In childhood, Attention Deficit Hyperactivity Disorder (ADHD) is reliably associated with reduced volume of the striatum. In contrast, striatal abnormalities are infrequently detected in voxel-based morphometry (VBM) neuroimaging studies of adults with ADHD. This discrepancy has been suggested to reflect normalisation of striatal morphology with age and prolonged treatment of symptoms. If so, this would indicate that while striatal abnormalities are linked to symptom expression in childhood, they cannot explain the persistence of these symptoms in adulthood. However, this may not be case. Instead, we hypothesized that the lack of evidence for striatal abnormalities in adult ADHD may reflect poor sensitivity of typical (T1-weighted) neuroimaging to detect subcortical differences. To address this, we acquired both magnetisation transfer (MT) saturation maps optimised for subcortical contrast, and conventional T1-weighted images in 30 adults with ADHD and 30 age, IQ, gender and handedness-matched controls. Using VBM of both datasets, we demonstrate volumetric reductions within the left ventral striatum on MT that are not observed on identically pre-processed T1-weighted images from the same participants. Nevertheless, both techniques reported similar sensitivity to cortical abnormalities in the right inferior parietal lobe. Additionally, we show that differences in striatal iron may potentially explain this reduced sensitivity of T1-weighted images in adults. Together, these findings indicate that prior VBM studies reporting no abnormalities in striatal volume in adult ADHD might have been compromised by the methodological insensitivity of T1-weighted VBM to subcortical differences, and that structural abnormalities of the striatum in ADHD do indeed persist into adulthood. •Striatal grey matter changes in adult ADHD are robustly observed using VBM of MT maps.•These changes are not detectable when using typical T1 images in the same subjects.•Prior negative findings using T1-VBM in adults were thought to reflect therapeutic volumetric normalisation.•We suggest that such findings reflect the insensitivity of T1-VBM to these changes, not structural normalisation. Symptoms fully persist into adulthood in around 15% of individuals, with partial persistence observed in around 65% . RecentlTheoretical accounts of ADHD highlight a central role for deficiencies in dopaminergic signalling within the striatum . AberranWhile striatal abnormalities are reliably observed in VBM studies of childhood ADHD, similar abnormalities are often not detected in studies of adult patients . Meta-anHowever, methodological explanations could potentially account for the absence of strong empirical support from neuroimaging data linking abnormal structural integrity of the striatum to adult ADHD. For example, less statistically stringent region-of-interest (ROI) based approaches can detect striatal changes in adults with ADHD, even though threshold significance for whole-brain statistical correction is not met . ImportaPoor methodological sensitivity may represent a previously unexplored explanation for these apparently contradictory findings: meta-analyses typically have fewer and smaller adult studies to draw on, and the T1-weighted neuroimaging sequences used in these VBM studies have poor subcortical contrast. Consequently the automated segmentation of these structures is less accurate. Problematically, inaccuracy of subcortical segmentation using T1-weighted images may also increase with age. Iron accumulates within the striatum over the lifespan , and hasPrevious data suggest that magnetization Transfer (MT) saturation maps derived from Fast Low Angle Shot (FLASH) 3D multi-parameter provide better subcortical contrast than T1-weighted images traditionally used in VBM . This is22.1Thirty adult patients with ADHD and 30 healthy control participants were included in the study. Patients with ADHD were recruited from specialist ADHD clinics at Sussex Partnership NHS Foundation Trust (SPFT). All had a DSM-IV confirmed diagnosis of ADHD following comprehensive assessment by a consultant psychiatrist specializing in ADHD management. Assessments included semi-structured clinical interview, self-report CAARS, informant  history and completion of Wender-Utah questionnaire, and wherever possible review of school reports. Age, sex and IQ matched controls were recruited though classified advertisements and university mailing lists. All participants were aged 18–65 years. Exclusion criteria included past or current history of any neurological or psychiatric history other than anxiety and/or unipolar depressive disorder currently in remission and past history of significant head injury.ADHD is associated with increased susceptibility to substance use disorders  which haParticipants gave written, informed consent following full explanation of the experimental procedures. Local and national ethical approvals were obtained from Brighton and Sussex Medical School Research Governance and Ethics Committee (12/131/HAR) and the East of England (Hertfordshire) National Research Ethics Committee (12/EE/0256).2.2The Conner's self-report Adult ADHD Rating Scale (CAARS)  was used2.33 resolution . Proton density weighted volumes were acquired with TR = 24 ms, TE from 2.51 to 21.9 ms (eight equidistant bipolar echoes collected), and flip angle (α) of 6°. T1 weighted volumes were acquired with a TR of 19 ms, a TE ranging from 2.51 to 10.82 ms (four equidistant bipolar echoes collected), and a flip angle of 20°. MT weighted volumes were acquired at TR = 30 ms, with the same 4 TEs as the T1-weighted volumes, a flip angle of 12°, and with magnetisation transfer contrast on. These parameters were selected to maximise the contrast between the white matter and the substantia nigra as described elsewhere , with a TE of 3.57 ms, TR of 27.3 ms and a flip angle of 7°. The total acquisition time for the 3 sequences required for MT saturation mapping was approximately 18 min, while the duration of the MPRAGE acquisition was 6 min.All neuroimaging was performed on a 1.5T MRI scanner  with a body coil used for RF transmission, and a 32-channel head coil for NMR signal reception. MT maps were calculated using a multi-parameter protocol adapted from a 3D multi-echo FLASH sequence . Three clsewhere . Standarlsewhere  along th2.4MT) presented in (MT − 1)R1TR − α2/2 where: A is the amplitude of the echo at TE, R1 is the inverse of T1, and α is the imaging flip angle. The resulting maps show a contrast similar to T1-weighted scans and can be entered directly into a VBM pipeline.Based on the model of the signal measured in a MT-weighted spoiled gradient echo acquisition  using a standard pipeline. Images were bias field corrected and segmented into grey matter (GM), white matter (WM) and cerebrospinal fluid (CSF). The segmentation algorithm used adaptive maximum a priori estimations  for each modality. Treatment effects within the ADHD group were examined in SPM using regression analyses, with length of time on medication (in months) as the variable of interest and age, total intracranial volume, BDI and STAI trait scores as covariates. The results of the whole-brain analyses were accepted as significant at p values < 0.05, after false discovery rate (FDR) correction at cluster level .Group comparisons were performed according to the General Linear Model (GLM), controlling for age, total intracranial volumes , and BDI and STAI trait scores. First, we entered both sets of images into a single 2 (imaging sequence) × 2 (diagnosis) second-level mixed measures ANOVA , to test for the presence of a significant interaction, indicating different sensitivity for the 2 modalities ([Control MT > ADHD MT] > [Control T1 < ADHD T1]). In case of significant interaction, we performed 2 separate group comparisons  = 0.20, p = 0.66), IQ  = 0.40, p = 0.53), gender and handedness  = 17.01, p < 0.001) and STAI trait anxiety  = 36.51, p < 0.001).Groups were matched for age  years, ndedness . Mean psndedness . ADHD pa3.2Comparison of MT saturation maps and T1-weighted images highlighted the augmented MT contrast in subcortical regions. The difference of averaged segmentations from each modality also demonstrated enhanced subcortical segmentation accuracy using the MT images  > (Control T1 > ADHD T1)] within a large cluster centred on the striatum and extending into the insula (FDR p < 0.05) and right inferior parietal lobe in ADHD compared to controls (FDR p < 0.05)  ; Table 2 < 0.05) ; Table 2 < 0.05) ; Table 23.4F = 5.60, p = 0.002). Finally, we examined whether these iron differences in ADHD might be influenced by aging or medication use. Left ventral striatal R2* correlated with age , but not time on medication (p > 0.05).To examine whether abnormal iron content may contribute to the methodological insensitivity of T1 to the left hemispheric ventral striatal abnormalities reported, we used a ROI approach to assess R2* as an index for brain iron content. We report increased R2* in ADHD (14.20 ± 2.89) compared to controls (13.26 ± 1.56) in the left ventral striatum  are approximately three times longer than for conventional MPRAGE (with isotropic resolution of 1 mm3). This must of course be factored in when planning a study.Before generalising our results a couple of factors need to be considered. First, this study was conducted at 1.5T and neuroimaging research has increasingly shifted to 3T. T1-weighted image contrast is dependent on field strength. Performance advantages of MT over MPRAGE might therefore differ at 3T. Additionally, MPRAGE is not the only sequence used for high-resolution 3D T1 weighted imaging. Other manufacturers use inversion recovery spoiled GRASS (IR-SPGR). Like MPRAGE, this is based on spoiled fast gradient echoes during inversion recovery. However it differs on acquisition timing and other details, which result in slightly different contrast. Results of automatic segmentation may therefore differ for IR-SPGR. In addition, it is worth noting that scan times for acquisition of full MT saturation mapping  as the current study and those previous to it  have excIn addition to ventral striatal abnormalities, we also report volumetric reduction of the right inferior parietal lobe. These findings support the importance of attention networks in ADHD. In particular, these reductions appeared to be localised within the PFm of the supramarginal gyrus  which co5The present findings encourage caution in the interpretation of evidence acquired from T1-weighted imaging studies of the basal ganglia. We show that, rather than reflecting age or treatment related normalisation, the failure of previous studies to detect striatal differences is likely to reflect, not their absence but methodological weaknesses of using T1-weighted volumes. We show that these striatal abnormalities in adult ADHD are readily detectable using MT saturation VBM. Adoption of such MT neuroimaging protocols offers the potential for enhanced subcortical segmentation, and increased statistical sensitivity to detect subcortical abnormalities. However, before recommending broader adoption of this approach, our findings would benefit from replication in a larger population. Ultimately, leveraging these benefits may prove essential to characterizing the role of subcortical structures in ADHD, as both age and disorder -related iron content changes can systematically bias analyses using typical T1-weighted images.Wellcome Trust Intermediate Fellowship (WT093881MA) to NAH, Brighton & Sussex Medical School & the Dr. Mortimer and Dame Theresa Sackler Foundation. The authors declare no conflict of interest.This work was supported by a


Complex genomic rearrangements (CGRs) consisting of interstitial triplications in conjunction with uniparental isodisomy (isoUPD) have rarely been reported in patients with multiple congenital anomalies (MCA)/intellectual disability (ID). One-ended DNA break repair coupled with microhomology-mediated break-induced replication (MMBIR) has been recently proposed as a possible mechanism giving rise to interstitial copy number gains and distal isoUPD, although only a few cases providing supportive evidence in human congenital diseases with MCA have been documented.Here, we report on the chromosomal microarray (CMA)-based identification of the first known case with concurrent interstitial duplication at 1q42.12-q42.2 and triplication at 1q42.2-q43 followed by isoUPD for the remainder of chromosome 1q (at 1q43-qter). In distal 1q duplication/triplication overlapping with 1q42.12-q43, variable clinical features have been reported, and our 25-year-old patient with MCA/ID presented with some of these frequently described features. Further analyses including the precise mapping of breakpoint junctions within the CGR in a sequence level suggested that the CGR found in association with isoUPD in our case is a triplication with flanking duplications, characterized as a triplication with a particularly long duplication-inverted triplication-duplication (DUP-TRP/INV-DUP) structure. Because microhomology was observed in both junctions between the triplicated region and the flanking duplicated regions, our case provides supportive evidence for recently proposed replication-based mechanisms, such as MMBIR, underlying the formation of CGRs + isoUPD implicated in chromosomal disorders.To the best of our knowledge, this is the first case of CGRs + isoUPD observed in 1q and having DUP-TRP/INV-DUP structure with a long proximal duplication, which supports MMBIR-based model for genomic rearrangements. Molecular cytogenetic analyses using CMA containing single-nucleotide polymorphism probes with further analyses of the breakpoint junctions are recommended in cases suspected of having complex chromosomal abnormalities based on discrepancies between clinical and conventional cytogenetic findings.The online version of this article (doi:10.1186/s13039-017-0316-6) contains supplementary material, which is available to authorized users. Complex genomic rearrangements (CGRs) consisting of two or more breakpoint junctions have been frequently observed during the characterization of nonrecurrent microduplications associated with genomic disorders , 2. The Here, we report on a patient with the co-occurrence of interstitial trisomy at 1q42.12-q42.2 and tetrasomy at 1q42.2-q43, followed by a segmental isoUPD for 1q43-qter, as additional evidence for an MMBIR-based model generating DUP-TRP/INV-DUP rearrangement followed by isoUPD. Detailed molecular genetic analyses at the sequence level revealed the presence of microhomology at two breakpoint junctions of the CGR, probably underlying the formation of the complicated genomic alteration (CGR + isoUPD). Notably, this is the first case of CGR + isoUPD detected in the long arm of chromosome 1. In addition, the pattern of flanking duplications experimentally documented in the present case, namely, a long duplicated segment with a size on the order of megabases at the centromeric junction observed by CMA with a short duplication at the telomeric junction only identified by sequencing of the breakpoint, has not been reported previously.The 25-year-old Japanese male reported on here was the first child of a non-consanguineous healthy mother  and father (details are unclear due to a divorce) with no notable family disease history. After an uncomplicated pregnancy, he had been born at 38 weeks of gestation by a normal delivery. His birth weight was 1958 g (−2.52 SD) and he was introduced into a neonatal incubator to treat intrauterine growth retardation (IUGR) and poor sucking by tube feeding for 20 days, although detailed medical records of his physique are not available. Physical examination at the age of 1 month showed height 46 cm (−3.4 SD), weight 2715 g (−2.6 SD), and head circumference 29.8 cm (−4.6 SD). The abilities to hold up his head, eat solid food, imitate the behaviors of others, and walk alone were recognized at 6 months, 18 months, 2 years and 6 months, and 3 years of age, respectively. The patient had never been able to speak until now, and his comprehension was limited to simple signs, but he recognized various sounds. At 3 years of age, he was diagnosed with the congenital heart defect of tetralogy of Fallot (TOF) but was not treated surgically, although he showed frequent squatting and cyanotic attacks. On physical examination at 24 years and 6 months of age, he showed growth retardation with height 136 cm (−6 SD), weight 28.1 kg (−3.3 SD), and severe mental retardation with a developmental quotient of 5. At 25 years of age, he had TOF, bilateral congenital inguinal hernia, bilateral cryptorchidism, club feet, scoliosis, Chilaiditi’s syndrome, and several facial anomalies, such as thinning of the hair, strabismus, widely spaced eyes, a down-slanted palpebral fissure, low-set ears, a prominent forehead, and a coarse face. He has some missing teeth due to having suffered from periodontal disease. Serial complete blood counts showed thrombocytopenia, and magnetic resonance imaging showed cerebral atrophy especially of the frontal lobe, with enlargement of the ventricles. His karyotype at birth was reported to be normal, but repeatedly performed karyotyping revealed 46,XY,dup(1)(q32.1q42.1),inv(9)(p12q13).This research protocol for this study was approved by the local ethics committee of Tokushima University. Written informed consent for the participation of the patient in this study was obtained from the patient’s mother DNA was extracted from a peripheral blood sample.A high-resolution CMA using the CytoScan HD array  with Chromosome Analysis Suite software  to process the raw data detected a 9.2-Mb trisomy at 1q42.12-q42.2, a 6.7-Mb tetrasomy consisting of the duplication of two haplotypes, each of which probably derives from either the father or the mother, at 1q42.2-q43, and a 8.2-Mb segment with the absence of heterozygosity at 1q43-qter consistent with isoUPD x3,1q42.2q43x4,1q43qterx2 hmz, Fig. in situ hybridization (FISH) studies using bacterial artificial chromosome (BAC) clones located around the region . Two recurrent structural variations within 1q42.12-1qter were identified from the discordant read pairs around the estimated boundary areas by the expected number of reads per region and visual inspection using the Integrative Genomics Viewer. Long-range polymerase chain reaction (PCR) using primers designed around the estimated boundaries , we first performed mate pair next-generation sequencing using the Nextera Mate Pair Sample Preparation Kit and Illumina HiSeq 1500 with 100 paired-end cycles according to the manufacturer instructions . Reads were aligned to the human genome sequence using the Burrows-Wheeler Alignment tool 0.7.12.  and the Catalogue of Parent of Origin Effects , indicated that there are no known imprinting genes within this isoUPD region.Within the isoUPD region, three genes were associated with four autosomal recessive diseases, as determined by a search of the Online Mendelian Inheritance in Man database  and isoUPD, which may be explained by the MMBIR-based mechanism, detected by CMA containing SNP probes, have been reported –7. HowevBecause partial 1q trisomy is a rare disorder and unbalanced chromosomal translocations are often observed with this alteration –16, it iWe report the first case with concurrent CGR (duplications and triplication) + isoUPD in 1q42.12-qter, from an initial diagnosis of interstitial trisomy 1q by conventional karyotyping. Comprehensive cytogenetic and molecular analyses provide additional evidence that DUP-TRP/INV-DUP rearrangement having a unique long proximal DUP structure followed by isoUPD may be generated by an MMBIR-based mechanism. Because it is almost impossible to quantify precise chromosomal copy numbers and detect UPD by conventional karyotyping, molecular cytogenetic analyses using CMA containing SNP probes with additional detailed analyses of the breakpoint junctions in a sequence level are recommended in cases suspected of having complex chromosomal abnormalities based on clinical and cytogenetic findings.Additional file 1: Table S1.BAC clones used in FISH experiments. (DOCX 14 kb)Additional file 2: Table S2.List of primer sets used in PCR and sequencing for junctions of the CGR. (DOCX 14 kb)Additional file 3: Table S3.Autosomal recessive diseases and causative genes aound the isoUPD region. (DOCX 14 kb)


N,N-dimethyl-N-dodecylammonium bromide) C6 and pentamethylene-1,5-bis- C5, and their two neutral analogs, hexamethylene-1,6-bis-(N-methyl-N-dodecylamine) (A6) and pentamethylene-1,5-bis-(N-methyl-N-dodecylamine) (A5). Experiments were performed with Aspergillus brasiliensis, which is used in the standard tests for biocides. The minimal inhibitory concentration (MIC) values for conidia and mycelium were determined using the dilution method. The viability of the conidia was evaluated using the plate count method. The dry mass of the mycelium was determined using the thermogravimetric method. Ergosterol was extracted from the mycelium and evaluated by gas chromatography. The effect of gemini surfactants on fungal morphology was observed using scanning electron microscopy. Cationic gemini surfactants were found to be active at lower concentrations compared to their non-ionic analogues, rapidly reducing the total number of conidia that were able to grow. They also decreased both the ergosterol content in the mycelium and its dry weight. These results suggest that cationic gemini surfactants C6 and C5 could have a wide range of practical applications as active compounds. However, it should be remembered that usage at too low concentrations, below the MIC, will only lead to short-term disturbances in the development of conidia and mycelium.This study investigates the biological activity of two cationic gemini surfactants, hexamethylene-1,6-bis-( Gemini surfactants are amphiphilies composed of two hydrophilic head groups and two hydrophobic hydrocarbon tails, linked by a spacer at the head groups or nearby. They cover a wide hydrophilic-lipophilic balance (HLB) range, and are characterized by very low critical micelle concentrations (CMC), low surface tension (γ) and low minimal inhibitory concentrations (MIC) ,2,3,4,5.In procaryotic or eucaryotic cells, there are several areas susceptible to the activity of biocides, including the cell membrane ,17, whicAspergillus brasiliensis ATCC 16404 of cationic gemini surfactants hexamethylene-1,6-bis- (C6) and pentamethylene-1,5-bis- (C5), as well as that of their neutral analogues, hexamethylene-1,6-bis-(N-methyl-N-dodecylamine) (A6) and pentamethylene-1,5-bis-(N-methyl-N-dodecylamine) (A5). Particular attention is given to conidia and hyphae morphology, growth and ergosterol content.The aim of the present study is to describe the antifungal activity against Aspergillus brasiliensis ATCC 16404 treatment was examined by determining the minimal inhibitory concentration (MIC) for conidial growth. The effectiveness of cationic  and neutral  gemini surfactants on The minimum concentrations at which the cationic surfactants inhibited conidia formation were three-fold lower (0.12 mM) compared to their neutral analogues (0.38 mM). A substantial difference can be observed in the case of mycelium. The ionic compounds suppressed the formation of mycelium at concentrations nearly a 100-fold lower (0.31 mM) than the MICs of the neutral compounds (25–30 mM). The MIC values of the four tested compounds clearly show that conidia are more sensitive than the other morphological forms of mould. The MICs of alkanediyl-α,ω-bis(dodecyldimethyloammonium bromides)  for conidia were 2.5-fold lower than those for mycelium, while the MICs of the neutral surfactants were over 60-fold lower for conidia than for mycelium .In the first stage of the study, the viability of planktonic cells was analysed in medium containing surfactants.p < 0.05) reduction in the number of conidia of 0.8 log. However, after 8 h of biocide activity, their number was reduced significantly, by up to a maximum of 1.3 log  C6 was found to be the most reactive. Its application at 2 MIC concentration prevented the germination of conidia soon after 8 h. In contrast, the other cationic surfactant, C5, caused a 4.3 log reduction in the number of viable conidia. The least effective compound was pentamethylene-1,5-bis-(N-methyl-N-dodecylamine)– A5. Active germination of conidia was detected even after 24 h. Long-term exposure to all gemini surfactants resulted in A. brasiliensis losing the ability to germinate after 48 h. Similar results were obtained by SEM analysis. 1.3 log . The forWhile observing changes in the dry weight of the mycelium, it was noted that after treatment with the surfactants the growth of the strains was inhibited, even at the lowest concentrations (½ MIC). The neutral surfactants A5 and A6 were least effective, as after 4 h of treatment there were no clear differences in mycelial dry mass compared to the samples without biocide. As the concentrations of all the compounds was increased (MIC and 2 MIC), the mycelial dry weight gradually decreased, which may indicate that they had lethal impact . The greAnother important marker associated with the formation of mycelium is ergosterol. Exposure to gemini surfactants, both cationic and neutral, at ½ MIC had little impact on ergosterol content, which was comparable to that in the control sample A,B. The A comparison of the content of ergosterol and the mycelial dry mass over 48 h shows a strong linear correlation (>0.8). However, a negative Pearson’s coefficient was determined for the cultures containing neutral gemini surfactants at MIC . TherefoThe effect of A5 and A6 compounds on mycelium growth was also examined. The results revealed that these compounds did not increase the biomass yield, while an increase was observed in the concentration of ergosterol. Moreover, the mycelium had stiffer membranes, which indicates that they were defending against the penetration of the surfactants into the cells.Binding test results, showing the ability of gemini surfactant to bind to ergosterol, are presented in In the binding assay, the MICs were two times lower in the presence of ergosterolin in the case of neutral gemini surfactants and three time higher in the case of their cationic analogues, in comparison to the control. This may indicate that cationic gemini surfactants, like several monomeric quaternary ammonium salts , possessA. niger and with surfactants at ½ MIC. After 4 h, swelling of the conidia was observed , the species of fungus used in this study, is recommended as a candidate quality control microorganism for an antifungal susceptibility test (EN 1650). The strain was stored on Malt Extract Agar (MEA)  slants at 4 °C. Prior to each experiment, the strain was subcultured in MEA medium at 28 °C for 4–5 days until the conidia were fully mature. Spore suspensions were prepared by washing the conidia from the agar slants using deionized sterilized water with 0.1% Tween 80, and then stirred. The spore concentrations in the initial water suspensions were checked using a Thoma chamber and the concentrations were adjusted to 1.0–2.0 × 107 conidia/mL.N,N-dimethyl-N-dodecylammonium bromide) (C6) and pentamethylene-1,5-bis- (C5), as well as their neutral analogues, heksyleno-1,6-bis(dodecylometyloamina) (A6) and pentyleno-1,5-bis(dodecylometyloamina) (A5). The gemini surfactants were obtained according to procedures described in the literature [The antimicrobial agents used in the study were the gemini surfactants (GS) hexamethylene-1,6-bis- medium , inoculated with 2 mL of conidia and incubated for 48 h at 28 °C. Aliquots of 2 mL of the cationic gemini surfactants 0.05–15 μM/mL) and neutral gemini surfactants (0.75–150 μM/mL) were added. The samples were incubated for another 48 h. A sample without biocide was treated as a control. After 24 and 48 h, the morphology of the mycelium was evaluated macroscopically. The MIC values were defined as the lowest concentrations of the compounds at which the development of mycelium was inhibited in comparison to the control sample –15 μM/mL. The ana6 cfu/mL. The samples were incubated at 28 °C for 2 days. After 4, 8, 24 and 48 h, 1 mL of each mixture was transferred to 9 mL of 0.85% (w/v) saline with a mixture of neutralizers  [A. brasiliensis were counted. The analysis was performed in triplicate.The growth of conidia was evaluated in a liquid medium of 17.8 mL MEB with the addition of 2 mL of gemini surfactant at ¼ MIC, ½ MIC and MIC concentrations, previously designated. In the control sample, 2 mL of sterile, distilled water was used instead of antimicrobial agents. Each test flask was inoculated with 0.2 mL of standardized conidia suspension to obtain a conidia level of 1–2 × 10sulfate) . ViabileThe samples were prepared analogously to those used for MIC determination. Each sample was then supplemented with gemini surfactants at ½ MIC, MIC or 2 MIC. A sample without gemini surfactants served as the control. In order to measure the fungal biomass after 48 h of incubation at 28 °C, the mycelium was filtered through filter paper and dried to a constant weight. The mycelium was placed in a moisture analyzer and the dry weight determined using the thermogravimetric method. The results were expressed as dry weight (in grams) per medium volume. The experiment was performed in triplicate.m/z 337 and 363 ions used for ergosterol quantification. As an internal standard, 7-hydrocholesterol was used to monitor the instrument response and retention time stability.Extraction of ergosterol was performed on untreated mycelium and mycelium exposed to gemini surfactants at ½ MIC, MIC and 2 MIC concentrations, according to the methodology developed by Seitz et al.  and furtQuantitative analysis was performed using Agilent MassHunter software . The detection limit of ergosterol was LOD = 0.0603 µg/mL. The ergosterol concentrations were expressed as micrograms of ergosterol per gram of dry weight. The analysis was performed in triplicate. A. brasiliensis were determined in the presence or absence of 400 μg/mL ergosterol . The MIC values for conidia were determined according to the method described above. The binding assay showed the ability of the surfactants to bind to ergosterol [To find out whether the gemini surfactants bind to sterols in fungal membranes, their MIC values substances against conidia of gosterol .The morphological changes in the conidia and mycelium were observed 4 and 24 h after the addition of the gemini surfactants to the cultures. Samples were analyzed using a Quanta 200  scanning electron microscope. Tests were performed in environmental mode, which allows samples to be studied in their natural state.p < 0.05).The mean results from three independent experiments were calculated, together with their standard deviations. Statistical differences in the data were compared using a one-way repeated measures analysis of variance . Statistical significance was set at 5% . The Pearson correlation has a value between +1 and −1, where −1 is a total negative linear correlation, 1 is total positive linear correlation and 0 is no linear correlation.N,N-dimethyl-N-dodecylammonium bromide) C6 and pentamethylene-1,5-bis- C5 were found to exhibit high antimicrobial efficacy against Aspergillus brasiliensis—one of the moulds least sensitive to the action of antimicrobial agents. Even at a very low concentration of 0.12 mM, these compounds reduced the number of viable conidia. The mycelium was less sensitive to biocides than the conidia. The cationic GS inhibited development of mycelium at 0.31 mM. A reduction in dry weight and a decrease in ergosterol were observed. However, when investigating the effects of antimicrobial agents, it should be noted that ergosterol is not necessarily a good indicator of the effectiveness of antifungals on moulds.In this study, the cationic gemini surfactants hexamethylene-1,6-bis-(The results of this study suggest that cationic gemini surfactants are superior to their non-ionic analogues and could have a wide range of practical applications as active compounds.


Objective: Knowledge and attitude of parents about attention deficit/hyperactivity disorder (ADHD) is an important factor in management of the disorder in children. This study investigates the parents’ knowledge and attitude towards ADHD, its symptoms, diagnosis, treatment and prognosis.Method: In this cross-sectional descriptive study, the subjects were 150 parents (120 mother and 30 father) of ADHD children who were referred to a child psychiatry clinic affiliated in university of social welfare and rehabilitation sciences in Tehran. The diagnosis was made by a child psychiatrist according to DSM-IV TR criteria. The parents completed a 40 items questionnaire that was prepared by the authors and assessed their knowledge and attitude towards ADHD and source of their information.Results: The most common source of parent’s information about ADHD was TV. The parent’s knowledge about the symptoms of the disorder was relatively good. But in regard to diagnosis, treatment and prognosis of the disorder, they have very low knowledge and even incorrect beliefs. The parent’s knowledge significantly correlated with their educational level (p=0.01).Conclusion: In general, knowledge of the parents was low and it can lead to misdiagnosis or mismanagement of this common and important disorder and need to further consideration in terms of educating parents about the disorder in media specially TV. Attention Deficit Hyperactivity Disorder (ADHD) is one of the most common problems among the children who are referred to mental health facilities . This di It is estimated that about 5 to 10% of all the school-age children are diagnosed with ADHD . ParentsIn a study, the commitment of families with an ADHD child was compared in three groups: One group receiving methylphenidate alone, the second, receiving methylphenidate and behavioral interventions, and the third group using placebo and behavioral interventions. Of these three groups of families, 20% discontinued medication within four months and 45% within 10 months and 51% did not complete their behavioral intervention plans : This questionnaire included age and gender of the child, age and education of the parents.The Kiddie-schedule for Affective Disorders and Schizophrenia (K-SADS): This semi-structured interview allows the interviewer to make judgment during the interview . The relThe Knowledge and Attitude Questionnaire: It is a 40-item questionnaire, designed by the researcher, and includes the source or sources of information of parents about ADHD, their knowledge about the symptoms and their attitudes toward the cause, prognosis, treatment and possible side effects of the therapy.Given that this questionnaire was not already present in a standard form, it was prepared as follows:Questionnaire Compilation MethodAfter collecting the survey questionnaires, content and statistical analysis was performed on the opinions, and the necessary changes were applied based on the results.The next step was the pilot study. The edited questionnaire was given to 15 parents of the target population. The purpose of this step was to determine the potential cultural and linguistic ambiguities and inconsistencies of the questionnaire for the audience and to identify the implementation problems. Then the necessary modifications were made to the questionnaire.The final step in compiling the questionnaire was to test its reliability. Cronbach’s alpha method was used to determine the reliability of the questionnaire wherethe value was calculated to be 0.667, indicating acceptable reliability.Statistical AnalysisThe data and the variables were entered into SPSS 18 for statistical analysis. Spearman test was used to evaluate the possible relationship between the demographic factors such as gender and parents’ education level and awareness and attitude. The significance level was set at p ≤ 0.05.EthicsWritten consent was obtained from the parents, and adequate explanation was provided about the study while handing out the questionnaires.The sample included 150 children aged 6-13 years and their parents. Boys constituted 50% of the sample. The average age of children was 9±1.9 years, and 80% of the parents were mothers. Age range of the parents was 25-64 years and the age mode was 33-44 years old (52.1%). Parents’ level of education ranged from elementary education to doctorate. The highest and lowest frequencies were related to diploma (45.1%) and doctorate (0.7%), respectively. In 48.6% of the children, more than 6 months had passed since the diagnosis of ADHD. Among the parents, 88% were familiar with this disorder before participating in the study. TV was the main source of information for the parents (52%). Other sources are shown in In 82.2% of the cases, the teacher had complained to the parents about hyperactivity or inattention of the child. In 74.5% of the cases, children had gone to the physician before participating in the study because of symptoms of hyperactivity. Table 1 shows the rate of false beliefs. 40% of parents believed that Children with ADHD were always hyperactive and 71.3% of them believed that Hyperactivity released the energy of these children and reduced their symptoms.46% of parents reported that Heredity had no role in this disorder, 38.7% of them reported that ADHD resulted from bad parenting practices and 38% of them reported that symptoms of the disorder resulted from the IQ score. 28% said that ADHD disappeared as the child growing up. More than 45% of parents thought that if these children behaved inappropriately, they couldn’t be punished like other children. More than 52% believed that psychological testing was necessary to diagnose this disorder. 11.3% of parents reported that ADHD could be controlled by herbal medicines and 26% of them believed that Ritalin had serious side effects.Table 2 demonstrates the rate of true beliefs about ADHD. More than 66% of parents agreed that if ADHD was not properly treated, other problems could arise, 51.3% of them said that the risk for delinquency is higher in these children and 58% reported that the risk of addiction and other psychiatric problems is higher in children with ADHD. 60% believed that these children could be as successful in their lives as others. 45.3% of them agreed that the symptoms of ADHD could be control by drugs.In 64% of the cases, Ritalin was prescribed for these children, among whom 97.8% had taken the drug and 83.1% were satisfied with its effect. Table 3 shows parents’ level of awareness of ADHD symptoms. Parents’ level of awareness of poor attention and distractibility in ADHD was 84.7%, difficulty in doing or finishing homework was about 86%, difficulty in organizing tasks and activities was about 80% and reluctance to do homework was 84.7%. More than 92% of parents believed that children with ADHD need to monitoring them while doing their homework and 88% of them said that these children only pay attention to what they were highly interested in. 86% of parents said that ADHD associated with hyperactivity and 90% said that these children were restless and fidgety. More than 86% of them reported that children with this disorder were unable to sit still, about 80% believed that they had difficulty in waiting their turn and 73.3% reported that they were unable to play quietly.Given that the items of the questionnaire assessed parents’ level of awareness in various areas such as symptoms, diagnosis, treatment, and prognosis, the subjects’ scores in each are calculated and shown in diagrams .As the diagram displays, the parents’ lowest level of awareness was related to the diagnosis and treatment of the disorder.The Spearman’s correlation test showed that parents’ awareness of and attitude toward ADHD are associated with their level of education. In other words, the higher the education level, the higher the level of awareness (p = 0.01).Moreover, it was found that level of education had no relationship with the level of awareness about the symptoms, etiology and diagnosis of the disorder.However, level of education had a significant relationship with the level of awareness of prognosis and treatment of the disorder. In other words, the higher the level of education, the higher the level of awareness will be .The results revealed that most parents do not have enough information about the disorder. However, awareness about the symptoms of the disorder was relatively good. The rates of correct answers about the symptoms ranged from 73.3% to 92.7%. However, about one third of parents did not believe in the existence of this disorder in their children. More than two-thirds of parents believed that this disorder always accompanies hyperactivity, andthis attitude leads to ignore ADHD, predominantly inattentive type. Parental knowledge of the etiology, diagnosis, prognosis, management and treatment of this disorder was lower than their knowledge of the symptoms.Less than one third of parents believed that this disorder is hereditary. More than one third of the parents believed that bad parenting caused the disorder. In a study in 2007 in Shiraz , 52% andIn addition, teachers of one-fifth of these children did not recognize their problem. In another study by Ghanizadeh in 2006, Iranian teachers' awareness of ADHD has been reported to be low . In a study in Rasht, the majority of primary school teachers had moderate knowledge about ADHD . SimilarIn general, results of this study highlight the importance of informing parents about the symptoms, etiology, prognosis and treatment of ADHD. Increasing parents' knowledge and correcting their myths decrease treatment resistance, leading to ADHD prognosis improvement and complications decrease .This study had some limitations. First, the participating parents themselves detected a problem in their children and asked for an evaluation; therefore, they were probably more knowledgeable than parents in the general population. Second, information level may differ in various therapeutic phases. Therefore performing studies on the general population with larger sample size is required. Furthermore conducting an interventional study is recommended in this area, in which parents' knowledge and attitude are investigated and compared, before and after the provision of a training package. Another limitation was that the study was conducted in one center; therefore, the participants may have the same social class, so the results could not be generalized to the general population, and community sample studies are necessary.


SLC6A3 rs2550948, DRD4 promoter duplication, SNAP25 rs3746544, and ADGRL3 rs1868790. Interactions between the response over time and SLC6A3 and DRD2 were found in the CGI-S and CGAS scales, respectively. The proportion of the variance explained by the models was 18% for the CGI-S and 22% for the CGAS. In this long-term study, the effects of SLC6A3, DRD4, SNAP25, and ADGRL3 on response to treatment reflect those observed in previous studies. In addition, 2 previously unreported interactions with response to treatment over a 12-month period were found (SLC6A3 and DRD2).Attention-deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder in which a significant proportion of patients do not respond to treatment. The objective of this study was to examine the role of genetic risk variants in the response to treatment with methylphenidate (MPH). The effectiveness of MPH was evaluated based on variations in the CGI-S and CGAS scales over a 12-month treatment period using linear mixed effects models. A total of 208 ADHD patients and 34 polymorphisms were included in the analysis. For both scales, the response was associated with time, extended-release MPH/both formulations, and previous MPH treatment. For the CGI-S scale, response was associated with  About 65% of ADHD children are also symptomatic in adulthood, thus suggesting that the disease is chronic3. The symptoms of ADHD include inappropriate levels of attention and/or hyperactivity and impulsivity. In addition, over 65% of ADHD patients present psychiatric comorbidities, such as depression, anxiety, and learning disorders5, all of which affect academic performance and family life, with huge social and economic repercussions7.Attention-deficit/hyperactivity disorder (ADHD) is a very common neurodevelopment condition in children, involving about 5% of children and adolescents10. However, although the clinical condition of most patients treated with MPH improves, a considerable proportion (35%) do not respond to treatment or present adverse effects, thereby making response to MPH variable and difficult to predict12. As a result, clinicians often use a trial-and-error approach based on different types of medication or on titration of dosages to find the best fit for each patient13. It seems clear that identifying accurate predictors of response to medication would be beneficial for clinical practice16.Stimulants are the most effective medications for improvement of ADHD symptoms, and methylphenidate (MPH) is often the first choice owing to its effectiveness and safety, as demonstrated in several studies17, suggesting that ADHD is among the most heritable neuropsychiatric disorders.ADHD is a heterogeneous and complex disorder involving environmental and genetic risk factors. The strong genetic component of ADHD is supported by family, twin, and adoption studies, which have found a mean estimated heritability of 76%18. As many of these genes play a role in the mechanisms of action of psychostimulants, there is a high probability that they are also associated with response to treatment19. The results of pharmacogenetic studies of ADHD are variable and inconclusive19. The objective of this study was to examine the role of risk genes in the response to MPH in children with ADHD and to evaluate the effectiveness of the drug over 12 months of follow-up.Predictions based on genetic factors are the basis of pharmacogenetic testing. Numerous candidate genes have been associated with an increased risk of ADHDA total of 238 Caucasian ADHD patients were included in the initial step of the study. After the quality control procedure, 208 patients remained in the final analysis, and 176 completed the 12-month follow-up. Fifty-seven (27%) were treatment-naive. Among the MPH-experienced patients (151), 41% reported a poor or partial response at the time they entered in the study. The demographic and clinical characteristics of the cohort are shown in Table For all variants, the call rates of genotype per-marker were higher than 96%; therefore, no polymorphisms were excluded from the analysis. The fixed effects of the models are summarized in Table Sex, age, and ADHD subtype were not significant as covariates for any of the efficacy models. We found a significant improvement in the response over time in the CGI-S and CGAS scales. In addition, previous MPH treatment and extended-release MPH alone/both formulations were positive predictors of response according to CGI-S and CGAS. Dosage was a significant factor only in the CGAS effectiveness model.DRD4 promoter duplication, and ADGRL3 rs1868790, which were associated with significant impairment, and a dominant effect in SLC6A3 rs2550948 and SNAP25 rs3746544, with a significant improvement in the symptoms. Moreover, an interaction was found between the SLC6A3 intron 8 VNTR and treatment over time . Treatment-experienced patients were better responders, possibly because of selection bias: they continued on treatment because they were responding to the treatment, with no safety issues. Adjusting models by treatment status, we controlled which polymorphisms were associated with the response regardless of the confounding variable. The resulting data suggest that the extended-release formulation could have led to an improvement in the response to MPH, for example, by improving adherence to treatment21. Dosage was only a significant factor in the CGAS model. In CGI-S, statistical significance may not have been reached because of insufficient sample size.The values for CGI-S and CGAS improved significantly over time, although with a modest effect size, maybe because 73% of patients were on treatment before the beginning of the study. ADHD subtype was not a significant factor in response to treatment, according to previously studiesSLC6A3 and DRD4, which have been widely associated with changes in response to MPH22. On the other hand, we failed to replicate the most studied variant in SLC6A3 (VNTR 3′ UTR), thus reflecting previous negative results on the implication of this variant and the pharmacogenetics of ADHD23. Consequently, other polymorphisms of this gene must be explored. In this context, we found a previously unreported association between a promoter variant (rs2550948) and response to MPH. In the case of DRD4, once again, we did not replicate the most studied association, which was with the VNTR exon 3 variant19. The results of studies that investigate the implication of this variant in response to MPH are also conflicting19. However, we did find an association between the DRD4 tandem duplication polymorphism in the promoter region and response to MPH. No significant effect on response to MPH was found with this variant in 2 small-scale studies of children24 and adults25.As for the genetic component, our results showed the implication of the genes SNAP25 and ADGRL3. Although the role of these genes in susceptibility to ADHD is widely studied, they have been less largely studied in terms of the effectiveness of MPH. Contini et al.26 evaluated the same polymorphism in SNAP25 (rs3746544) but identified no effect in adults with ADHD. Elsewhere in the literature, inconsistent findings were found between ADGRL3 and response to MPH. The marker rs6551665 had previously been associated with the response to MPH28. Arcos-Burgos et al.27 reported that the G allele was associated with a good response, whereas Labbe et al.25 found that it was associated with a poor response. The divergent results could be explained by differences in sample subtypes or outcome measures29. In addition, the marker rs6858066 was associated with a better response30. In the present study, the associations between rs6551665 and rs6858066 and response to MPH were not statistically significant. In contrast, we established a previously unreported statistically significant association between rs1868790 and response to MPH.A statistically significant association with response to MPH was found in the neurodevelopmental genes SLC6A3 and DRD2 genotypes were associated with different degrees of improvement in ADHD symptoms. For both genes, a faster response effect was observed during the first 3 months, respectively, in patients with the genotypes 6/− or −/− and T/C or C/C. To our knowledge, this is the first report of differences in the response to MPH over time in these genes.Furthermore, our pharmacogenetic study suggested that 32. Results are also difficult to reproduce because of the definition of response in assessment scales. In fact, there is no clear consensus on the best approach to find objective and reliable measures of response to treatment33.Our study reflects the considerable difficulty in replicating pharmacogenetic association studies in ADHD. The results reported are conditioned by polymorphisms analyzed per gene or model of inheritance evaluated. Furthermore, they depend on factors that influence the response, such as phenotype, concomitant treatment, and sample characteristics , which are not always taken into account34. Ours is the first 12-month study of the pharmacogenetic of response to MPH in children, and we provide much more relevant clinical information than short-term studies. The literature contains little evidence of the long-term effects of medication owing to the difficulty in follow-up and the low persistence on therapy rate35.The strengths of our study are that we evaluated response using 2 scales that provide more detailed information and thus reveal the heterogeneity in response effect. Moreover, we evaluated the response to MPH under conditions of routine clinical practice, thus highlighting the role of genetic factors in real-world situations36. By contrast, this approach has to cope with the risk of serious discrepancies between informants, which ultimately will hinder patient’s assessment37.Some limitations of this study should be considered. First, the determination of the scores for the clinical response through the scales CGI-S and CGAS, which are recorded by doctors, despite are based on what parents and children report, is not free of subjectivity risk of the doctor. However, having scales assessing what parents and patients believe with regards to the overall functioning of patients would be of interest in the assessment of the treatment effectiveness, since it would be free of evaluator’s biasAnother limitation of the study to be considered is that 73% of patients were on treatment before beginning the study. Although we have adjusted the models by previous treatment status, experienced patients started with better response at baseline visit, and for that reason they had limited clinical improvement at the end of the study period.SLC6A3, DRD4, SNAP25, and ADGRL3 in the response to MPH, thereby supporting several previous studies of these genes. We also found interactions between response to treatment over 12 months and genotypes of SLC6A3 and DRD2. When all the covariates are taken into account, the models explain around 20% of the response to MPH. Therefore, other genetic or non-genetic factors must be involved in the variability of response to MPH. More research is required to find pharmacogenetic variants that could help to establish the best treatment regimen.In conclusion, we report moderate effects of the genes 38. All patients were evaluated, taking into account different sources of information .We performed a prospective, observational study of unrelated Spanish Caucasian patients with ADHD aged 6 to 18 years who were enrolled and clinically assessed by psychiatrists and pediatricians at Fundación Jiménez Díaz University Hospital. ADHD was diagnosed following the Diagnostic and Statistical Manual of Mental DisordersTo be included, patients had to have ADHD, be Spanish and Caucasian, be treatment-naive or have been treated with only MPH at baseline, and have been receiving MPH at least from baseline onwards. Patients could receive one of 2 formulations: (a) the immediate-release formulation (Rubifen) or (b) the extended-release formulation (Medikinet and Concerta)/ both formulations. Patients treated with medication (in addition or instead of) other than MPH were excluded.Doses and type of MPH formulation were individually prescribed according to the summary of product characteristics and the clinical criteria of the psychiatrist and were adjusted during follow-up visits until the desired therapeutic effects were obtained.39 and the Children’s Global Assessment Scale (CGAS)40. CGI-S provides a global evaluation of the severity of illness at the time of evaluation using a 7-point scale ranging from 1  to 7 (maximum impairment). CGAS is used to rate the general functional status in children and adolescents using a numerical scale, with values ranging from 1 (need for constant supervision) to 100 (superior functioning).Clinical effectiveness was evaluated using the Clinical Global Impression-Severity (CGI-S) scaleDuring the assessment period, the following side effects were evaluated: loss of appetite, insomnia, gastrointestinal problems, headaches, cognitive, emotional and behavioral disturbances.Clinical assessments were performed at baseline and after 3, 6, and 12 months of treatment with MPH.The study protocol was reviewed and authorized by the Research Ethics Committee of the IIS-Fundación Jiménez Díaz University Hospital. The study was carried out in accordance with the ethical principles that are reflected in the Declaration of Helsinki. Before recruitment, once the study objectives and procedures had been detailed, parents or legal guardians signed a written informed consent.Peripheral blood lymphocytes or saliva were used to obtain genomic DNA, employing an automatic DNA extractor  or the Oragene DNA self-collection kit , respectively, according to the manufacturer’s recommendations. DNA concentration and sample quality were evaluated through a spectrophotometer .Thirty-four polymorphisms from 18 genes were chosen according to their significance in the literature. All genes were previously associated with ADHD Table .Table 3D® 480 software, version 1.5. .Single-nucleotide polymorphisms (SNPs) were genotyped through the TaqMan on-demand or pre-designed SNP genotyping assays system, according to the company’s instructions . We run PCR and allelic discrimination assays in a LightCycler 480  and we analyzed them using the LightCyclerVariable number tandem repeat (VNTR) polymorphisms were identified using fragment analysis. PCR products were displayed on an ABI Prism 3130xl DNA sequencer , and we analyzed the results by means of GeneMapper software, version 4.0 . Primer sequences and PCR conditions can be provided upon request.18, as follows: SLC6A3 3′UTR VNTR , SLC6A3 intron8 VNTR , DRD4 promoter duplication , DRD4 exon3 VNTR , SLC6A4 promoter VNTR , and SLC6A4 intron2 VNTR .For each VNTR polymorphism, subjects were categorized into 3 genotypes according to the previously described risk allele41. Models were constructed using the lme function from the nlme package in R.During the quality control procedure, genotype call rates per sample and per polymorphism < 80% were excluded from the analysis. The outcome measures of treatment with MPH were evaluated as quantitative data according to CGI-S and CGAS. Analyses of the effects of different genotypes on response to treatment over time were performed using linear mixed-effects models. These mixed-models are useful for repeated-measures analyses where follow-up times are not uniform across all subjectsHTR2C and STS genes) were analyzed taking X inactivation into account according to Clayton’s approach42.As in other genetically complex diseases in which the model of inheritance is uncertain, the analyses were performed under the assumption of dominant, recessive, codominant, and additive models. The best model was selected based on the one with the lowest Akaike information criterion (AIC). Data for variants located on chromosome X , and dosage were also entered into the models as potential explanatory covariates. Statistical significance for main effects and interactions was assessed using the ANOVA F-test and set at a 2-tailed p value of 0.05. In these multivariable models, the effect size of the associations was measured by the coefficients of the models “β”.43.The proportion of the variance explained by the model was assessed using Omega Squared


The SLY1 gene encodes the F-box subunit of an SCF E3 ubiquitin ligase needed for GA-triggered proteolysis of DELLA repressors of seed germination. DELLA overaccumulation in sly1-2 seeds leads to increased dormancy that can be rescued without DELLA protein destruction either by overexpression of the GA receptor, GA-INSENSITIVE DWARF1b (GID1b-OE) (74% germination) or by extended dry after-ripening . After-ripening of sly1 resulted in different transcriptional changes in early versus late Phase II of germination that were consistent with the processes known to occur. Approximately half of the transcriptome changes with after-ripening appear to depend on SLY1-triggered DELLA proteolysis. Given that many of these SLY1/GA-dependent changes are genes involved in protein translation, it appears that GA signaling increases germination capacity in part by activating translation. While sly1-2 after-ripening was associated with transcript-level changes in 4594 genes over two imbibition timepoints, rescue of sly1-2 germination by GID1b-OE was associated with changes in only 23 genes. Thus, a big change in sly1-2 germination phenotype can occur with relatively little change in the global pattern of gene expression during the process of germination. Most GID1b-OE-responsive transcripts showed similar changes with after-ripening in early Phase II of imbibition, but opposite changes with after-ripening by late Phase II. This suggests that GID1b-OE stimulates germination early in imbibition, but may later trigger negative feedback regulation.While widespread transcriptome changes were previously observed with seed dormancy loss, this study specifically characterized transcriptional changes associated with the increased seed dormancy and dormancy loss of the gibberellin (GA) hormone-insensitive  The evolution of seeds that carry plant embryos in a state of arrested growth was critical to the success of land plants and to agriculture . Becauseper se, embryo emergence from the seed, and includes the post-germinative processes of cell division, seedling growth, and the majority of nutrient mobilization. Dormant seeds undergo the Phase II processes of cellular restoration, DNA repair, initiation of respiration, and some RNA transcription and protein translation, but never reach Phase III. Differences in dormant seed Phase II processes likely prevent germination per se. Since dormant seeds will not complete the germination process, we will refer to ungerminated seeds undergoing Phase I and II as “imbibing seeds.” While imbibing in the cold breaks dormancy, Arabidopsis thaliana seeds do not reach Phase III during this cold stratification and eventually enter secondary dormancy ,15per sega1-3, DELLA protein levels are high and block GA responses including stem elongation, cell division, the transition to flowering, and seed germination . Th. Thsly1-sly1-2 after-ripening were examined at the two imbibition timepoints by TAGGIT gene ontology analysis using categories defined by relevance to seed germination [sly1-2 after-ripening was differentially enriched for categories associated with early Phase II, including DNA repair, protein degradation/inhibition of protein degradation (involved in mobilization of seed storage proteins), and protein translation , cell division, cell-wall modification (needed for seedling emergence and growth), and photosynthesis  between the 0h and 12h timepoints, suggesting that negative regulation of germination sly1-2 ARvsD datasets using the Plant GeneSet Enrichment Analysis (PlantGSEA) tool that identifies TFs based on enrichment of their experimentally identified targets in a differentially-regulated gene set (Drosophila melanogastera complex) and PIF1/PIL5 targets at both 0h and 12h. E2Fa–DPa may promote germination per se because it is a positive regulator of the cell division cycle, and stimulates gene expression involved in DNA replication, nitrate assimilation, and cell wall biosynthesis [Transcription factors (TFs) possibly controlling after-ripening-regulated transcription were identified in the ynthesis ,55. PIF1ynthesis ,57. Therynthesis . In addiynthesis . Interessly1, GA, or DELLA  are called GA-DOWN, and those up-regulated by DELLA (higher in ga1-3 than in the ga1-3 della) are called DELLA-UP. DELLA-UP transcripts are generally also GA-DOWN [sly1-2 mutants over-accumulate DELLA, DELLA-UP genes are expected to be up-regulated in sly1-2 (sly1-UP), and vice versa. Indeed, out of the 3213 DELLA-regulated and the 3559 0h sly1-regulated genes, only 72 genes showed opposite DELLA and sly1-regulation. If sly1-2 after-ripening leads to down-regulation of DELLA by a non-proteolytic mechanism, then we would expect DELLA-UP genes to be after-ripening (AR)-DOWN, and vice versa. Of the 18 putative DELLA targets, 8 showed significant differential AR-regulation, and of these 6 AR-DOWN targets were also DELLA-UP and/or sly1-UP at 12h. Moreover, the DELLA target GID1a was AR-DOWN/DELLA-UP at 0h and 12h. The DELLA-UP target XERICO was AR-UP at 0h and AR-DOWN at 12h of imbibition, suggesting that its regulation changes over imbibition time. The MYB TF At3g11280 was DELLA-UP and AR-UP.Zentella et al.  identifior DELLA . Our rea-3 della . For con GA-DOWN ,49. SincGID1b-OE rescued sly1-2 germination as well as after-ripening, it was associated with far fewer changes in transcript accumulation, including only 23 transcripts over the 0h and 12h timepoints , and At2g46250. Given that it is overexpressed on the 35S promoter, GID1b served as an internal control, showing the strongest up-regulation in GIDvsD at 0h and 12h  and At2g46250, may share a promoter as they are oriented head-to-tail on chromosome 2. The putative EF hand domain protein At1g21630 was GID1b-UP at 0h and 12h in both the GIDvsD and sly1-2 GIDvsAR comparisons (logFC > 2.68).Although mepoints . In the GID1b overexpression rescues germination by down-regulating DELLA repressors via protein-protein interaction [GID1b-OE was expected to mainly cause changes in DELLA-regulated transcript levels. Indeed, only 3 of the 22 GID1b-OE-regulated genes were not found to be GA-, DELLA-, or sly1-regulated  and At2g44800 are MYB-type TF GL1 targets, while putative nuclear transport factor 2 family member At5g01740 is a TF GL3 target based on PlantGSEA. Both GL1 and GL3 are known DELLA-interacting proteins [GID1b-OE may alter expression of these genes by down-regulating DELLA.In the non-proteolytic GA signaling model, eraction ,42. Thusegulated ; S4 Fig.pression . The EXPproteins , supportGID1b-OE both rescue sly1-2 germination without DELLA destruction, we examined whether they were associated with similar transcriptome changes. Many more genes were differentially regulated with after-ripening than with GID1b-OE (GID1b-OE-regulated transcripts (GIDvsD) showed similar AR-regulation (sly1-2 ARvsD) at the 0h than at the 12h imbibition timepoint. Of the 13 GID1b-OE-regulated genes at 0h, 1 GID1b-OE-UP and 7 GID1b-OE-DOWN genes showed similar AR-regulation  showed similar AR-regulation  were GID1b-OE-DOWN, but up-regulated in 12h WTvsD, and with sly1-2 after-ripening. Thus, many of the 12h GID1b-OE differentially-regulated genes actually showed opposite regulation from other germinating versus non-germinating comparisons. This may suggest a negative feedback response.We expected to find agreement between comparisons of lines that germinate to lines that do not germinate in late Phase II, including the 12h GIDvsD, the 12h WTvsD, and both the 12h parisons . Indeed,HBT , a constitutive control gene [er ARvsD, GASA2 and HSFA9 were AR-DOWN both in the 12h sly1-2 ARvsD microarray and RT-qPCR analyses. RT-qPCR also confirmed differential regulation of GASA2 and HSFA9 by GID1b-OE at 12h  and XERICO significantly AR-DOWN  in the Ler ARvsD comparison [GID1b-OE-down-regulation of XERICO and GASA4 at 24 h of imbibition following cold stratification [GID1b-OE-regulated in our microarray study based on an FDR adjusted-p-value < 0.05, they both exhibited a downward trend at the 12h timepoint. By RT-qPCR analysis at 12h, XERICO showed a non-significant downward trend, whereas GASA4 was significantly GID1b-OE-DOWN  expression levels were positively correlated with higher seed dormancy [DOG1 mRNA levels were lower with Ler after-ripening , and higher in dormant sly1-2 than in WT at 0h . Although DOG1 expression at 12h was very low, a similar expression pattern was observed by RT-qPCR.In previous studies, dormancy ,62. ConsWT at 0h ; S6 Fig.SLY1 (logFC: 5.70 (0h), 5.34 (12h); Figs sly1-2 transcript did not interfere with detection by RT-PCR primers or by the 11 SLY1 Affymetrix ATH1 probes. The only gene with a SLY1-like expression pattern was DDB1b, a subunit of the ultraviolet-damaged DNA-binding protein complex (logFC: 3.90 (0h), 3.69 (12h)) [SLY1 and DDB1b were more highly expressed in WT and in sly1-2(AR) than in sly1-2(D), suggesting that SLY1 and after-ripening stimulate expression of these genes. Both of these transcripts were DELLA-UP (The gene with the largest fold increase with after-ripening was 9 (12h)) . Both SLsly1-2 were designed to ask specific questions about DELLA-regulated and DELLA-independent seed dormancy loss. Although sly1-2 cannot degrade DELLA repressors via the ubiquitin-proteasome pathway, non-proteolytic GA signaling allows some rescue of seed dormancy by long after-ripening or by GID1b overexpression [sly1-2 ARvsD to Ler ARvsD revealed that over half of the transcriptome changes with after-ripening depend upon DELLA destruction  or wild-type Ler (33%) were known GA-regulated genes, in spite of the larger total number of transcriptome changes in Ler  the DELLA-interacting TFs, PIF1/PIL5 and PIF3, 2) the GA- and light-regulated TF, HY5  GID1b-OE rescues sly1-2 germination via a non-transcriptional mechanism, 2) GID1b-OE rescue of sly1-2 germination results from transcriptional changes during seed development rather than during seed imbibition, or 3) the 22 GID1b-OE-regulated transcripts are particularly critical regulators of dormancy loss. While future work will need to examine whether loss or gain of GID1b-OE-regulated gene function alters seed germination potential, it is interesting to consider the functional categories of these GID1b-OE-regulated transcripts.Very few transcriptional changes occurred with ripening . This reGID1b-OE-regulated transcripts encode ABA-responsive seed-related regulatory genes  RAB28 were GID1b-OE-up-regulated. Both play roles in seed desiccation tolerance and are regulated by the seed-specific TF ABI3 of the ABA signaling pathway [RAB28 increased germination rates under both standard and osmotic stress conditions [HSFA9 is not heat stress-responsive, and is the only one of 21 Arabidopsis HSFs that is solely expressed in seeds starting late in seed development [GID1b-OE-up-regulated transcript, At2g34740, encodes a protein phosphatase type 2C (PP2C). Although not well characterized, At2g34740 was induced by ABA and repressed in the ABA-signaling SNF1-related protein kinase 2 (SnRK2) gene triple mutant, srk2D srk2e srk2i [Several of the ry genes . The TF  pathway –77. Consnditions . HSFA9 ielopment . HSFA9 a2e srk2i . This su2e srk2i ,81.SLY1 expression in seedlings [GID1b-OE-down-regulated non-symbiotic hemoglobin AHb1 modulates NO accumulation in seeds and regulates expression of ROS metabolism and ABA signaling genes [GID1b-OE-down-regulated genes were implicated in oxidative stress regulation, including GASA4, AHb1, BAG6, CYSa, KUOX1, and the oxidoreductase At2g44800 [Both nitric oxide (NO) and reactive oxygen species (ROS) have been implicated in stimulating seed germination (reviewed in ). NO regeedlings . The GIDng genes ,86. ROS t2g44800 . Oxidatit2g44800 ,90–92. Mt2g44800 ).GID1b-OE-regulated transcripts  was GID1b-OE-DOWN, while the calcium-binding EF hand family protein (At1g21630) was one of the most strongly GID1b-OE-UP transcripts , whereas most of the genes differentially regulated with GID1b-OE showed the opposite regulation with after-ripening in late Phase II (12h)  Figs  and 8. Tbidopsis . The nining Figs  and 8.GID1b-OE rescue sly1-2 seed germination by down-regulating DELLA through increased GID1-GA-DELLA complex formation, then we would expect GID1b-OE- and AR-regulated genes to be similarly regulated by GA and inversely by DELLA. While this held true for both sly1-2 and Ler AR-regulated genes, it did not always hold true for GID1b-OE particularly in late Phase II  . Only 7LLA Figs , 7 and 8LLA Figs  and 8. ILLA Figs . Thus, msly1-dependent and independent aspects of seed dormancy and dormancy release. Previous work also found that there were ABA-dependent and independent transcriptome changes associated with dormancy and after-ripening [vice versa [XERICO, a positive regulator of ABA biosynthesis [There are many genetic mechanisms contributing to seed dormancy and dormancy release. This study specifically defined DELLA/ripening . Of the ripening ,8). Cleace versa ,42. Moreynthesis . Transcrynthesis ,7. The rsly1-2 seeds  wild-type, sly1-2 and sly1-2 GID1b-OE in the Ler background were previously described [GID1b-OE allele is an HA:GID1b translational fusion driven by the strong cauliflower mosaic virus 35S promoter in the sly1-2 background. Arabidopsis used for all experiments were cultivated side-by-side in a Conviron® growth chamber according to McGinnis et al. [sly1-2(AR) sample was a separate sly1-2 batch grown in advance under the same conditions and stored at room temperature for 19 mo.escribed ,41. The N-morpholino)ethanesulfonic acid), pH 5.5. The germination of the seed batches used for microarray analysis were scored daily for 8 d during incubation in the light at 22°C following cold stratification for 4 d at 4°C. Germination and testa rupture were also scored at the 12 h imbibition timepoint used for microarray analysis.For the germination screen , 80 to 1For RNA extraction, 20 mg of dry seed per sample was sown on filter paper moistened with sterile 0.5× MS salts buffered with 5mM MES, pH 5.5. Seeds were collected at two timepoints: 1) a 0h timepoint harvested immediately after cold stratification for 4 d at 4°C in the dark, and 2) a 12h timepoint incubated 12h in the light at 22°C following the 4 d cold stratification at 4°C. RNA isolation was performed using a phenol-chloroform extraction developed for tough tissues such as Arabidopsis seeds based on Oñate-Sánchez and Vicente-Carbajosa  with an er wt (2 wk old), dormant sly1-2 (2 wk old), after-ripened sly1-2 (19 mo old), and sly1-2 GID1b-OE (2 wk old). For each biological replicate, 2 μg of RNA was submitted to the Molecular Biology and Genomics Core Laboratory at Washington State University for synthesis of biotin-labeled cRNA, hybridization to ATH1 Arabidopsis microarray chips (Affymetrix), and chip scanning (http://crb.wsu.edu/core-laboratories/molecular-biology-and-genomics-core). Data analysis of raw CEL files was performed using the LIMMA package as part of the Bioconductor suite of tools in the R statistical programming environment [http://www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-4782 [1) measurement of skew was calculated using the equation: G1= g1* sqrt(n(n-1)) / (n-2) [Affymetrix ATH1 chip  oligonucleotide-based DNA microarray analysis was performed using three biological replicates at the 0h and 12h imbibition timepoints, for each genotype: Lironment –112. MicTAB-4782 . BackgroTAB-4782 ,115. For / (n-2) .http://affymetrix.arabidopsis.info), respectively, and reanalyzed by the same methods to allow fair comparisons to the current study. In the Cao et al. [er were freshly harvested from yellow siliques, and the after-ripened Ler were stored dry for 2 mo. Note that the freshly harvested Ler were dormant, whereas the 2 week after-ripened Ler wt seeds in Raw datasets from Cao et al.  and Carro et al.  study, so et al.  study, tMicroarray analysis measures relative steady-state transcript levels, not active gene transcription. Thus, differential regulation could result from differences at the level of gene transcription or at the level of transcript stability. References to differential regulation or expression indicate differential regulation, but do not necessarily indicate differences in gene transcription. When referring to the differential regulation in A relative to B, or AvsB, up in AvsB means up-regulated in A (or down-regulated in B), whereas down in AvsB means down-regulated in A (or up-regulated in B).GID1b was not causing artifactually low significance for other genes as a result of the multiple comparisons adjustment, significance testing was repeated with GID1b removed. This reanalysis of the data did not result in any change to the number of differentially-regulated genes in GIDvsD comparison at either imbibition timepoint. To determine whether differences in signal intensity might account for differences in the number of differentially-regulated genes, we also graphed signal intensities for sly1-2 ARvsD and GIDvsD at both imbibition timepoints . The microarrayTools package includes four functions: getProbeID, venndia, TAGGITontology, and TAGGITplot. getProbeID can be used to convert a gene or list of genes from AGI identifiers (e.g. At4g24210) or short gene names (e.g. SLY1) to ATH1 Affymetrix probe ids. Overlaps between comparisons were identified using the venndia function , which identifies gene categories based on enrichment within a set of differentially regulated genes, TAGGIT quantifies enrichment for genes in a set of 26 specific categories defined for their involvement in seed dormancy and germination . This lihttp://structuralbiology.cau.edu.cn/PlantGSEA/) with the Transcription Factor Targets (TFT) dataset identified known gene targets of transcription factors within differentially regulated genesets [in vivo DNA-binding techniques such as ChIP coupled with DNA sequencing (ChIP-seq) and ChIP followed by microarray hybridization (ChIP-chip). The TFT dataset includes both targets of transcription factors and of transcriptional regulators that interact with a DNA-binding proteins, both referred to as TFs in this work. TFTs are considered “confirmed” if their function was proven by two or more approaches including in vivo evidence. Significantly enriched targets are presented in The web-based PlantGSEA toolkit , and RT-qPCR analysis was conducted using the Lightcycler LightCycler® FastStart DNA Master SYBR Green I kit (Roche). Primers were designed using the QuantPrime online tool (http://www.quantprime.de), with the exception of the previously published HSFA9 and DOG1 primers  control [HBT control was selected based on the Graeber et al. [RT-qPCR analysis for microarray validation was performed using gene-specific primers for select differentially regulated genes: and SLY1 ; S6 Fig. primers , and 10  control . The HBTr et al.  comparisr et al. ).S1 Figsly1-regulated at 0h (based on DvsWT) with those that are oppositely GA-regulated at 0h in Cao et al. [ga1-3 vs WT). (B) Overlap between genes differentially regulated with after-ripening of sly1-2 at 12h with those of Ler at 24 h (12h sly1-2 ARvsD ∩ 24 h Ler ARvsD). (C) Overlap between genes inversely regulated by the sly1 mutation at 0h with genes differentially regulated with sly1-2 after-ripening at 0h (0h WTvsD ∩ 0h sly1-2 ARvsD). (D) Overlap of 12h sly1-2 and 24 h Ler after-ripening-regulated genes with those inversely regulated by the sly1 mutation at 12h. The sly1-regulated genes are likely also DELLA-regulated. Throughout this study, up- (red) and down-regulated (blue) genes are based on FDR cutoff of p < 0.05.(A) Overlap between genes that are DELLA/o et al.  (0h DvsW(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Figer wt vs ga1-3 comparison, (B) DELLA-regulated genes determined by the ga1-3 vs ga1-3 4x della comparison, and (C) total GA-regulated and total DELLA-regulated genesets compared. The value on the x-axis shows the percentage of the total differentially regulated genes within a dataset.Using reanalyzed datasets from Cao et al.  with tim(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 FigSignificant values are indicated in black text. Significance is based on pairwise t-tests with Bonferroni-Holm correction for multiple comparisons with α = 0.07.(PDF)Click here for additional data file.S7 Figsly1-2 at 12h with those that are GA-regulated based on the Ler wt vs ga1-3 comparison from Cao et al. [sly1-2 ARvsD ∩ 0h WT vs ga1-3). (B) Overlap between genes differentially regulated with after-ripening of Ler wt at 24 h . (C) Overlap between genes differentially regulated in sly1-2(D) from 0h to 12h of imbibition with those of sly1-2(AR) from 0h to 12h of imbibition (sly1-2(AR) 12hvs0h ∩ sly1-2(D) 12hvs0h). Significance based on an FDR cutoff of p < 0.05.(A) Overlap between genes differentially regulated with after-ripening of o et al.   with th(PDF)Click here for additional data file.S8 Fig2 scale) were compared. A, 0h sly1-2(AR) (y-axis) versus 0h sly1-2(D) (x-axis). B, 12h sly1-2(AR) (y-axis) versus 12h sly1-2(D) (x-axis). C, 0h sly1-2 GID1b-OE (y-axis) versus 0h sly1-2(D) (x-axis). D, 12h sly1-2 GID1b-OE (y-axis) versus 12h sly1-2(D) (x-axis).Red circles indicate genes that were significantly differentially regulated . Black circles were plotted with transparency of 25% such that darker areas indicate a larger accumulation of dots at the same location. Normalized probe intensities (log(PDF)Click here for additional data file.S9 Fig(PDF)Click here for additional data file.S10 Fig(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Tableer ARvsD  the down-regulated fraction of 0h DvsWT, 2) the downregulated fraction of 12h DvsWT, and 3) up-regulated fraction of 24 h L et al., ).(XLSX)Click here for additional data file.S4 Tablesly1-2 after-ripening at 0h and 12h.Transcription factor targets differentially regulated with (XLSX)Click here for additional data file.S5 TableUsed by the TAGGITontology R function to classify genes into TAGGIT gene ontology categories based on lists of AGI locus identifiers, and lists of search terms corresponding to each category, respectively.(XLSX)Click here for additional data file.


To systematically review the literature on the prevalence and pharmacological treatment of ADHD in looked-after children (LAC).LAC are a very challenging population from a clinical and psychosocial standpoint, with higher mental health needs compared to non LAC. To date, no systematic review on the prevalence of ADHD, and its treatment, in LAC is available.We searched Pubmed, PsycInfo EMBASE + EMBASE CLASSIC, OVID Medline and Web of Science up to November 9 th, 2016. We found 24 papers meeting our criteria. The vast majority of the retained studies are from the USA and show rates of ADHD and of its pharmacological treatment substantially higher in LAC than those reported in national estimates. Future studies from countries other than the USA, aiming to understand the most cost-effective strategies, in the short as well as long term, to manage symptoms of ADHD in LAC are needed.The online version of this article (doi:10.1007/s40474-017-0116-z) contains supplementary material, which is available to authorized users. Attention-deficit/hyperactivity disorder (ADHD) is characterized by impairing, persistent and pervasive levels of inattention and/or hyperactivity/impulsivity . With anADHD is a complex and heterogeneous disorder in terms of brain correlates, characterized by a dysfunctional interplay among several neuronal networks . Its aetAvailable treatments for ADHD include pharmacological and non-pharmacological interventions. Medications for ADHD comprise psychostimulant (e.g. methylphenidate and amphetamine derivatives) and non-psychostimulant drugs . A large body of evidence shows that ADHD medications are efficacious, at least in the short and medium terms, to control ADHD core symptoms , 14. NonWhilst ADHD has been extensively investigated in particular populations, such as preterm children or individuals in prisons, it has been neglected in others, such as looked-after children (LAC).LAC are defined as those children who are being provided with substitute care . The terLAC are representative of a very challenging and complex population. Children in residential care settings are generally regarded as having greater mental health needs, in comparison to the general population of the same age . AccordiAmong the psychopathological risks that LAC are prone to, it has been reported that children in foster care are more prone to experiencing psychosocial  and biological risk factors  before and during their stay in care that makes them more susceptible to hyperactivity, impulsivity and inattention.To our knowledge, literature on the prevalence and characteristics of ADHD, including its treatment, in LAC has not been systematically reviewed. Gaining insight into the prevalence of the diagnosis of ADHD in LAC, as well as of the rates of prescription of ADHD medication, is of relevance from a clinical as well as public health standpoint. The aim of this paper is to fill this gap, in order to provide relevant and updated information to patients, clinicians and managers when designing clinical pathways for the care of LAC.Methods were developed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)  recommenWe searched the following electronic databases: PubMed, PsycInfo, Embase + Embase Classic, Ovid MEDLINE and Web of Sciences databases, with no language restrictions, from inception to November 9, 2016. The search terms and syntax for PubMed were as follows: (ADHD [tiab] OR Attention-deficit/hyperactivity disorder [tiab] OR attention deficit disorder with hyperactivity [tiab] OR Attention deficit [tiab] OR hyperkinetic disorder [tiab] OR hyperkinetic syndrome [tiab]) AND . The search terms and syntax were adapted for the other databases and are reported in the Supplemental Material. We also searched bibliographic references from relevant papers.We retained peer-reviewed, empirical quantitative studies providing information on the prevalence of ADHD, and/of its treatment, in LAC. We excluded case reports, case series, qualitative studies or non-peer-reviewed publications. As for the diagnosis of ADHD, we included studies based on a formal diagnosis of ADHD as per the Diagnostic and Statistical Manual of Mental Disorders (DSM), III, III-R, IV, IV-TR or 5 or the International Classification of Diseases (ICD), 10th edition, or studies where ADHD was defined based on scores above a cut-off on a validated ADHD questionnaire.From an initial pool of 350 possibly relevant references, we retained 24 studies meeting our criteria . The details of the selection process are reported in Fig. As for the prevalence of ADHD, only a minority of studies contrasted LAC and non-LAC, showing higher rates in LAC. The rest of the studies reported the prevalence of ADHD in LAC (without comparison to a non-LAC group), which was substantially higher than the national prevalence of ADHD in the study country . Indeed,To our knowledge, this is the first systematic review aimed at comprehensively assessing the literature on the prevalence of ADHD, and its pharmacological treatment, in LAC.Given the complex nature of the clinical presentation of LAC and their needs, some authors have reported concerns that ADHD might be under diagnosed in LAC, missing out on appropriate treatment . HoweverAnother concern is that ADHD is over diagnosed in LAC. This would particularly be favoured by misdiagnosing ADHD-like symptoms accounted for by disorders other than ADHD  as “real” ADHD. In this regard, the use of the ICD category of hyperkinetic syndrome, which is ruled out in the presence of other disorders such as anxiety that can manifest with ADHD-like symptoms, would tend to reduce the risk of over diagnosis. However, rather than under or over diagnosis, in our view, the main issue is to which extent ADHD is correctly diagnosed in LAC, limiting false-positive as well as false-negative cases. Comparative studies across countries will be of interest in this respect.Similarly to the prevalence of ADHD, the rate of ADHD medication prescription was general high across studies and higher than the national estimates of the use of ADHD drugs . Data onOur work should be considered in the light of study strengths and limitations. Among the strengths, we highlight that we conducted a comprehensive search across a large set of databases and with no language/date restrictions. The main limitation is represented by the heterogeneity in the methodology across studies, which prevented us from conducting a formal meta-analysis based on the data retrieved in the studies retained in our systematic review.In conclusion, this systematic review showed high prevalence of ADHD and high rates of ADHD pharmacological treatment in LAC, at least in the USA. Further methodologically sound research is needed from other countries. Perhaps more importantly, rather than assessing if ADHD is over or under diagnosed and treated in LAC, future research should aim to understand to which extent ADHD is appropriately diagnosed and treated in LAC and which are the most cost-effective strategies, in the short as well as long term, to manage symptoms of ADHD in LAC.ESM 1(DOC 39 kb)