This study aimed to assess:  tested the best factor structure. Complex logistic regressions assessed differential contributions of each symptom to clinical impairment. Receiver-operating characteristic (ROC) analyses tested which would be the best symptomatic cut-off in the number of symptoms for predicting impairment.The prevalence of DSM-5 ADHD was 3.55% [95% confidence interval (CI) 2.98–4.12]. The estimated prevalence of DSM-IV ADHD was 2.8%. CFA revealed that a bifactor model with a single general factor and two specific factors provided the best fit for DSM-5 symptoms. Inattentive symptoms continued to be the most important predictors of impairment in adults. The best cut-offs were five symptoms of inattention and four symptoms of hyperactivity/impulsivity.Our results, combined with previous findings, suggest a 27% increase in the expected prevalence of ADHD among young adults, comparing DSM-IV to DSM-5 criteria. The DSM-5 symptomatic organization derived a similar factor structure for adults as DSM-IV symptoms. Data using DSM-5 criteria support lowering the symptomatic threshold for diagnosing ADHD in adults. DSM-5 also proposed modifications that are relevant for all age ranges, such as defining pervasiveness as the presence of several ADHD symptoms (instead of impairment) in two or more settings, and the removal of autism/pervasive developmental disorder (PDD) as a specific exclusion  the factorial solution that provides the best fit for the DSM-5 ADHD symptoms, (b) the association of DSM-5 ADHD symptoms with clinical impairment, and (c) the best number (cut-off point) of DSM-5 ADHD symptoms to identify impaired adults in both ADHD dimensions.Thus, our main objective was to assess the prevalence of ADHD according to DSM-5 criteria in a large, non-referred, representative population of young adults. We also addressed the individual impact of new criteria (such as the extension of the age of onset to 12 years and lower symptom cut-offs) on ADHD prevalence rates. In addition, because DSM-5 criteria displayed some rewording and new clinical examples for ADHD symptoms in adults, we also tested: , a short semi-structured diagnostic interview for DSM-IV and ICD-10 psychiatric disorders that provided prevalence estimates of common mental disorders. Because of logistic issues (i.e. the psychiatric interview was part of a larger follow-up assessment), only some MINI sections were performed. The most prevalent mood (major depression and bipolar) and anxiety  disorders were assessed. The MINI has a previously validated Portuguese version Amorim, . In primwww.dsm5.org. Thus, we present here the performance of the current DSM-5 diagnostic criteria for ADHD . These questions were formulated exactly as proposed by the DSM-5 ADHD working group and were freely available during 2011 and 2012 at DHD APA, . DSM-5 det al.The DSM-5 ADHD symptoms were rated as present or absent. Considering that this was a large population study, we initially applied a screening questionnaire using the same structure as the World Health Organization Adult ADHD Self-Report Scale (ASRS) screener for all subjects. The ASRS includes six questions about ADHD symptoms . In a previous population study, the ASRS had 68.7% sensitivity, 99.5% specificity and 97.9% total classification accuracy, considering blind clinical assessment as the gold standard and using a cut-off of 4/6 screening symptoms  were assumed to be negative for ADHD. Moreover, we estimated a comparison between DSM-IV and DSM-5 ADHD prevalence in our sample. This was an indirect comparison because we could not assess either ADHD symptoms with DSM-IV wording or the presence of impairment of symptoms before 7 years (as DSM-IV required). To derive an ADHD diagnosis according to DSM-IV criteria, we considered data from this sample using the 6/9 cut-point proposed by DSM-IV. To deal with age-of-onset criterion modification while avoiding the effect of recall bias in a cross-sectional study asking for the exact date of beginning ADHD symptoms, we relied on a previous study documenting an increase of 0.1% in ADHD prevalence rates with this DSM criterion B modification. It is important to note that this rate was obtained in a longitudinal assessment (Polanczyk a) a one-factor model; (b) a correlated two-factor model; (c) a correlated three-factor model; (d) a second-order two-factor model; (e) a second-order three-factor model; (f) a bifactor model with one general and two specific factors; (g) a bifactor model with one general and three specific factors; and (h) an incomplete bifactor model with one general and two specific factors . This strategy allowed us to determine which factor solution provided the best fit for DSM-5 ADHD symptoms. CFA models were fitted to polychoric correlations among the symptoms using the weighted least squares means and variance adjusted (WLSMV) estimator implemented in Mplus 7.0 , the Comparative Fit Index (CFI), the Tucker–Lewis Index (TLI) and the root mean square error of approximation (RMSEA). To demonstrate good fit to the data, an estimated model should have a WRMR ⩽0.9  in the subsample that screened positive for ADHD (at least 2/6 positive screening questions) and completed the entire ADHD assessment. Several CFA models were fitted to the data: (χ2 test and unadjusted odds ratio (OR) estimates, which allowed ranking the DSM-5 symptoms according to their OR related to clinical impairment. Second, a binary stepwise logistic regression model was performed, considering clinical impairment as the dependent variable and all DSM-5 ADHD symptoms as independent variables. This analysis depicted how many, and which, DSM-5 symptoms were independently associated with clinical impairment after controlling for the other DSM-5 symptoms. Third, all-possible-subsets (APS) logistic regression analysis was used to confirm the set of DSM-5 symptoms that best predicted clinical impairment. The APS analysis helped to select the best subset from a larger set of predictors. In such situations, different subsets might have almost equivalent associations with the outcome, and conventional stepwise regression analysis might select a suboptimal subset because of minor differences in bivariate associations. The APS analysis avoids this problem as it generates results for a large number of different models with a fixed number of predictors, which were determined by the earlier stepwise logistic regression analysis. The APS analysis also ranks the best subsets according to their association with the outcome . Once the ranking of subsets is known, the researcher can select the predictors that are more consistent across the top-ranked subsets. In our APS analyses, we followed the procedures of Kessler et al.  . For children with no positive symptoms in the questions that were part of the screening instrument, the vast majority (86.9%) had no additional symptoms. Thus, we included subjects with no positive symptoms in the screening instrument in our first set of ROC analyses. However, we did not include subjects with one positive symptom among the six screening items in this first set of analyses because predictions of the number of other positive symptoms and impairment from the symptoms would be impossible. Next, we performed the same analyses only in the ADHD screening positive subsample.Receiver-operating characteristic (ROC) analyses tested which would be the best cut-off  for the number of DSM-5 ADHD symptoms to predict clinical impairment, for both inattention and hyperactivity/impulsivity dimensions. For these analyses, we initially assumed that those with no positive symptom among the six screening symptoms would be negative for the remaining 12 symptoms. To test this assumption, we simulated the performance of the screening questions in a real dataset that had all the 18 symptoms without any skipping rule for a sample of children from the same Brazilian region , Signal Detection Software ROC4 , Mplus version 7.0 (CFA) and IBM SPSS version 20 .All subjects were in the age range between 18 and 19 years at the time of the interview. A total of 4106 cohort members were located , and 4000 subjects provided information on ADHD, co-morbidities and sociodemographics. Of these, 1329 subjects (33.2% of this study sample) screened positive for ADHD and provided information about all ADHD criteria. Sociodemographic and co-morbidity profiles for ADHD cases and subjects without ADHD are shown in p = 0.73) and both groups differed from the subjects without ADHD (p < 0.001). Both groups of cases also presented similar male/female ratios (39.3% versus 40%) and rates of co-morbidity with anxiety disorders (37.1% and 40%). However, new cases identified only by DSM-5 had a higher prevalence of co-morbid mood disorders (16% and 7.7%).According to full DSM-5 criteria, the prevalence of ADHD was 3.55% [95% confidence interval (CI) 2.98–4.12]. The prevalence of inattentive, hyperactive/impulsive and combined presentations was 1.65, 0.45 and 1.45% respectively. The effects on ADHD prevalence rates of using different symptom cut-off points for assigning a diagnosis are displayed in The data fit indexes for several factorial models tested in CFA are presented in R2 = 0.123). In the APS regression, five inattention  and only two hyperactivity symptoms  were present in more than half of the top-10 subsets of seven symptoms. In all the analyses, inattention items performed better than hyperactivity/impulsivity items in predicting clinical impairment.The results for the performance of the 18 DSM-5 symptoms in predicting moderate to severe clinical impairment are presented in ROC analyses were performed with both inattention and hyperactivity/impulsivity symptoms, considering the presence of moderate to severe clinical impairment as the gold standard. In the model including subjects with no positive symptoms in the screening instrument, the best inattention cut-off for predicting clinical impairment was four symptoms . For hyperactivity/impulsivity, the best cut-off was one symptom . When only the ADHD screening positive subjects were included, the best inattention cut-off for predicting clinical impairment was five symptoms . For hyperactivity/impulsivity, the best cut-off was four symptoms . The sensitivity and specificity for other cut-off points are available upon request.v. 2.8%). However, the new criteria did not impact substantially on the factor structure of the disorder. In addition, our data support previous DSM-IV findings suggesting that inattentive symptoms are core in the ADHD diagnostic definition in adults, and that there are potential benefits in lowering the symptomatic threshold for diagnosing ADHD in adults.As far as we are aware, this is the first comprehensive evaluation of DSM-5 ADHD criteria in a large, representative, population-based sample of adult subjects. In general, our results suggest that the new DSM-5 ADHD criteria would determine a 27% increase in the prevalence rate of ADHD, compared with DSM-IV criteria  besides symptom threshold were used, the prevalence of ADHD ranged from 2.9% (with a cut-off of six symptoms in any dimension) to 3.8% (with a four-symptom cut-off). These prevalence rates are not very different from the rates found in previous DSM-IV-based population studies in the USA. A telephone survey of 966 adults . Adding the age-at-onset criterion alone  would decrease this rate to 5.4% (95% CI 4.7–6.1). As expected, a similar result emerged for the impairment criterion: adding the moderate impairment criterion alone would increase the endorsement rate to 15.1% (95% CI 13.99–16.21). These additional criteria were more relevant for making the prevalence closer to the one expected than any potential variations in symptom cut-off criterion. Taken together, these results might serve as clinical rationale for emphasizing the importance of carefully addressing ADHD additional criteria  during the diagnostic evaluation.et al.et al.et al.et al.et al.et al.et al.CFAs with the DSM-5 symptoms found a bifactor model with one general and two specific factors as the best fit for the data. A bifactor model was also the best fit in many DSM-IV-based previous studies (Dumenci p > 0.10). These results stress the relevance of inattention as a predictor of clinical impairment in adults. Although the specific list varies across studies, inattentive symptoms as a group are usually reported as the best predictors of ADHD-related impairment in previous investigations. Barkley et al. . For our prevalence assessment, we assumed that those who screened negative for ADHD did not have the disorder. This assumption allowed us to assess the prevalence of ADHD for the entire population sample, but it is theoretically possible  that our approach missed some ADHD cases. To deal with this possibility, we performed a mathematical modeling for imputing data for the 1833 subjects who had only one screening positive symptom based on the patterns of response of those that had two screening positive symptoms and therefore provided information on all 18 symptoms (data available upon request). The prevalence rate found was slightly higher, as expected . Finally, the AUC for our ROC analyses when only the ADHD screening positive subsample was included showed a diagnostic performance substantially lower than that detected when the entire sample was considered . In other words, these analyses are more likely to reflect the clinical reality of differentiating clinical cases from those with some level of symptoms including subthreshold cases.Our study has some strengths and also methodological limitations related to logistic issues. To the best of our knowledge, this is the first population study assessing DSM-5 ADHD criteria. We assessed young adults, and all previous DSM-IV and DSM-5 field trials in adults have not addressed ADHD. Our data were derived from a large, representative, population-based sample. Finally, we implemented assessment methods that could mimic clinical assessment in the real world, as far as possible in an epidemiological investigation. Regarding limitations, our subjects were all 18–19 years old and our data might not be fully generalized to older adults. Our diagnostic process was performed only with the subjects and did not aggregate information from significant others, a strategy that could have minimized the risk of under-reporting ADHD symptoms, and might have improved the reliability of our retrospective assessment of ADHD symptoms before age 12. Our impairment measure was based only on the subject's perspective; a rater-derived score based on functional correlates was not used. It is important to bear in mind that previous investigations have found high agreement between self and parent information for ADHD diagnosis in adults  are included in the evaluation process. The results reinforce the notion that using new wording and clinical examples would not change either the expected factorial structure of ADHD symptoms or the stronger association between inattention and clinical impairment found in adults. Moreover, our study reinforces the view that a lower cut-off point for the number of inattentive and/or hyperactive/impulsive symptoms is more suitable for adult patients.


Secale cereale L., has historically been a crop of major importance and is still a key cereal in many parts of Europe. Single populations of cultivated rye have been shown to capture a large proportion of the genetic diversity present in the species, but the distribution of genetic diversity in subspecies and across geographical areas is largely unknown. Here we explore the structure of genetic diversity in landrace rye and relate it to that of wild and feral relatives.Rye, S. strictum Presl. and S. africanum Stapf. Evaluation of effects from ascertainment bias suggests underestimation of diversity primarily in S. strictum and S. africanum. Levels of ascertainment bias, STRUCTURE and principal component analyses all supported the proposed classification of S. africanum and S. strictum as a separate species from S. cereale. S. afghanicum (Vav.) Roshev, S. ancestrale Zhuk., S. dighoricum (Vav.) Roshev, S. segetale (Zhuk.) Roshev and S. vavilovii Grossh. seemed, in contrast, to share the same gene pool as S. cereale and their genetic clustering was more dependent on geographical origin than taxonomic classification. S. vavilovii was found to be the most likely wild ancestor of cultivated rye. Among cultivated rye landraces from Europe, Asia and North Africa five geographically discrete genetic clusters were identified. These had only limited overlap with major agro-climatic zones. Slash-and-burn rye from the Finnmark area in Scandinavia formed a distinct cluster with little similarity to other landrace ryes. Regional studies of Northern and South-West Europe demonstrate different genetic distribution patterns as a result of varying cultivation intensity.A total of 567 SNPs were analysed in 434 individuals from 76 accessions of wild, feral and cultivated rye. Genetic diversity was highest in cultivated rye, slightly lower in feral rye taxa and significantly lower in the wild S. strictum and S. africanum different rye taxa share the majority of the genetic variation. Due to the vast sharing of genetic diversity within the S. cereale clade, ascertainment bias seems to be a lesser problem in rye than in predominantly selfing species. By exploiting within accession diversity geographic structure can be shown on a much finer scale than previously reported.With the exception of The online version of this article (doi:10.1186/s12870-016-0710-y) contains supplementary material, which is available to authorized users. Secale cereale L.) has the ability to thrive and to produce high yields also under adverse environmental conditions .Rye . These weedy forms, here called feral, occur as weeds in cereal fields, mostly in the Near East and Central Asia and are fully inter-fertile with cultivated rye  or from S. vavilovii in Eastern Turkey and Armenia [S. vavilovii is the most likely wild ancestor of cultivated rye.ral type . NonethePCA Fig.  and muston Table . Cultiva Armenia . Our res Armenia , 15 suggS. secale subspecies has been more inconclusive, particularly the relationship of feral and cultivated rye forms [S. afghanicum accession R 777 has a very low FST (0.096) from the Afghan landrace PI 220119; the Spanish S. segetale R 779 has one of its lowest pairwise FST (0.087) with the Spanish landrace R 785  mentioned above is described in the passport data as S. strictum subsp. anatolicum), from Turkey and clusters together with feral and cultivated rye from Turkey, Afghanistan and Pakistan and one cultivated rye from the same region  rather than with accessions of the same taxon.The classification of ye forms –9. Our aye forms , we concS. vavilovii and S. cereale landrace accessions. These results suggest a complex scenario of high introgression between the ryes in the S. cereale – vavilovii group, but with no detectable contribution from the S. strictum – S. africanum group.Feral rye phenotypes have been described to morphologically diverge quickly from cultivated rye progenitors, especially if cessation of rye cultivation in one region reproductively isolates feral types from and cultivated ones . In thisin silico pooled data. This supports the results of computer simulations [Previous studies of the distribution of genetic diversity in cultivated rye have mainly failed to find any clear geographic patterns , 23, 63.ulations .The five major groups of landrace rye detected, partly reflects the agro-climatic zones of Europe (as e.g. proposed by Bouma ). Howeveth century. It is well known that the Finnish immigrants practiced slash-and-burn cultivation of rye in this area and also brought seed with them from their native area [The smallest cluster, consisting of a single Norwegian and a single Swedish landrace, both from the Finnmarken area stood out as the geographically most local group. Most of the individuals in these accessions also shared the same, and amongst cultivated rye unique, chloroplast genotype. The distinctiveness of landrace rye from this area from the majority of Scandinavian rye was previously noted by Persson et al. . The ryeive area . The accive area . We wereive area . These aK = 9 STUCTURE model  types and northern European ryes being winter types. Ma et al.  found thThe STRUCTURE assignment of the three modern varieties reflected their breeding history. NGB 6431 ‘Kungs II’ clustered among landraces from Northern Europe. This variety was breed in Sweden from landraces from Northern Germany, possibly pollinated with Swedish landraces. PI428373 ‘Petkus’ spring rye, clustered among other spring rye landraces from Western Europe. PI 323382 ‘Imperial’ clustered among landraces from the Central Europe. ‘Imperial’ is an American variety selected from the landrace ‘Schlanstedt’, originating in central Germany.Carex [Arabidopsis [S. strictum and africanum, we observed no taxonomic clustering, again supporting the inclusion of all feral taxa, S. vavilovii and cultivated rye in the same species. Among the cultivated landraces, accessions from Western Europe, Northern Europe and South Eastern Europe formed three groups, suggesting seed exchange within these areas and supporting nuclear SNP geographic patterns. Among the accessions from Finnmarken all individuals of NGB13868 and half of the individuals in NGB14283, had a distinct cp-genotype not found elsewhere among cultivated rye. This suggests a foreign, but unknown, origin for these landraces. Screening additional landraces, especially from remote areas in Finland, with the cpSSR markers, or the full set of SNP markers, could shed light on the origin of these ryes and the possible connection to Finnish migration in the 16th century.Mitochondrial and chloroplast markers have been used to contrast maternal spread (in plants through seeds) with biparental spread (through both seed and pollen). In rye, exclusively maternal transmission of chloroplasts has been detected in some studies , 67; whiCarex , almond Carex  and Arabbidopsis . We careST and PC dispersion analysis that accessions in the Southern group were much more genetically distinct from each other than the majority of accessions in the Northern group .


Relative to controls, children with ADHD had a significant reduction in thalamic volume and dorsolateral prefrontal cortical area in both hemispheres. Furthermore, children with ADHD with smaller thalamic volumes were found to have greater reductions in surface area, a pattern not evident in the control children. Our results are further evidence of reduced lateral prefrontal cortical area in ADHD. Moreover, for the first time, we have also shown a direct association between thalamic anatomy and frontal anatomy in ADHD, suggesting the pathophysiological process that alters surface area maturation is likely to be linked to the development of the thalamus.Although previous morphological studies have demonstrated abnormalities in prefrontal cortical thickness in children with attention deficit/hyperactivity disorder (ADHD), studies investigating cortical surface area are lacking. As the development of cortical surface is closely linked to the establishment of thalam-ocortical connections, any abnormalities in the structure of the thalamus are likely to relate to altered cortical surface area. Using a clinically well-defined sample of children with ADHD ( •Few studies have investigated cortical surface area in ADHD.•Thalamic volume and cortical surface area is reduced in children with ADHD.•Morphological thalamic abnormalities are associated with reduced cortical surface area in ADHD.•There is an association between thalamic and frontal anatomy in ADHD. It is cADHD is associated with subtle abnormalities in brain structure and function. One of the earliest brain areas considered in the pathophysiology of the disorder was the thalamus  due to iMorphological studies that use surface based morphometric (SBM) approaches suggest that cortical surface area and thickness have developmentally distinct trajectories, with divergent genetic influences . SBM appEarly development of cortical surface is tightly linked to the establishment of thalamocortical connections . Animal Using a clinically well-defined sample of children with ADHD and typically developing controls in whom we have previously shown reduced cortical thickness , we stud22.12.1.1Participants were recruited as part of the MIDAS (Motivation Inhibition and Development in ADHD) multi-modal neuroimaging study of ADHD, reported elsewhere  and usin2.1.2n=6) were excluded.Letters detailing the study were sent to approximately 600 families of children and adolescents attending primary and secondary schools in Nottinghamshire. From the sample who volunteered to take part, a group of right-handed controls was selected, pair-wise matched for age (± 6 months), sex and socio-economic status (SES) to a member of the ADHD group. Parents completed a shortened version of the DAWBA and the same battery of questionnaires as the ADHD group. Exclusion criteria were as detailed for the ADHD group. In addition, any participants with attention scores>4 on the SDQ   and verbal assent (children/adolescents) was obtained. Neuroimaging data were available from 54 children. A quality control inspection assessed images for gross structural abnormalities, motion and other artefacts. Data from five subjects  were not included because of motion artefacts or poor registration of their scans. In the final sample, we report data from 49 participants; 25 ADHD  and 24 typically developing controls (12.93±1.62 years).2.2T1-weighted (T1W) brain images in the sagittal plane were obtained with a Philips Achieva 1.5-T MRI scanner with an eight-channel SENSE head coil using a 3D Turbo Field Echo (TFE) sequence with the following parameters: 160 contiguous slices; repetition time/echo time (TR/TE)=9.9/3.7 ms; matrix size=256×256; voxel size, 1×1×1 mm. Head movement was minimised by the use of foam pads placed within the head coil.2.3http://surfer.nmr.mgh.harvard.edu/). After completion of skull-stripping and intensity correction, the grey–white matter boundary for each cortical hemisphere was determined using tissue intensity and neighbourhood constraints. With the use of a deformable surface algorithm guided by the grey-cerebrospinal fluid (CSF) intensity gradient, the resulting grey-white interface was expanded to create the pial surface, followed by a spherical morphing procedure and registration based on sulcogyral alignment. The surface boundary was tessellated to generate multiple vertices (and triangles) across the whole brain. All surfaces were visually inspected following an automated topology fixation procedure, and remaining minor defects were manually corrected as recommended by the software guidelines. Automated subcortical segmentation using probabilistic information regarding the location of subcortical structures was carried out using Freesurfer v5.0, with the investigator being blind to the diagnostic group  tests for categorical variables between patients and controls (CTRL). Thalamic volume was examined using a General Linear Model (GLM), with hemisphere as a within-subjects factor, diagnosis as a between-subjects factor and age, and full-scale IQ as covariates. To ensure that the group differences in thalamic volume were not confounded by individual variations in total brain size, total brain volume  was also used as a covariate in the GLM. A significance level of p<0.05 was used for all tests. To compare the surface area between the two groups in a vertexwise fashion, we used the Query-Design-Estimate-Contrast (QDEC) statistical interface of Freesurfer v5.0. With the use of a GLM with diagnosis as a between-subjects factor, the relative surface area change was assessed separately for the right and the left hemispheric surfaces, with age and total brain volume as covariates. To study the effect of diagnosis on the relationship between thalamic volume and the cortical surface area, total thalamic volume  was added as a predictor variable and the effect of the interaction between diagnosis and thalamic volume was sought while adjusting for the effects of total brain volume and age. To correct for multiple vertexwise comparisons, we undertook a Monte Carlo permutation analysis  and thalamic volume and the surface area of frontal clusters. We also related the duration of stimulants use with thalamic volume and the surface area of frontal clusters. A statistical threshold of 33.1t (47)=3.66, p=0.001) and FSIQ was used as a covariate in all subsequent analyses.There were no significant betweein-group differences in age, sex or socio-economic status see . However3.2F = 5.8, p=0.02; adjusted mean thalamic volumes in mm3 in ADHD: Left (SD)=7284.05(528.20), Right=7266.22 (503.75); controls: Left=7615.49(528.23), Right=7608.56 (503.13)). Both total brain volume =29.7, p<0.001) and age =9.56, p=0.003) were significant positive predictors in the model, with older children and children with more total brain volume having higher thalamic volume. There was no. diagnosis × hemisphere interaction. The magnitude of these effects did not change and remained significant when the female subjects were removed from the analysis. Age and brain volume did not have any group-specific effects.Children with ADHD had a significant reduction in thalamic volume  compared with controls  9, 10 and 46 , Table 23.4r=0.60, df=23, p=0.001), while controls showed a trend towards a negative association with thalamic volume . No other cortical region showed an ADHD-specific relationship between thalamic volume and surface area.In the whole brain analysis seeking the brain regions showing differential covariance with thalamic volume in the two groups, a significant interaction between thalamic volume and the diagnosis of ADHD was seen in a cluster that included right inferior and mid-lateral prefrontal cortex , and a trend towards a positive correlation for the left prefrontal cluster . None of these clusters showed any relationship with thalamic volume in controls .To further examine the covariance between thalamus and the bilateral frontal clusters that showed the most significant surface area reduction in ADHD, mean surface area values were extracted from the clusters for all participants and plotted against thalamic volume after adjusting for age and total brain volume. The scatter plots are shown in ps>0.2).Correlational analysis did not show any relationship between either the duration of stimulant use or current (mg/kg) dose of stimulants and either thalamic volume or the surface area of frontal clusters (all 4Using a surface based morphometric approach, we have obtained further evidence of reduced lateral prefrontal cortical area in ADHD. Moreover, for the first time, we have also shown that this reduction in prefrontal cortical area covaries with smaller thalamic volume in patients with ADHD. Our findings suggest that the pathophysiological process that alters surface area maturation, especially in lateral prefrontal cortex, may well be linked to the development of the thalamus, highlighting the possibility of deviant thalamo-cortical connectivity in ADHD.Although the functional role of the thalamus in ADHD has previously been investigated, particularly with respect to CSTC dysfunction , few strIn contrast to the study of The reduction in lateral prefrontal area in ADHD has been ascribed to a developmental delay in the normal process of cortical maturation, in keeping with models that propose maturational delay as a key feature of the disorder . The detThe influence of thalamic connections on cortical maturation is likely to extend beyond the neonatal period. Preliminary evidence from diffusion tensor studies suggests that thalamocortical connectivity changes with normal development in children, with the most pronounced age-related increases in thalamocortical connectivity occurring in bilateral prefrontal areas . RestingOur cross-sectional observation is insufficient to support or refute the possibility that a reduction in the volume of the thalamus could be secondary to cortical surface changes seen in ADHD. Experimentally induced aberrations in the development of thalamo-cortical connectivity in animals result in a reduction in the volume of thalamic nuclei, in addition to a disruption in the cortical circuitry . FurtherOur observation of reduced lateral prefrontal area in ADHD is consistent with other reports  despite The specific strengths of this study include a clinically well-defined sample with carefully matched controls, and the use of a well-validated surface-based automated morphometric measure that obviates the poor reliability associated with manual delineation of cortical and subcortical regions. Furthermore, we used an unbiased whole brain vertexwise approach to locate the brain region showing the most significant surface area change in ADHD. Nonetheless, there are a number of limitations that need to be considered in interpreting our results. The size of the sample is modest and, as characteristic of the disorder, was predominantly composed of males. This limits the generalisability of our findings, but it also increases the homogeneity and clinical applicability of the study. Furthermore, as is typical of the disorder, the children with ADHD had a number of comorbid disorders, particularly externalising disorders, and was underpowered to examine their specific effects. The children with ADHD also had lower IQ scores than controls. Nonetheless, the effects remained robust when IQ was covaried.increased in children with ADHD taking medication relative to unmedicated children with ADHD (Our sample included children who were prescribed stimulant medications. Correlational analysis has not shown any relationship between either the duration of use or current dose of stimulants. Moreover, previous studies of the thalamus found that overall thalamic volume was ith ADHD . As suchAs the participants were selected on the basis of meeting diagnostic criteria for hyperkinetic disorder, the severity of ADHD symptoms in the clinical group were near ceiling, with a limited spread of symptoms. Thus, it was not possible to determine whether the severity of the morphological abnormalities was related to symptom severity. Future studies in children with a continuum of ADHD symptoms are needed to study the association between thalamo-frontal covariance and symptom profile.In summary, our findings implicate a role for abnormal thalamo-cortical connectivity in the pathophysiology of the neurodevelopmental defects underlying ADHD. This observation will help to focus the search for genetic markers that involve very early cortical development in ADHD. Evaluating experimental approaches targeting thalamo-cortical connectivity for the effects on the frontal structural deficits associated with clinical symptoms could be an important next step in translational neuroscience of ADHD.MJB and LP wrote the manuscript and carried out the statistical analysis. LP conducted the Freesurfer segmentation. All the authors read and contributed to the manuscript and the ideas presented in it. MJB and LP are joint first authors of the paper.Eli Lilly. In the past 5 years, PFL has received honoraria for academic presentations from Janssen-Cilag and Bristol Myers Squibb; and has taken part in advisory panels for Bristol Myers Squibb. MG has received a non-interventional ‘unrestricted’ research grant and conference support from Shire Pharmaceuticals Ltd. All other authors report no biomedical financial interests or potential conflicts of interest.LP has received a Young Investigator Travel Fellowship from


Attention deficit hyperactivity disorder (ADHD) is characterized by severe and age-inappropriate levels of hyperactivity, impulsivity and inattention. ADHD is a heterogeneous disorder, and the majority of patients show comorbid or associated problems from other psychiatric disorders. Also, ADHD is associated with cognitive and motivational problems as well as resting-state abnormalities, associated with impaired brain activity in distinct neuronal networks. This needs to be considered in a multimodal treatment, of which neurofeedback (NF) may be a promising component. During NF, specific brain activity is fed-back using visual or auditory signals, allowing the participants to gain control over these otherwise unaware neuronal processes. NF may be used to directly improve underlying neuronal deficits, and/or to establish more general self-regulatory skills that may be used to compensate behavioral difficulties. The current manuscript describes pathophysiological characteristics of ADHD, heterogeneity of ADHD subtypes and gender differences, as well as frequently associated behavioral problems such as oppositional defiant/conduct or tic disorder. It is discussed how NF may be helpful as a treatment approach within these contexts. Difficulties with Inattention or Hyperactivity and Impulsivity as the core symptoms of Attention deficit Hyperactivity disorder (ADHD) are a frequent psychosocial burden. With an early onset during childhood, ADHD is often persisting throughout life. It is a heterogeneous disorder, and a challenge to treat. In light of this heterogeneity, the most promising treatment approach should be multimodal in nature  and to decrease theta (4–8 Hz) activity, or to train slow cortical potentials (SCP) in order to modulate cortical excitability); and successful trials are positively reinforced.Neurofeedback (NF) as a non-pharmacological intervention for ADHD utilizes cognitive-behavioral therapeutic elements to gain access on and practice brain activity  may improve performance and may ameliorate neuropsychological deficits found in ADHD, but direct effects on ADHD symptoms may be limited  usually shows a fronto-central negativity peaking around 200–400 ms after onset of the stimulus which is larger for the Nogo than for the Go condition, particularly when the Nogo condition is rare. The same effect can be observed when the target is primed with either congruent or incongruent distractors. The so called N2 and the N2-enhancement were originally attributed to (response) inhibition Kok, , but recWhile several studies using Continous Performance Tests (CPT) or Go-Nogo-tasks in children did not find conflict-specific differences in N2 between ADHD and controls  and error processing (error negativity) may operate with theta (or maybe even lower delta) frequency  NF of brain activity in the bilateral prefrontal cortex (implicated in executive functions and response inhibition) showed effectiveness in behavioral symptom ratings and executive functions, but may require fewer sessions than EEG or EMG NF  evoke a centrally negative SCP that terminates with the presentation of the next stimulus , frontal cortex, thalamus and midbrain dopaminergic nuclei  revealed coherent activity fluctuations with low frequency  in ADHD should be equal to the prevalence in the total population—in fact it is at least 20-times higher, and the reasons for this are still under debate. Research indicates that some comorbidities may in fact be a separate clinical entity , whilst others may in many ways an addition of difficulties present in either disorder . In any case, heterogeneity in ADHD may further complicate treatment.The Diagnostic and Statistical Manual distinguishes Hyperactive/Impulsive (ADHD-H) and Inattention (ADHD-I) symptom clusters in the diagnosis of ADHD APA, , but it However, on the neuropsychological level children with ADHD-I show similar performance problems as ADHD-C in a wide range of demands . In a large multicenter randomized controlled trial of a combination of theta-beta/SCP NF training for children with ADHD, we found comparable effects for symptoms of inattention as well as hyperactivity/impulsivity  axis and expressed in the release of cortisol, which was moderately inverse associated with aggressive symptoms  resulting from dysfunctions in the paralimbic system including orbito-frontal and superior-temporal areas and the ACC as well as the limbic system , whilst problems with “cool” attentional orienting and response preparation may be less severe than expected from an additive model of impairments found in both pure ADHD and ODD/CD.There is evidence that children with comorbid ADHD + ODD/CD may show particularly problems with self-regulation, whilst cognitive deficits may be less severe than expected from typical findings from ADHD and ODD/CD, suggesting that NF interventions may use these cognitive resources and may dwell on the enhancement or compensation of diminished self-regulation skills accordingly. Since ADHD may precede ODD/CD in child development, early interventions may be particularly promising. Moreover, combined NF SCP and theta/beta training yielded, besides particular reductions of teacher and parent-rated ADHD symptoms, also effects on parent-rated oppositional behavior and conduct problems when compared to a standardized computer attention training  are characterized by involuntary, sudden, short, repetitive and non-rhythmic fragments of usual movements and/or vocal expressions. Tics are ranging from mild  to severe  intensity and simple  to complex  extent that do not fulfil any subjective purpose Leckman, . TD are Tics do often occur in bouts and are particularly present during stress or in positive or negative emotional situations. Particularly patients older than 10 years may realize sensory-motor phenomena  before and/or after tic, and are often able to suppress their tics for a limited period of time  additive in ADHD and TD (Roessner et al., TD is probably associated with disturbances in cortico-striato-thalamo-cortical neuronal networks which may be partly compensated by increased prefrontal activity instrumental in tic suppression (Leckman et al., A recent imaging study in adults generally replicated an earlier study of Bohlhalter by further elucidating the progression of brain activity in the cortico-striato-thalamo-cortical circuit preceding a tic and additionally suggesting the role of DMN activity in tic generation (Bohlhalter et al., Moreover, studies on excitability of the motor system using transcranial magnetic stimulation indicated reduced motor inhibition in individuals with TD (Ziemann et al., Studies on higher order cognitive functions in TD suggest that patients may not show general impairments, but may at most be affected as a consequence of their tics or potential compensatory mechanisms; studies addressing the co-existence of TD + ADHD often found deficits explained by ADHD following an additive model (Schuerholz et al., Functional imaging studies particularly in adults with TD yielded mixed results, and the interpretation of functional data in TD may be complicated by the interplay of both activity reflecting pathophysiological mechanisms and potential compensatory activity required during tic control (Gerard and Peterson, In sum, ADHD + TD is probably not a separate clinical entity as it is the case for ADHD + ODD/CD. Individuals with ADHD + TD may share difficulties associated with both disorders often following an additive model, which may however be a special challenge to treat. SCP-NF may be used to ameliorate diminished slow-wave activity during preparation, which may be a common psychopathological impairment in both ADHD and Tic disorders. Typical interventions used in the treatment of TD that rely on intact cognitive control mechanisms or executive functions such as habit reversal may be less effective when ADHD is associated.Nevertheless, NF may be a promising component of a multi-modal therapy, as it combines training of potentially problematic brain activity with direct feedback and reward.In previous intervention studies, Theta-SMR-protocols have been applied in Tic-/Tourette disorder, from a traditionally point of view aiming at inhibiting over-activity in senso-motor cortical regions Tansey, . There iADHD as a neurodevelopmental disorder is associated with pathophysiological problems during cognitive demands, reward processing and during rest. It is further complicated by a number of heterogeneities regarding clinical characteristics, sex differences, and frequent comorbid disorders. The current manuscript introduced associated pathophysiological characteristics and discussed their potential relevance for NF intervention in ADHD.It is argued that cognitive deficits during preparation for an upcoming event and response inhibition problems associated with deficient activity in the prefrontal cortex and in the ACC, as well as impaired resting-state brain activity may be ameliorated by respective NF trainings. Tomographic NF interventions using high density EEG recordings, fMRI or NIRS targeting specific brain areas may allow more direct training of brain activity impaired in ADHD. Motivational problems during reward processing may rather be compensated by the acquisition of self-regulation skills.NF interventions may be used for hyperactive/impulsive and inattentive subtypes, and may not require sex-specific adaptations. It may in general be useful for children with comorbid ADHD and conduct disorders characterized in many ways by sub-additive cognitive deficits of both pure disorders but pronounced self-regulation deficits, as well as for ADHD and associated Tic disorder characterized largely by additive impairments.Taken together, NF intervention in ADHD may be applied in order to ameliorate specific deficits, and/or to acquire self-regulation skills to use them for the compensation of difficulties in other domains.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Children with attention deficit and hyperactivity disorder (ADHD) suffer from a variety of emotional and social difficulties including high levels of depression, anxiety, poor regulation, other executive malfunctions, and lack of satisfying social experiences  Riva, . VR is aRecently, we have utilized a self-face recognition paradigm to enhance the verisimilitude of a VR-generated substituted reality , ten children participated in a single arm prospective study to determine the efficacy of a combined intervention consisting of biofeedback based techniques of relaxation coupled to a VR system. The VR algorithm was designed so patients were led by their virtual self from a painful state to a relaxed pain free state. Biofeedback was based on a reduction of galvanic skin response which is a measure of relaxation. The subjects underwent 10 sessions, and at the end, several outcomes were measured including measurements of pain, quality of life, and a survey related to the efficacy of the intervention (Shiri et al., Based on the preliminary results from these pilot studies, we suggest that implementing a self-face viewing paradigm may be particularly effective in drawing patients' attention and in generating sound neural processing of the virtual scenery, especially neural networks within the mirror neuron system (Uddin et al., The virtual classroom used for evaluation and treatment of ADHD is a familiar and well studied model used to examine various neuropsychological abilities in an environment that simulates a real classroom (Rizzo et al., In future studies we would like to examine the utility of a VR-based intervention to enhance the self-image of children and adolescents with ADHD. Picturing oneself performing a desired behavior from a third-person perspective has the potential of causing individuals to adopt attitudes that are concordant and harmonious with the content drawn in the pictures (Libby et al., In conclusion, elevating hope among children with ADHD utilizing a self- face recognition paradigm specifically designed for the needs of children with ADHD has the potential for providing an emotionally positive experience that is therapeutically beneficial in treating the cognitive impairments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sixteen adult patients with attention deficit/hyperactivity disorder and 16 matched healthy control subjects were positron emission tomography and magnetic resonance imaging scanned and tested on a computerized sustained attention task after oral methylphenidate (0.5 mg/kg) and placebo administration in a within-subject, double-blind, cross-over design. Although patients with attention deficit/hyperactivity disorder as a group showed significant attentional deficits and reduced grey matter volume in fronto-striato-cerebellar and limbic networks, they had equivalent D2/D3 receptor availability and equivalent increases in endogenous dopamine after methylphenidate treatment to that observed in healthy control subjects. However, poor attentional performers drawn from both the attention deficit/hyperactivity disorder and the control groups had significantly reduced left caudate dopamine activity. Methylphenidate significantly increased dopamine levels in all nigro-striatal regions, thereby normalizing dopamine levels in the left caudate in low performers. Behaviourally, methylphenidate improved sustained attention in a baseline performance-dependent manner, irrespective of diagnosis. This finding was accompanied by an equally performance-dependent effect of the drug on dopamine release in the midbrain, whereby low performers showed reduced dopamine release in this region. Collectively, these findings support a dimensional model of attentional deficits and underlying nigro-striatal dopaminergic mechanisms of attention deficit/hyperactivity disorder that extends into the healthy population. Moreover, they confer midbrain dopamine autoreceptors a hitherto neglected role in the therapeutic effects of oral methylphenidate in attention deficit/hyperactivity disorder. The absence of significant case–control differences in D2/D3 receptor availability (despite the observed relationships between dopamine activity and attention) suggests that dopamine dysregulation per se is unlikely to be the primary cause underlying attention deficit/hyperactivity disorder pathology in adults. This conclusion is reinforced by evidence of neuroanatomical changes in the same set of patients with attention deficit/hyperactivity disorder.Through the combined use of  Attention deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by symptoms of inattention and/or hyperactivity/impulsivity. Despite its declining prevalence with age, ADHD symptoms in some affected patients persist into adulthood, affecting ∼2.5% of the adult population (in vivo nuclear imaging techniques such as PET and single photon emission computed tomography (SPECT) have often yielded inconsistent findings regarding the state of the dopamine system in this patient population  and is thus better suited for simultaneous measurement of D2/D3 receptor availability in striatal regions and the midbrain , the following questions regarding the neurochemical underpinnings of ADHD were addressed.To investigate nigro-striatal mechanisms underlying ADHD and its treatment with methylphenidate, we used PET imaging of 2/D3 receptor availability in the striatum and the midbrain compared with healthy volunteers? Second, how is D2/D3 receptor availability in these regions associated with inattention, a core feature of the disorder  were ADHD medication-naïve. Patients on methylphenidate treatment at the time of scanning were asked to discontinue their medication at least 3 days before each PET session.Patients were recruited from the Adult ADHD Research Clinic Cambridge and through the Attention Deficit Disorder Information and Support Service (ADDISS), UK. Diagnosis was contingent upon six of nine DSM-IV  inattention and/or hyperactivity/impulsivity criteria being met during childhood and the previous 6 months (1994). Clinical assessments were performed by two psychiatrists specialized in adult ADHD  as described previously   and curr18F-fallypride PET scans at least a week apart and one MRI scan. On each PET session, subjects were administered a capsule containing either 0.5 mg/kg of methylphenidate or placebo 75 min before the 18F-fallypride injection  to assess compliance with drug discontinuation in medicated patients and account for between-subject differences in methylphenidate plasma levels. Cardiovascular measures were regularly monitored throughout the study.Participants were enrolled into a randomized, double-blind placebo-controlled cross-over design, consisting of two njection . The doswww.camcog.com). RVP primarily measures sustained attention, defined as the ability to maintain attention on a series of numerical stimuli over a period of time, to detect infrequent targets . Response latencies were also measured. To minimize practice effects, all subjects performed the task once before their first study session.On both PET sessions, participants performed the Rapid Visual Information Processing (RVP) task from CANTAB  isAll participants completed the adult ADHD self-rating scale (v1.1), a short self-rating test addressing the severity of ADHD symptoms in adults consistent with DSM-IV criteria  – posterior commissure (PC) line. A T2-weighted image was also acquired for each subject .Magnetic resonance images were acquired for all subjects on a Siemens Trio 3 T system  with gradient coils capable of 45 mT/m and 200 T/m/s slew rate. A 12-channel total-imaging matrix head-coil was used to transmit/receive radio-frequency magnetic resonance signals. T18F-fallypride PET data were acquired in 3D mode on a GE Advance scanner . Before 18F-fallypride injection, a 15 min transmission scan was acquired. 18F-fallypride was injected intravenously over 30 s, with a mean activity of 103.5 MBq (range: 52.2–119.8 MBq), and a fallypride mass of 1.3 ± 0.7 nmols.18F-fallypride injection PET emission data were continuously acquired for 75 min in 55 frames . Before analysis, volumes were preprocessed, and then spatially normalized and segmented into grey and white matter tissue using the unified segmentation model in SPM5 (http://www-bmu.psychiatry.cam.ac.uk/software/)  template image (where voxels/region > 20)  as previously described  as previously described  with group and medication status as between-subject factors. Where assumptions of sphericity were violated, degrees of freedom were corrected by applying Greenhouse-Geisser corrections. Significant differences were further examined using post hoc t-tests or, where appropriate, the equivalent versions for non-parametric data (Wilcoxon signed-rank test and Mann-Whitney U test).Group effects on ADHD self-rating scale scores and both group and methylphenidate effects on task performance and BPND and BPND % change. Where relevant, methylphenidate plasma concentrations were partialled out. Relationships were investigated for left and right hemispheres separately, in-line with previous observations of lateralization differences in dopaminergic parameters in the healthy population  and performed significantly worse on the RVP A’ signal detection parameter . There were no between session practice effects on task performance (P > 0.1).Demographic characteristics of the sample and mean scores on psychological measures are shown in P > 0.1). However, patients with ADHD showed significantly reduced grey matter volume in left prefrontal cortical areas , and in bilateral putamen, amygdala, hippocampus, fusiform, insula and cerebellum  are subsequently presented split by experimental session (placebo then methylphenidate). Supporting whole-brain voxel-wise PET results are presented and discussed in the ND values were not significantly different between patients with ADHD and control subjects across all regions , with bilateral values for the latter shown in ND values pooled across all subjects are shown in ND values on placebo were in the same range as those previously reported in a study examining test–retest variability of baseline 18F-fallypride BPND across the same brain regions .The mean BPND both in patients  and control subjects   although not in patients   A but did = 0.33) B.FigureP > 0.1). For patients and control subjects combined, inattention scores were negatively correlated with BPND in the portions of the associative striatum corresponding to pre-commissural dorsal caudate and post-commissural caudate  and in sensorimotor striatum . Hyperactivity scores for all subjects on the other hand were associated with BPND in left substantia nigra/ventral tegmental area  but not in any other region.Correlation coefficients for left and right striatal subregions and substantia nigra/ventral tegmental area were similar in patients and control subjects  and right post-commissural caudate . Although the correlation coefficients in control subjects were not significant, they were in the same direction as those observed in patients for all regions and were not significantly different from them (P > 0.1). Across the entire subject group, low A’ scores were associated with low BPND in left pre-commissural dorsal caudate  as well as in left and right post-commissural caudate .In patients, low A’ scores were associated with low BPND values on attentional performance  revealed a significant best-fit model with the left pre-commissural dorsal caudate as independent variable .Stepwise linear regression of all regional BP = 4.5; P < 0.001] and heart rate  . Mean methylphenidate plasma concentrations were similar in patients with ADHD and control subjects (P > 0.05).Plasma level analysis of methylphenidate confirmed randomization and compliance with discontinuation of therapeutic medication. Methylphenidate produced significant increases in systolic blood pressure , improving A’ scores in low  but not in high performers   = 30.51, P < 0.001], ranging from −4.0 to −7.8% depending on anatomical region {region of interest by treatment interaction }, being greatest in substantia nigra/ventral tegmental area and least in ventral striatum  = 8.34, P = 0.002], revealing a preferential effect in sensorimotor striatum. The magnitude of regional BPND % change was similar in patients with ADHD and control subjects (main effect of group and group by drug interaction: P > 0.05).Methylphenidate significantly reduced BPstriatum . MethylpND % change in ventral striatum  and positively with substantia nigra/ventral tegmental area , with the correlation coefficients being highly significantly different .Methylphenidate-induced % change in A’ was negatively correlated with BPND % change in right ventral striatum  and lower BPND % change in substantia nigra/ventral tegmental area . CorrelSupplementary material).The above analyses were also calculated after excluding ceiling performers on the attentional task (five control subjects and one patient), yielding similar results  had no impact on the correlation between methylphenidate-induced changes in A’ scores and BPND % change in ventral striatum . Therefore, these effects were not baseline-dependent.We hypothesized that the relationship between A’ % change and BPND % change in substantia nigra/ventral tegmental area were dependent on baseline A’ scores, the correlations for this region no longer being significant . Corroborating this finding, repeated measures ANOVA revealed lower BPND % change in low compared with high performers in substantia nigra/ventral tegmental area  = 9.351, P = 0.005; F = 5.46, P = 0.027 and F = 5.2, P = 0.030 for bilateral, left and right substantia nigra/ventral tegmental area, respectively], but not in any other region.On the contrary, changes in sustained attention associated with BPtal area  . A further regression model revealed that baseline performance levels on A’ predicted BPND changes specifically in the left midbrain , confirming the above correlational results.Stepwise linear regression modelling to determine the relative impact of methylphenidate-induced BPND in left pre-commissural dorsal caudate  = 4.63, P = 0.04]. As shown in F = 2.97, P = 0.096].On placebo, low performers had reduced BP caudate A [F in the left pre-commissural caudate than high-performing subjects, though independent of diagnosis. This result is consistent with previous evidence implicating the left caudate in inattentiveness, not only in patients with ADHD, but also in healthy subjects , the alternative first-line treatment for ADHD which also increases catecholamine levels, albeit via different cellular mechanisms -ergic projections from the same striatal subregion they project to through reciprocal connections, giving rise to topographically organized striato-nigro-striatal circuits A. GABAerWe hypothesize that the inverse relationship in dopamine release between substantia nigra/ventral tegmental area and ventral striatum but not associative striatum or sensorimotor striatum following methylphenidate reflects the asymmetrical nigro-striatal projection pattern. Ventral striatum is the only striatal region whose dopamine input is unaffected by non-reciprocal connections, its dopaminergic input thus being regulated to a greater extent by synthesis- and release-regulating midbrain dopamine autoreceptors. The observed inverse correlation may thus reflect the inhibitory influences exerted by midbrain dopamine neurons to regulate dopamine levels in ventral striatum. This inverse relationship did not hold for associative striatum and sensorimotor striatum, which can be explained by the fact that, via the aforementioned feed–forward spiralling connections, associative striatum receives additional inhibitory influences from ventral striatum and sensorimotor striatum from both ventral striatum and associative striatum. These findings possibly constitute the first direct evidence in humans supporting the notion that the doses used clinically to treat ADHD exert their therapeutic actions through presynaptic mechanisms. A key finding of this study is that midbrain dopamine autoreceptor regulation was reduced in low performers, as evidenced by the significantly smaller methylphenidate-induced increases in midbrain dopamine levels observed in low compared to high performers C.How are the changes in midbrain dopamine receptor availability related to the attention-enhancing effects of methylphenidate, whether in patients with ADHD or healthy control subjects? A plausible explanation is provided by the classical model of catecholamine neurotransmission regulation and psychostimulant action postulated by Grace , 2001. AThe idea that the clinical effects of stimulants are mediated by the ability of small increases in extracellular dopamine to produce a net decrease in phasic catecholamine release by the selective activation of dopamine autoreceptors (following an increase in tonic dopamine levels)  is suppoIn an attempt to identify putative biomarkers of ADHD, we used, for the first time, an interdisciplinary approach encompassing both PET and MRI in combination with neuropsychopharmacological assessment which allowed characterization of the disorder at clinical, cognitive, neurochemical and neuroanatomical levels. The current paper focused on the neuropsychopharmacological findings; a companion paper will address in more detail grey and white matter tissue in relation to the behavioural changes.2/D3 receptor density . Values were largely comparable in terms of both magnitude and rank order, suggesting that the regional BPND values reported here following placebo administration were within the expected baseline ranges. One way future research might help to disentangle undesired effects of drug-order and stimulant carry-over effects is through implementation of a double-blind randomized design with parallel groups, as used by A further strength was the experimental design,  which differs from the fixed design  used in previous studies investigating the effects of methylphenidate in ADHD  or their state of affinity for dopamine. It is important to note also that the slow kinetics of 18F-fallypride necessitated a long acquisition period (3.15 h) and thus did not allow us to differentiate tonic from phasic dopamine activity in the midbrain, which can only be inferred.A caveat of dopamine receptor imaging technique as the one reported here is that receptor availability and dopamine release are inferred from estimates of BPlization  or chang18F-fallypride PET imaging of the striatum satisfies both these criteria and hence partial volume effects are theoretically an issue of concern in this study. Moreover, our finding of reduced grey matter volume in the putamen in patients with ADHD compared with control subjects implies that partial volume effects may have had a differential effect on sub-striatal BPND estimation in patients and control subjects, potentially masking group differences in regional D2/D3 receptor availability. In order to rule this out, an additional set of analyses was carried out to estimate partial volume error using an in-house implementation of a well-validated method . Importantly, to ensure complete washout at the time of scanning among patients under current treatment, compliance with treatment discontinuation 3 days before each PET scan was verified through urine screening. Moreover, medication status was controlled for in all repeated measures ANOVAs. There is a lack of clarity regarding chronic effects of ADHD medication, with different studies reporting contradicting results with regard to its impact on dopaminergic markers  dose and time course of methylphenidate actions; (ii) level of responsivity of the dopamine system determined by tonic dopamine levels; and (iii) baseline level of performance, possibly reflecting basal dopamine function in the striatum. The findings of reduced methylphenidate-induced midbrain dopamine increases and improved sustained attention in low performers (mostly patients with ADHD) implicate a hitherto neglected role of midbrain dopamine in the mediation of therapeutic effects of methylphenidate.We have shown the considerable potential of combining the complementary strengths of structural MRI and neuroreceptor PET imaging in identifying and characterizing cognitive endophenotypes of ADHD, which may help our understanding of the diagnosis, prognosis and treatment of ADHD.


Bats can be potent reservoirs of human bacterial pathogens. Bartonella. Bartonella spp. also were either detected or isolated from the peripheral blood of Daubenton's, northern, and whiskered bats and were detected in the ectoparasites of Daubenton's, northern, and Brandt's bats. The blood isolates belong to the Candidatus-status species B. mayotimonensis, a recently identified etiologic agent of endocarditis in humans, and a new Bartonella species . Phylogenetic analysis of bat-colonizing Bartonella spp. throughout the world demonstrates a distinct B. mayotimonensis cluster in the Northern Hemisphere. The findings of this field study highlight bats as potent reservoirs of human bacterial pathogens.A plethora of pathogenic viruses colonize bats. However, bat bacterial flora and its zoonotic threat remain ill defined. In a study initially conducted as a quantitative metagenomic analysis of the fecal bacterial flora of the Daubenton’s bat in Finland, we unexpectedly detected DNA of several hemotrophic and ectoparasite-transmitted bacterial genera, including  The 1,100 species of bats (Myotis daubentonii) in Finland. Unexpectedly, we found that the fecal material contained DNA of several hemothrophic and ectoparasite-transmitted bacterial genera, such as Bartonella. This DNA may originate either from bleeding into the intestine or from the insect prey of the bats that includes the abundant bloodfeeding bat ectoparasites. Therefore, the study further focused on detecting and isolating Bartonella spp. from peripheral blood and ectoparasites of several bat species in Finland in 2012.The development of next-generation sequencing techniques has revolutionized biological science. It is now possible—and cost-friendly—to gain access to massive amounts of qualitative and quantitative sequencing data in a short time without a priori knowledge of the sequence . We have described the detailed protocols, including bat sampling for peripheral blood, fecal droppings, and ectoparasites; metagenomic analysis of fecal DNA; isolation of Bartonella from peripheral blood; extraction of DNA from bat blood, ectoparasites, and Bartonella isolates; Bartonella and ectoparasite PCR analyses; transmission electron microscopy; and nucleotide sequence and phylogenetic analyses in the >50 bp) were assigned on the basis of best E-value BLASTN scores (http://blast.ncbi.nlm.nih.gov/blast.cgi) in GenBank. The most abundant non-metazoan sequence matches were with bacteria. The genera Leuconostoc, Enterobacter, Lactococcus, and Chlamydia dominated  (Eptesicus nilssonii), and 1 Brandt’s bat (Myotis brandtii) , were B. tamiae Th339 (GenBank accession no. JN029780) and Th307 strains (GenBank accession no. JN029778) isolated from 2 humans in Thailand  from DNA sequencing of fecal material from a Daubenton’s bat  Figure 1Bartonella spp. by sequencing a PCR-amplified 485-bp fragment containing the hypervariable regions V6–V8 of the 16S rRNA gene. Overall health of the bats as analyzed by body condition indexing was not affected by the Bartonella infection , citrate synthase gene (gltA), filamenting temperature-sensitive mutant Z gene (ftsZ), VirB type IV secretion system VirB4 component gene (virB4), hypervariable region 2 of the 16S-23S rRNA intergenic spacer region (ISR), and ssrA , 1 rpoB-2 allele isolate , and 1 rpoB-3 allele isolate . Thin-section transmission electron micrographs of these isolates are shown in the The 16S rRNA gene sequences are highly conserved within the genus and ssrA  Table 2.entified  Table 1.Bartonella  clearly diverges from the other bat isolates. species  Table 5.Bartonella rpoB. The blood isolate rpoB alleles 1 and 2 were detected in samples from 1 flea and 2 flies, respectively , which is higher than with the isolates 1157/3  and 1160/1 . The data further support the conclusion that bats are reservoir hosts of B. mayotimonensis and indicate that the bat flies and fleas transmit Bartonella spp. to new hosts.We sequenced a PCR-amplified fragment of the mitochondrial cytochrome c oxidase subunit I  Table 1.ectively  Table 1.gltA sequences obtained from Bartonella that infect bats in the United Kingdom  does not belong to the Northern Hemisphere B. mayotimonensis cluster. Remarkably, the 253-bp gltA fragment of 1 of the Bartonella-like bacteria detected in minced heart tissue of a common noctule   yielded a 95.3% (241/253 bp) sequence identity score, compared with the corresponding fragment (FJ376732) of Candidatus B. mayotimonensis. This value is significantly higher than those obtained with corresponding gltA fragments of Finland bat isolates 1157/3  or 1160/1 . The UK gltA data further support the conclusion that bats are reservoir hosts of B. mayotimonensis. Most importantly, bats appear to be reservoir hosts of B. mayotimonensis only in the Northern Hemisphere.A maximum composite likelihood–based neighbor-joining tree  was consBartonella naantaliensis  is the name proposed to highlight the municipality where the bat was trapped from which the type strain was isolated. The type strain is 2574/1. Its partial 16S rRNA gene nucleotide sequence is deposited in GenBank (accession no. KF003116).Bartonella spp. are facultative intracellular bacteria that typically cause long-lasting hemotrophic bacteremia in their mammalian reservoir hosts, such as rodents  was isolated from the blood and detected from the ectoparasites.Bartonella spp. discriminatory marker, the ISR, were used . These numbers probably reflect the frequency of bat bites in most countries of the Northern Hemisphere. We propose that fecal droppings of blood-fed bat ectoparasites might transmit Bartonella spp. into the human host, assisted by superficial scratching or tissue trauma of the skin. The presence of viable bacteria in feces of body lice (Pediculus humanus) that have been feeding on B. quintana–infected rabbits is well documented  (B. henselae–infected cat led to bacteremia in a pathogen-free cat (Cimex spp.) known to also feed on humans were not analyzed in the current study.Findings of the study raised an interesting question: how could The reported metagenomic analysis of bat fecal material indicates that bats are reservoir hosts for several pathogenic bacterial genera. No comprehensive study has been published on the bacterial flora of bats in light of its zoonotic threat to humans. The major research focus has been on viruses, and several deadly viruses have been detected or isolated (Materials and methods, Technical Appendix Tables 1–5 and Technical Appendix Figures 1 and 2.


Dimethyl (β-substituted) vinylphosphonates do not readily undergo cross metathesis reactions with Grubbs catalyst and terminal alkenes. However, the corresponding mono- or diallyl vinylphosphonate esters undergo facile cross metathesis reactions. The improved reactivity is attributed to a relay step in the cross metathesis reaction mechanism. A . A n-but19 was treated with 5 equivalents of allyl bromide and 5 mol % TBAI in refluxing toluene for 36 hours to give a 76% conversion with 1.8:1 ratio of mono- to diallylated vinyl phosphonates 20a and 20b  and Hβ, 6.2  were examined . The monster 16b  was formphole 22 . An impufication . Howeverructures  giving csters 23  and 24, 21a with substituted styrenes. p-Substituted styrenes are type I substrates and should readily engage in the metathesis reaction. Thus, reaction of the mono-allyl vinylphosphonate 21a with 5 equivalents of styrene using 10 mol % Grubbs second generation catalyst and 10 mol % CuI in refluxing CH2Cl2 for two hours gave tetrahydropyran 25 in 82% isolated yield   requireded yield . Similargenation .27 was isolated in small amounts (~20%) from the reaction of vinylphosphonate 21a with styrenes. The dimeric product 27 was not observed during the cross metathesis of the vinylphosphonate 21a with methyl acrylate.Surprisingly, the dimer 28) are reported to undergo ring closing metathesis to give either 7-membered (29) or 5-membered (30) phosphorus heterocycles (E) diallyl vinylphosphonates would prefer to form the 5-membered ring oxaphosphole 30 and therefore, like the corresponding mono-allyl phosphonates, should engage in relay cross metathesis reactions.Diallyl vinylphosphonates (rocycles  23–24].–24.28) a14b was subjected to cross metathesis with methyl acrylate using standard conditions  to generate the oxaphosphole 22 and a new metal alkylidene 34. The sequence is completed by reaction of the metal alkylidene 34 with the metathesis partner (styrene) to give the tetrahydropyran 25. The formation of the dimeric product 27 is probably the result of a competitive cross metathesis reaction between the tetrahydropyran 25 and the metal alkylidene 34  that dimethyl vinylphosphonate 12b reacted with neat methylimidazole at 100 °C to give the imidazolium salt 37 (Analysis of the TBAI allylation mechanism suggested that a good approach to mono-allyl phosphonate ve yield . We were salt 37 . TreatmeThe experiments presented above have demonstrated that whereas the dimethyl esters of substituted vinylphosphonates are characterized as type IV substrates in alkene cross metathesis reactions and are unreactive, the corresponding allyl esters show significantly improved reactivity. The improved reactivity is attributed to relay step in the cross metathesis reaction mechanism.File 11H and 13C spectra for all new compounds.Experimental procedures, characterization data,


Purpose. To evaluate the effectiveness of presence of desmopressin in treating primary enuresis (PE) for children with attention deficit-hyperactivity disorder (ADHD) symptoms. Materials and Methods. Children aged from 5 to 12 years with the chief complaint of PE treated with desmopressin were enrolled in pediatric urology clinics. The parent-reported SNAP-IV questionnaire was used to evaluate ADHD symptoms . Voiding symptoms were assessed by the Dysfunctional Voiding Scoring System (DVSS) questionnaire. The responses to desmopressin were analyzed in children with and without ADHD symptoms. Results. The study sample comprised 68 children; 27 (39.7%) presented with ADHD symptoms and 41 (60.3%) with non-ADHD symptoms. The children collected from a tertiary referral center may explain the high prevalence of ADHD symptoms in the present study. The total DVSS score in the ADHD symptoms group was significantly higher than in the non-ADHD symptoms group . In the ADHD symptoms group, there were significantly higher score in the “pee 1-2 times/day” and “can't wait” subscales of DVSS and lower sleep quality based on the Pediatric Sleep Quality questionnaire, as well as significantly lower peak flow rate and voided volume. The responses to desmopressin for enuresis were comparable between children with ADHD and non-ADHD symptoms. Conclusions. Approximately 39.7% of PE children presented with ADHD symptoms at urologic clinics. PE children with ADHD symptoms had higher risk of daytime LUTS and comparable response to desmopressin treatment for PE. To evaluate ADHD symptoms and daytime voiding symptoms is important in children with PE. Pediatric enuresis or nocturnal incontinence defined by International Children's Continence Society  is a comDesmopressin is one of the two standard treatments for primary enuresis (PE), the other being the alarm , but theThe study protocol was approved by the Institutional Research Ethics (no. 200907060R). Between September 2010 and August 2011, we consecutively collected children aged from 5 to 12 years whose chief complaint was PE (wetting the bed three or more times/week) for more than one month at the urology clinics of the National Taiwan University Pediatric Hospital. Subjects were excluded if they had (1) concurrent neurologic or urogenital disorders, (2) congenital disorders, (3) prior previous pelvic surgery, or (4) concurrent use of medications which are known to interfere with bladder or sphincter function. All study subjects received bladder and renal ultrasonography and urinalysis to exclude other genitourinary tract anomalies and urinary tract infection. All enrollee first received 4-week bladder training program at the beginning of the treatment protocol, including evenly distributed fluid intake throughout the day, the avoidance of drinks precipitating bladder overactivity, timed voiding every 2-3 hours, fluid restriction in the evening, and emptying bladder before bedtime. For subjects with good compliance, if enuresis did not demonstrate significant improvement, we proceeded with medical therapy. We administered 12-week desmopressin orally, starting with a standard dose of (0.2 mg) and increasing after 2 weeks to 0.4 mg in subjects if the episodes of enuresis did not become completely dry. We attempted to wean the subjects from the medication since 10th week by administration of one-half of the dose for two weeks before discontinuation. Patients were followed up 1 month after cessation of medications.The validated Chinese language version of the SNAP-IV was completed by the parent(s) of each enrollee –14. The Each subject, together with parents, answered the 10 questions of the validated Chinese language version of the Dysfunctional Voiding Symptom Scale (DVSS) questionnaire, which is commonly used to evaluate LUTS in children , 16. TheThe demographic data were elicited by a specially trained interviewer. Uroflowmetric studies were then conducted. The subjects were instructed to drink water until a strong desire to void was achieved, and they were then instructed to void into a uroflowmetry.During the whole course of treatment, the numbers of wet nights per week were recorded. The number of wet nights during the 2 weeks before desmopressin treatment and the last 2 weeks at 4th month follow-up was compared to evaluate the effectiveness of desmopressin treatment. Nonresponse was defined as a 0–49% decrease, partial/complete response as more than 50% decrease in the number of wet nights .t-test, paired t-test, or nonparametric test. The chi-square or Fisher's exact test was used to compare the categorical data. Two-sided tests were used, and a P value less than 0.05 was considered to be statistically significant. All data in the present study were analyzed with commercial statistical software .The numerical data were described by means and standard deviations (SD). The categorical data were expressed as counts and percentages. The numerical data were compared with Student's A total of sixty-eight children (51 boys and 17 girls) aged 5 to 12 years were enrolled; 27 (39.7%) presented with ADHD symptoms and 41 (60.3%) with non-ADHD symptoms, based on norm of the Chinese SNAP-IV parents form. Demographic data of the subjects are listed in Qmax) and voided volume in two groups are shown in P = 0.019). In terms of mean scores of all DVSS subscales, the items “I only go to the bathroom one or two times each day” and “I cannot wait when I have to pee” were significantly higher in the ADHD symptoms group . ADHD symptoms children suffered more from initial urgency than non-ADHD symptoms children .The daytime LUTS and peak flow rate (Qmax in uroflowmetry (UFM) was significantly lower in ADHD symptoms group  as well as lower voided volume . The sleep quality of ADHD symptoms group was significantly worse compared to non-ADHD symptoms group . There was no difference in constipation between the two groups.The P = 0.42). The responses between non-ADHD symptom subjects and ADHD subtypes were comparable as well . No serious adverse effect was encountered for both groups during treatment.The responses to desmopressin for enuresis were shown in Qmax and voided volume, were all comparable between partial/full response and poor response groups .Most pediatricians and pediatric urologists have mentioned that ADHD children disproportionately suffered from various forms of voiding issues, such as enuresis, urgent incontinence, and dysfunctional voiding –8, 20. IDesmopressin and alarm are standard treatment for primary monosymptomatic enuresis. Desmopressin alone accounts for 40 to 81% success rate and effectively reduces voids at night during treatment , 22. NevChertin et al. combined desmopressin and oxybutynin for enuresis in ADHD subjects and showed significant improvement in DVSS compared to that in the imipramine group, and the incidence of enuresis was significantly decreased as well . It had The correlations of enuresis with ADHD symptoms have not been investigated in children visiting pediatric urologic clinics, where the prevalence of ADHD diagnosed by the SNAP-IV MTA rating scale was high 39.7%) in the present study. Previous studies have found a higher prevalence of ADHD symptoms in enuretic children and linked the relationship between ADHD and voiding dysfunction % in the , 25. ComIn the present study, the percentage of urgency as the initial presentation and the items “I only go to the bathroom one or two times each day” (infrequent voiding) and “I cannot wait when I have to pee” (urgency) were significantly higher in the ADHD symptoms group, similar to results from other series , 8. ThisP = 0.017), which should have been evaluated and treated in pediatric urology clinics. Some of the deficits in attention and hyperactivity may be alleviated after treatment [Common sleep problems were related to ADHD such as shorter sleep duration, difficulties getting up, and more daytime sleep and account for behavior difficulties . Sleep rreatment .There are some limitations in the present study. First, the number of study subjects is limited. Since subjects of non-ADHD symptoms seem to have more favorable response compared to those of ADHD symptoms, further studies with larger sample size are warranted to clarify the result. Second, the relapse of enuresis and the coexisted daytime voiding symptoms is not evaluated after desmopressin treatment. As previously reported, the relapse rate after cessation of treatment is high (30–50%) and relapse might follow within a short period of time . Third, The prevalence of ADHD symptoms is high in children with PE at urologic clinics. Enuretic children with ADHD symptoms were more likely to have daytime LUTS. Desmopressin was effective in treating PE children regardless of the presence of ADHD symptoms. Further investigations are required to clarify the detailed voiding patterns in children with ADHD symptoms and long-term desmopressin response to PE.


In this work, we present the updated version (i.e. ZCURVE 3.0). Using 422 prokaryotic genomes, the average accuracy was 93.7% with the updated version, compared with 88.7% with the original version. Such results also demonstrate that ZCURVE 3.0 is comparable with Glimmer 3.02 and may provide complementary predictions to it. In fact, the joint application of the two programs generated better results by correctly finding more annotated genes while also containing fewer false-positive predictions. As the exclusive function, ZCURVE 3.0 contains one post-processing program that can identify essential genes with high accuracy . We hope ZCURVE 3.0 will receive wide use with the web-based running mode. The updated ZCURVE can be freely accessed from http://cefg.uestc.edu.cn/zcurve/ or http://tubic.tju.edu.cn/zcurveb/ without any restrictions.In 2003, we developed an  To extract more information and achieve higher accuracy, currently we further consider the short-term correlation among three or four adjacent nucleotides. That is to say, frequencies of 192 (43 × 3) phase-specific trinucleotides are transformed into 144 (192 × 3/4) Z variables and frequencies of 768 (44 × 3) phase-specific tetranucleotides are transformed into 576 (768 × 3/4) Z variables. In addition, the frequencies of phase-specific dinucleotides occurring at the codon positions 3–1 are also taken into account and there will be 36 (42 × 3 × 3/4) corresponding variables for dinucleotides. Hence, the number of characteristic variables changed from 33 to 765 (9 + 36 + 144 + 576) in ZCURVE 3.0. The Z transformation is responsible to change the four nucleotides into three distributions of purine versus pyrimidine, amino versus keto, and weak hydrogen bonds versus strong hydrogen bonds. If you want to learn more details and see the mathematical formula about the transformation, please refer to  genome on our ZCURVE web server, which has a very small genome size that enables a quick loading time. When annotating one anonymous genome, the users must prepare a standard FASTA sequence file as well as the example. Also, our server can handle multiple FASTA-formatted sequences, which is especially useful when users submit contigs of a draft genome. Our program can output four files by selecting each of the four output options. One is the chromosomal coordinates of all predicted genes. The second file includes DNA sequences of all genes in FASTA format. The third file corresponds to amino acid sequences encoded by each predicted gene. The last output file contains the essentiality of all predicted genes provided by our post-processing program Geptop. The essentiality score indicates the importance of the gene function: the higher the essentiality score, the more important the gene function. The first three files are output immediately , whereas the essentiality information needs over one hour to output. Therefore, users must record the outputting link and view the results two or three hours later. Figure For the input, we provide an example of the  NC_02083 genome oThe performance of ZCURVE 3.0 in the prediction of coding potentials and essentiality was evaluated in this work. To measure coding potentials prediction, we used the following indexes:Because negative samples could not be determined in a test set that was used to evaluate coding potential, the precision index was adopted instead of the specificity index. Hence, we define the accuracy index as follows:We also defined the following index to denote the percentage of additional prediction:Note that the prediction is better when the former three indexes are higher. However, a low value is better for additional positive rate.E. coli MG1655 genome; however, the number of non-essential genes is as large as 3852. For this special case, we primarily used the integrated index to evaluate the essentiality prediction of our program.The numbers of essential genes and non-essential genes are extremely imbalanced. For example, there are only 288 essential genes in the The commonly used indexes, sensitivity and specificity, are also used as additional references.A gene would only be considered a correct prediction if ZCURVE 3.0 not only found the gene but also correctly decided its essentiality. Experimentally determined essential genes in optimal growing conditions were taken as the benchmark of testing our program.ab initio prokaryotic gene finder, has an accuracy of 93.0%. In detail, Glimmer 3.0 has 1.0% higher sensitivity than ZCURVE 3.0. However, the latter has 2.5% higher precision than the former. Therefore, we can safely conclude that our program gives similar performance with Glimmer in the identification of protein-coding genes. Note that the standard deviation of the accuracies for Glimmer 3.02 is 1.3% higher than that of ZCURVE 3.0, indicating that ZCURVE may have more robust results than Glimmer. In fact, Glimmer produced poor results for a few genomes, whereas ZCURVE did not. For example, the sensitivity of Glimmer 3.02 was as low as 56.9% for the genome Anaeromyxobacter dehalogenans (NC_007760), and the precision was only 62.4%. In contrast, ZCURVE 3.0 gave stable results for this genome, with a sensitivity of 94.3% and precision of 95.7%. As another example, the precision of Glimmer 3.02 was only 44.8% for the Trichodesmium erythraeum (NC_008312) genome, whereas ZCURVE yielded a precision of 87.2%; both programs yielded a sensitivity of ∼94%.To evaluate the performance of ZCURVE 3.0 in the prediction of coding potentials of prokaryotic genes, we selected 422 genomes that were sequenced before 2007 that did not use the ZCURVE program in the annotating process so that the genomes could be used as a strict test set. For these genomes, we downloaded ‘*.fna’ and ‘*.ptt’ files from the RefSeq site at the National Center for Biotechnology Information (NCBI) . A gene We also used ZCURVE 3.0 to analyze 2787 bacterial and archaeal genomes that were sequenced between the year 2007 and 2013. Consequently, the average sensitivity of ZCURVE 3.0 was 95.1%, and the precision was 92.6% (Supplementary Table S2). The accuracy, which is the mean value of the above two indexes, should be 93.9%. Therefore, ZCURVE 3.0 gives stable results using large test sets. Finally, the quality of the annotation for each of the 422 genomes differs because of the applied annotation strategy. Therefore, the factual accuracy of both Glimmer and ZCURVE might be higher than that reported here because a few errors  may exist in the RefSeq annotation files for some genomes, which have been used as the gold standard by us.Pyrococcus abyssi GE5 (NC_000868), we excluded the genus Pyrococcus (taxid:2260) from the nr database. This logic simulates the factual case when annotating an anonymous genome because, in that case, the nr database will not contain any genes from itself and relative species.We do not intend to replace the Glimmer system with our ZCURVE program. Instead, we wish to provide an alternative solution for the issue of prokaryotic gene finding and hope it could be complementary to Glimmer. In fact, ZCURVE and Glimmer are based on different principles. Glimmer is a Markov-chain–based method, which reflects local statistical characteristics of coding sequences, whereas ZCURVE is based mainly on global statistical characteristics of coding sequences . Therefoab initio gene finders to minimize annotating errors. Many researchers have adopted such tactics. For example, in well-annotated genomes such as Tistrella mobilis KA081020-065 . Here, the set of predicted genes was taken as the initial set when it was integrated as the newly added function of ZCURVE 3.0. To evaluate the performance of the integrated program, seven bacteria were used in which genome-wide essentiality was determined after the publishing date of Geptop. As Table ab initio gene finding in prokaryotes. A higher accuracy was illustrated with the latest version, and it can give close accuracy with the commonly used program Glimmer 3.02. As the most prominent advantage, ZCURVE 3.0 can automatically select essential genes from the list of protein-coding genes, whereas none of the other ab initio gene-finding programs can provide such convenience. We hope that this feature will assist with identifying drug targets to select against pathogens. To facilitate its use, we provided a web-based automatic service for ZCURVE 3.0. The users can use the web service to analyze genomes without providing any user information.We updated ZCURVE for http://cefg.uestc.edu.cn/zcurve/ or http://tubic.tju.edu.cn/zcurveb/.Supplementary Data are available at NAR Online.