestimated the degree of impairment in relationships if people with hyperactivity lack insight into any problems that may be present. This explanation would not, of course, diminish the significance of the differences we found in adults who showed diagnosable ADHD.The question arises why milder degrees of childhood hyperactivity are not associated with relationship dissatisfaction in adulthood in most cases. This may be due to the overall decrease of hyperactivity from childhood to adulthood. In a previous follow-up of this sample, hyperactivity was found to diminish between the ages of 7 and 17 years . ConsistThe association of hyperactivity and antisocial behavior has been documented in clinic-referred samples . HoweverTherefore, it may only be possible to examine the impact of hyperactivity accurately in epidemiological studies. To our knowledge, there exist only two larger epidemiological studies. The Christchurch Health and Development Study  examinedIt has to be noted that none of the children in our sample had received medication for symptoms of ADHD. At that time, and in this part of London, the diagnosis was not used and medication was not prescribed to any of the children. If a referral to a mental health service was made, then the offer was of family therapy, which was usually unacceptable to the parents. Hence, the course of hyperactivity was not confounded by the effects of specific therapeutic intervention. It is uncertain whether medication is a protective factor in the longer term, and more research is needed to clarify its effect on the long-term course of ADHD symptoms. Our results do not support the treatment of young people with subdiagnostic levels of hyperactivity. Neither do they provide evidence for screening for hyperactivity in the general population. The majority of untreated children in our sample appeared to have a positive outcome regarding social relationships. However, reverse causality cannot be ruled out. Relationship problems may influence emotional well-being and increase the likelihood of affected individuals continuing to display high levels of hyperactivity. Whether this can happen in adulthood is not known. To date, little is known about the long-term developmental course of hyperactivity and accurate knowledge about factors involved in negative and positive outcome is needed. Future well-designed, long-term studies should contribute to gaining a better understanding of these factors, which in turn would help to define high-risk groups and eventually lead the way to new targets for intervention.This study has controlled for antisocial and emotional problems, but as in other long-term epidemiological studies there remains the possibility for other unmeasured, uncontrolled confounding factors. The rather small sample size entails limited power, and the attrition rate of around 30% might have had an effect on the validity of the results. Social relationships were documented through self-reports, which could have exaggerated, or understated, the associations with hyperactivity.


Weight at birth is a good indicator of the newborn's chances for survival, growth, long-term health and psychosocial development. Low birth weight (LBW) babies are significantly at risk of death, contributing to the high perinatal morbidity and mortality in developing countries. Hence, this study aims to assess the incidence and associated factors of low birth weight (LBW) in Gondar University Hospital deliveries.A cross-sectional study, conducted on 305 live births from May 1- July 30, 2010. Information on independent variables was collected from the mothers just before discharge using a structured interview questionnaire. Neonatal weight was measured using standard beam balance. Both interviews and weight measurements were done by two trained midwives. Gestational age was determined by last normal menstrual period and/or ultrasound examinations.The mean and standard deviations of the birth weights were 2976 ±476 grams. Incidence of LBW (birth weight <2500 grams) was 17.1% . LBW was associated with first delivery (AOR=2.85), lack of antenatal care follow up (AOR= 5.68) or infrequent visits and being HIV positive (AOR=3.22). More female newborns were with low birth weight than males though the difference was not significant after controlling for potential confounders in the multivariate analysis.There is a high incidence of LBW. Efforts should to enhance national antenatal care utilization in general, and particularly in Gondar, should be encouraged as its absence is closely associated with LBW. The World Health Organization (WHO) defines Low Birth Weight (LBW) as having a weight of less than 2500 grams at birth . LBW andBabies born with a LBW are more likely to have health problems and slower development from immediately after birth to later in life , 6. LifeAn estimated 18 million babies worldwide are born each year with low birth weight of which about 3.1 million are in sub-Saharan Africa , 9. AbouEthiopia, having an infant mortality rate of 59/1000 live births has limiThe institution based cross-sectional study was conducted at the University of Gondar Referral Hospital from May 1, 2010 through July 30, 2010. The study was conducted at the Maternity ward, Department of Gynecology and Obstetrics, University of Gondar Referral Hospital in Gondar town, Northwest Ethiopia.The hospital is located in North Gondar Zone at about 727 kilometers Northwest of Addis Ababa . The hospital provides delivery services 24 hours a day, 7 days a week with its staff including midwives, intern doctors, eight general practitioners and three obstetricians.A sample size of 309 was calculated using single proportion formula assuming a 22.5% proportion of LBW . Maternal information was collected just before discharge. Birth weights and their codes were recorded together so as to provide a link to the maternal interview results.The interview questionnaire consisted of three parts . Mothers were asked about their obstetric and gynecologic histories, morbidity experiences and other relevant information using a structured interview questionnaire.Relevant data such as history of medical or obstetric illnesses and laboratory profiles were retrieved from the antenatal (ANC) follow up charts when available, otherwise self reports by the mother were taken. Each mother was asked about her last normal menstrual period (LNMP) to calculate gestational age. Efforts were made to help mothers remember their LNMP dates by associating them with traditional and local events. For mothers who failed to remember or having unknown LNMP, gestational age estimated by ultrasound during pregnancy was taken.Previous history of delivery of LBW babies was only subjectively assessed from the mothers speaking of “small or very small baby”. Dietary history was grossly assessed if the woman said she had additional dietary intake during the pregnancy and whether she was given counseling about the importance and types of additional food intake during pregnancy. Pretest was done on 15 deliveries just a day before the actual data collection. Weighing scales were checked and adjusted at zero level between each measurement.Data were entered and analyzed by SPSS version 16.0 statistical package for windows . Logistic regression analyses were conducted to determine the effect of factor(s) on the outcome variable and to control possible confounders. Factors with a p-value < 0.05 were taken as statistically significant.Ethical approval was obtained from the Institutional Review Board of the University of Gondar. Permission to conduct this study was also obtained from the medical director's office of the hospital. The purpose of the study was explained to the mothers and consent was obtained from them regarding their agreement to participate in the study. Names were not recorded in order to keep the identity of respondents anonymous.A total of 305 neonate/mother pairs were involved in this study with a response rate of 98.7%. About 93% of the mothers were aged 18-35 years, the range between 15-41 years with a mean and SD of 25.37±5.29 years. More than three quarters were urban dwellers. Majorities were Amhara (97.0%) ethnics, married (93.8%) and Orthodox Christians (85.2%) .Almost half of the mothers (53.4%) were primiparous. Of the multiparous (n=142) women, 107 (81.7%) delivered more than three years ago. A satisfactory number of participants (86.2%) had history of ANC follow up, of which 181 (59.1%) had at least four visits during the current pregnancy. Nearly half (46.2%) of women said they were given dietary counseling during the current pregnancy and 57.7% were taking additional nutrients during pregnancy .Of the 305 deliveries , about 80% (n=244) were spontaneous vaginal deliveries and 16.4% (n= 50) by caesarian section and the rest 3.6% (n= 11) were assisted deliveries for example by vacuum and forceps. Two hundred fifty one (82.3%) of the mothers knew their LNMP or had it recorded on their follow up chart. For the rest 54(17.7%) the ultrasound estimate was taken from their follow up charts.2=1.78 & p=0.09).However, a total of 23 women failed to remember their LNMP and had no ultrasound gestational age estimates documented, hence they were excluded from further analysis to maintain the unbiased effect of gestational age. Babies of the 23 excluded women had a mean birth weight of 2931 grams and it was not significantly different from the 305 included for further analysis (XMajority (80.1% or n=260) of newborns from those mothers who remembered their LNMP were delivered at term, 31(12.4%) post term (after 42 completed weeks of gestation) while the rest 19(7.6%) were preterm ones (delivered before 37 completed weeks of gestation). Maternal obstetric and medical illnesses during pregnancy were also assessed for association with LBW. A total of 64 women had either medical or obstetric problems during pregnancy. Of the 44 (14.4%) women with medical problems 13 had HIV/AIDS and 11 had malaria. Similarly out of the 19 (6.2%) women with obstetric related problems 6 women had pre-eclampsia and 2 had eclampsia.The mean and standard deviation of the birth weights was 2972±476 grams (range 1500-4800 and median 3000 grams). Overall, 52 newborns  had LBW. The incidence of LBW was 61.9%, 14.3% and 9.4% among the preterm, term and post-term babies respectively.Female newborns were having smaller birth weight than males (21.5% vs 12.6%) though the difference was statistically significant in the bivariate analysis only . The results of multiple logistic regression analysis  indicateThere was a statistically significant difference in the birth weights between primiparous  and multiparous mothers. Five of the eight (62.5%) mothers with pre-eclampsia/eclampsia delivered LBW babies. However, owing to the small number of the outcome of interest in each disease category, associations were not tested. More than half (53.8%) of the HIV positive mothers delivered LBW babies. HIV positive mothers had a fivefold increased likelihood of delivering LBW babies  when compared to HIV negative mothers. In the multivariate analysis factors such as maternal age, residence and sex of the newborn were not significantly associated with LBW .The incidence of LBW in this study 17.1%) is within the range of the country's 2005 DHS values of LBW by measurement (14.6%) and by maternal reports of “very small or small” babies (21.5%) . It is c.1% is wiHowever, this study found out a higher prevalence of LBW compared to other hospital based studies in other countries which reported a LBW incidence ranging between 9.0% and 11.8% like Iran, Turkey, Pakistan, Nigeria, Gambia and Ethiopia , 19, 20.In the analysis of associated factors absence or infrequent ANC follow up, first delivery, premature delivery and being HIV positive were independently and significantly associated with LBW. The odds of no ANC follow up among mothers who delivered LBW babies is about 3 times higher than those who had normal weight babies. This may be ascribed to the routine provisions of nutritional and medical advice or care and supplementations offered during ANC visits.Factors that are associated with LBW such as first time delivery, lack of ANC follow up and being HIV positive were similar to other studies so far reported in Ethiopia , 16, 18 In the bivariate analysis, maternal age less than 20 years and being female neonate were associated with LBW. However, unlike other studies , 19, theThe fact that other potential factors considered in this study such as maternal education, marital status, ethnicity, occupation and mode of delivery failed to be associated with LBW can be due to the similar nature of mothers included in the study with majority being illiterate, belonging to one religious or ethnic group.This study clearly shares the limitations of cross-sectional studies and hence does not show seasonal variations of LBW. Additionally, being a referral hospital the study area can hardly be restricted to Gondar town. This study did not consider some potential risk factors for LBW such as placental factors, congenital syndromes and intra uterine infections.In this study, there is a high incidence of LBW, compared to similar hospital based studies in sub-Saharan African. LBW was associated with lack of or infrequent ANC follow up, first delivery and maternal HIV/AIDS. In general, LBW is still a public health problem, so it is essential to provide the necessary facilities for prenatal health care. At the same time, it is necessary to screen pregnant mothers for the important risk factors of LBW such maternal HIV/AIDS and other medical/obstetric problems to provide them with essential prenatal health care services. Efforts to enhance national ANC utilization should be encouraged.For further research on LBW in this country, it is recommended that other factors be considered, such as home delivery, maternal anthropometry and seasonal variations.


Childhood Attention Deficit Hyperactivity Disorder (ADHD) is a growing mental health concern worldwide. Effective, accessible and low-cost therapeutics for the disorder are urgently needed. Here we introduce a novel internet-based cognitive training intervention: Online Neuroplasticity-based Training for the Remediation of ADHD in Children (ONTRAC). The intervention is deployed in the home setting; it is customized to the cognitive capacities of each child and progressively improves performance in the specific neuro-cognitive domains deficient in ADHD. A feasibility trial of ONTRAC is being conducted in a resource limited clinical setting in New Delhi, India and is an exemplar of hi-tech global psychiatry. Nearly 10% of children worldwide are being diagnosed with ADHD . With thStimulant medications remain the conventional first line of treatment in many countries, partly due to unavailability of any other reasonable treatment option -7. HowevThe advantage of modern CT is that it is internet-based and hence accessible from any remote setting. Scientifically, we can now develop CT that is specifically targeted to the core neuropsychological and cognitive impairments observed in a neuropsychiatric disorder or clinical population -17. UsinScientifically, ONTRAC features cognitive personalization as the training challenges are adaptively modified in real-time based on the individual’s current performance capabilities. Most importantly, ONTRAC is custom-designed to selectively target ADHD-specific deficits in five critical neuro-cognitive domains of alertness, sustained and selective attention, working memory, impulsive response control and suppression of distracting interference. No prior ADHD CT program has comprehensively targeted all of these cognitive domains; especially, the key deficits observed in suppression of distracting information have not been targeted.ONTRAC is being currently evaluated in a recently initiated randomized controlled feasibility-efficacy trial in New Delhi, India . All stuResults pending, the ONTRAC trial illustrates in principle how modern technology and clinical neuroscience can be integrated for the development and evaluation of a 21st century personalized neurotherapeutic that may benefit many individuals with neuropsychiatric impairments in remote and resource-limited settings. We are aware that we may not achieve positive long-term real-life impacts in a first trial. Yet, the technology base of ONTRAC allows rapid iterative software development cycles. When rapid technology development is coupled with systematic and well-controlled clinical trial evaluations, it is envisaged that research efforts would ultimately converge onto an efficacious cognitive training program in the near future that has global positive and sustaining impact on ADHD in children.ADHD: Attention Deficit Hyperactivity Disorder; CT: Cognitive training; ONTRAC: Online Neuroplasticity-based Training for the Remediation of ADHD in Children.JM is a part-time scientist at the Brain Plasticity Institute, PositScience and MM is President and Founder of Brain Plasticity Institute, PositScience, a company that develops cognitive training software.All authors contributed to the writing and approval of this letter.