Cyclosporine A (CsA) is a potent immunosuppressant for treating ulcerative colitis (UC). However, owing to severe systemic side effects, CsA application in UC therapy remains limited. Herein, a colon-targeted drug delivery system consisting of CsA crystals (CsAc)-loaded, Eudragit S 100 (ES)-coated alginate microparticles (CsAc-EAMPs) was established to minimize systemic side effects and enhance the therapeutic efficacy of CsA. Homogeneously-sized CsAs (3.1 ± 0.9 μm) were prepared by anti-solvent precipitation, followed by the fabrication of 47.1 ± 6.5 μm-sized CsAc-EAMPs via ionic gelation and ES coating. CsAc-EAMPs exhibited a high drug loading capacity (48 ± 5%) and a CsA encapsulation efficacy of 77 ± 9%. The in vitro drug release study revealed that CsA release from CsAc-EAMPs was suppressed under conditions simulating the stomach and small intestine, resulting in minimized systemic absorption and side effects. Following exposure to the simulated colon conditions, along with ES dissolution and disintegration of alginate microparticles, CsA was released from CsAc-EAMPs, exhibiting a sustained-release profile for up to 24 h after administration. Given the effective colonic delivery of CsA molecules, CsAc-EAMPs conferred enhanced anti-inflammatory activity in mouse model of dextran sulfate sodium (DSS)-induced colitis. These findings suggest that CsAc-EAMPs is a promising drug delivery system for treating UC. Aspergillus terreus and Tolypocladium inflatum, is widely employed as an immunosuppressant owing to its potent anti-inflammatory effects [Ulcerative colitis (UC) is a chronic, relapsing disease of the gastrointestinal tract (GIT) that causes inflammation and ulcers in the mucosal lining of the colon and rectum . This mu effects . Notably effects . However effects ,9. In ad effects . Therefo effects ,12,13.co-glycolic acid) and poly methacrylate-based copolymers, prevents the initial burst release of CsA in the stomach and small intestine, thus resulting in reduced systemic absorption. However, although these systems allow the successful delivery of CsA to the colon during UC therapy, intrinsic limitations persist, including low drug loading capacity, complicated fabrication processes, low cost-effectiveness, and excipient-associated toxicities [As colon-targeted delivery systems, several CsA-loaded polymeric nano- and microparticles have been developed to minimize systemic absorption of the drug and enhance drug accumulation in inflamed colonic tissues ,11,12,14xicities ,16. In pxicities ,18. Unlixicities . In addixicities ,21. AccoAs an attractive drug carrier for the CsA crystal delivery system, alginate-based microparticles have gained momentum given the favorable characteristics of alginate, including low toxicity, biodegradability, relatively low cost, and gel-forming ability . In addiIn the present study, we developed CsA crystals-loaded, ES-coated alginate microparticles (CsAc-EAMPs) as a colon-targeted drug delivery system to enhance the therapeutic efficacy and reduce systemic side effects of CsA during UC therapy. After characterization of CsAc-EAMPs, the drug release profile was determined in pH-changing media. Furthermore, the enhanced in vivo therapeutic efficacy of CsAc-EAMPs was evaluated in a dextran sulfate sodium (DSS)-induced colitis mouse model.CsA, chitosan , sodium alginate , and Mayer’s hematoxylin solution were purchased from Sigma-Aldrich . ES  was generously gifted by Evonik . DSS  was obtained from MP Biomedicals . Eosin-Y solution was purchased from Daejung Chemicals & Metals . Primary anti-E-cadherin was purchased from BD Bioscience . Alexa Fluor 488-conjugated AffiniPure goat anti-mouse IgG was purchased from Jackson ImmunoResearch Laboratories . All other chemicals, reagents, and solvents were of the highest commercially available purity grade.g for 10 min, and washed three times with distilled water to remove ethanol.CsA crystals (CsAc) were prepared by employing an anti-solvent precipitation method, using sonication and ethanol as a water-miscible solvent . In briew/v) calcium chloride solution was freshly prepared as a cross-linker. The above suspension was filled in the encapsulator and injected into the solution , thereby forming CsAc-AMPs after 1 h. For preparing CsAc-EAMPs, CsAc-AMPs were coated with chitosan and ES using physical electrostatic interactions as a coating mechanism. Briefly, CsAc-AMPs were introduced into 2 mg/mL of chitosan in 0.1 M acetic acid solution. After stirring for 30 min at 4 °C, the CsAc-AMPs were recovered by centrifugation (2000× g for 10 min). Then, the microparticles were suspended in an ES solution  for 30 min at room temperature. After incubation, CsAc-AMPs were obtained by centrifugation (2000× g for 10 min).CsAc-EAMPs were fabricated via two sequential processes: fabrication of CsAc-loaded alginate microparticles (CsAc-AMPs), followed by enteric-coating of the particle surface using chitosan and ES. First, CsAc-AMPs were prepared using an ionic gelation method with an encapsulator . The shape of prepared CsAc was analyzed using scanning electron microscopy . Raw CsA powder and CsAc were suspended in water, dropped on a carbon tape, and air-dried at room temperature before visualization by SEM at an acceleration voltage of 1–5 kV. The particle shape of the CsAc-EAMPs was analyzed by light microscopy . A specific amount of CsAc-EAMPs was placed on a glass slide and covered prior to imaging. The particle size of CsAc was determined using ImageJ software  ,29.The amount of CsA in CsAc-EAMPs was determined using high-performance liquid chromatography (HPLC) . Prior to HPLC analysis, CsAc-AMPs and CsAc-EAMPs were placed in vials containing ethanol and shaken vigorously at room temperature for 24 h for the complete extraction of CsA from microparticles. The samples were centrifuged, and the supernatants were filtered through a 0.22-μm filter. The HPLC analysis conditions were as follows: detection wavelength, 210 nm; mobile phase, a mixture of acetonitrile and water (75:25); column, VDSpher 100 C18-E column ; injection volume, 20 μL; flow rate, 1.5 mL/min; oven temperature, 65 °C . Encapsug for 20 min, and the drug content in the supernatants was analyzed using HPLC with the method mentioned above.The pH-dependent drug release profiles of CsAc, CsAc-AMPs, and CsAc-EAMPs were examined using the dialysis bag diffusion method, with some modifications ,32. In bw/v) in water provided ad libitum for seven days to induce colitis. Age-matched male ICR mice provided with normal tap water served as the healthy control group. After colitis induction, CsA formulations  were daily administered orally by using a flexible plastic oral gavage at dose of 15 mg/kg for seven consecutive days.The animal study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of Pusan National University Institutional Animal Care and Use Committee (protocol code PNU-2019-2420 on 23 October 2019). DSS-induced colitis was induced in mice using a previously reported method with some modifications [For assessing the therapeutic effects of CsAc-EAMPs, the disease activity index (DAI) of mice was calculated based on changes in body weight, stool state, and rectal bleeding, recorded from day 1 to day 14 of the experiment . In brieFor direct monitoring of colitis severity in live mice, endoscopic analysis was performed using a Veterinary Endoscopy System , following the manufacturer’s instructions. Briefly, on the last day of the experiment, mice were anesthetized with isoflurane, and the colon was visualized after air inflation of the colon. For quantitative analysis of the colitis severity, the murine endoscopic index of colitis severity (MEICS) was used as previously described . The scoFor histological analysis, paraffin-embedded tissue samples were prepared following a previously reported method with some modifications ,36. BrieIn order to evaluate the E-cadherin expression in each colon sample, immunofluorescence staining was performed as previously reported . Brieflyg at 4 °C for 6 min), 7 µL supernatant was added to 200 µL of 0.167% (w/v) mg/mL o-dianisidine hydrochloride with 0.0005% (v/v) hydrogen peroxide in a 96-well plate. The absorbance of each sample was measured at 450 nm using a microplate reader .MPO activity in colon tissues was measured following a previously reported method . Brieflyt-test, one-way ANOVA, or two-way ANOVA were performed to assess differences between groups. Statistical significance was set at p ≤ 0.05.Data were analyzed using GraphPad Prism version 5.0 for Windows . Two-tailed CsAc-EAMPs were successfully fabricated by employing three sequential processes: fabrication of CsAc, incorporation of CsAc into alginate microparticles, and ES coating on the microparticle surface A. First,After fabrication of CsAc, CsAc-AMPs were fabricated via ionic gelation of alginate in the presence of calcium ions using an encapsulator. Globular-shaped, 46.4 ± 8.8 µm-sized CsAc-AMPs were obtained and exhibited 49 ± 9% CsA loading and 78 ± 7% EE . To fabrn = 100 for particle size analysis, n = 3 for encapsulation efficacy and drug loading measurement). CsAc, cyclosporine A crystals; CsAc-AMPs, CsA crystal-loaded alginate microparticles; CsAc-EAMPs, CsA crystal-loaded, Eudragit S 100 coated alginate microparticles.The results are expressed as mean ± standard deviation , all mice were sacrificed; then, the colons were excised to measure colon length as a parameter of colitis severity B,C. OwinWe next evaluated colitis severity by directly observing the colon in live mice by performing endoscopic analysis on the last day of the experiment A. The coTo further investigate the enhanced anti-inflammatory effects of CsAc-EAMPs, colon tissue samples were histologically assessed by microscopic examination of H&E-stained sections . In the The severity of colitis in colon tissue samples treated with or without CsAc formulations was further evaluated by E-cadherin immunostaining . As E-caTo confirm colonic inflammation, activity of MPO, a representative inflammatory marker, was assayed on the last day of the experiment. ® and coating techniques such as Duocoat®, and Colpulse® have been developed for increasing drug concentration in the colon [Although CsA is a potent anti-inflammatory drug for the treatment of UC, only a limited success has been achieved due to the severe side effects resulted from the unwanted systemic distribution of CsA. For the reason, development of targeted delivery system that could minimize systemic distribution of CsA and increase drug accumulation at the target site is needed. Until now, various colon-targeted drug delivery systems including SmPillhe colon ,42,43. Hhe colon . In this® and OPTICORE™ have been successfully commercialized [After successful fabrication of CsAc-EAMPs, their enteric protective effects were confirmed by examining drug release profiles in the pH-changing media that simulate the pH conditions of the GI tract. As shown in cialized ,46. In acialized . AccordiNext, the enhanced in vivo therapeutic efficacy of CsAc-EAMPs was evaluated in a DSS-induced colitis mouse model, which demonstrates characteristics similar to those of human UC . FollowiAs a result of rapid colitis relief, the colon cavity, as observed endoscopically, exhibited a higher number of ameliorated features in the CsAc-EAMP-treated group than in the untreated colitis group . ConversAfter macroscopic and endoscopic analysis, epithelium recovery in colon tissues treated with or without CsA formulations was examined by assessing E-cadherin expression in the colon epithelium. E-cadherin plays a major role in maintaining colon tissue architecture and adhesion between epithelial cells . E-cadheFinally, MPO activity was measured in colon tissues to evaluate the severity of inflammation . MPO, a Collectively, the enhanced therapeutic effects of CsAc-EAMPs were attributed to the colon-targeted delivery that enhanced CsA accumulation in the inflamed colonic regions to mitigate inflammation. Given the absence of colon-targeted delivery in the CsAc and CsAc-AMP-treated groups, CsA accumulation in the colitis area was insufficient to relieve inflammation, resulting in therapeutic failure in DSS-induced colitis mice.In the present study, CsAc-EAMPs were developed for targeted delivery of CsA to enhance therapeutic efficacy and minimize systemic side effects of CsA. Herein, 47.1 ± 6.5 µm-sized CsAc-EAMPs were successfully fabricated with 48 ± 5% CsA loading and 77 ± 9% EE via three sequential processes: fabrication of CsAc, incorporation of CsAc into alginate microparticles, and ES coating on the microparticle surface. In addition, CsA release from CsAc-EAMPs was effectively suppressed under simulated pH conditions of the stomach (pH 1.2) and small intestine (pH 6.8), indicating that CsA absorption could be restricted to reduce systemic side effects. Finally, CsAc-EAMPs showed enhanced anti-inflammatory effects in a DSS-induced colitis mouse model. Collectively, these results suggest that CsAc-EAMPs could be a potent colon-targeted CsA delivery system for the treatment of UC.


Is there an association of grandmother prepregnancy body mass index or gestational weight gain with risk of attention-deficit/hyperactivity disorder (ADHD) among grandchildren?This cohort study of 19 835 grandmother-mother dyads indicated independent, significant associations of grandmother underweight and mother overweight or obesity prior to pregnancy with higher odds of ADHD among 44 720 children in the following generation.The present study findings suggest that underweight grandmaternal periconceptional body mass index may be associated with ADHD among grandchildren, potentially via the germline. Neurodevelopmental disorders have been proposed to involve alterations to epigenetic regulation, and epigenetic effects may extend to germline cells to affect later generations. Weight status may affect DNA methylation, and maternal weight before and during pregnancy has been associated with offspring DNA methylation as well as attention-deficit/hyperactivity disorder (ADHD).To assess whether a woman’s weight before and during pregnancy is associated with ADHD in her grandchild.This cohort study analyzed data from 19 835 grandmother-mother dyads and 44 720 grandchildren in the Nurses’ Health Study II (NHS-II) cohort (2001-2013), a population-based prospective cohort study. Cluster-weighted generalized estimating equations were modeled to estimate the association of grandmother’s prepregnancy body mass index (BMI) and gestational weight gain with grandchild risk of ADHD. Data analyses were conducted from May 2018 to April 2021. Grandmothers reported their height and weight before, and weight gain during, their pregnancy with the NHS-II participants. Mothers self-reported height and weight prior to pregnancy. From those data, grandmother BMI and mother BMI were calculated as weight in kilograms divided by height in meters squared and categorized as underweight (<18.5), healthy/normal (18.5-24.9), overweight (25.0-29.9), or obese (≥30).Cases of ADHD identified by maternal report of having a child with a diagnosis of ADHD.In total, 19 835 grandmothers  were included in this cohort study. Of 44 720 grandchildren, 3593 (8%) received a diagnosis of ADHD. Higher odds of ADHD among grandchildren were found for those whose grandmother was underweight compared with healthy weight prior to pregnancy with the NHS-II participant . By contrast, grandmother gestational weight gain was not significantly associated with risk of grandchild ADHD . Mother prepregnancy BMI showed an association with ADHD among offspring, with a stronger association detected for obese status  than for overweight status  compared with normal weight as a reference group. The positive association between grandmother prepregnancy underweight and ADHD risk among the grandchildren remained unchanged after further adjustment for potential mediators, including maternal prepregnancy BMI.The results of this cohort study indicate that grandmother underweight prior to pregnancy is associated with an increased risk of ADHD among grandchildren, independent of grandmother gestational weight gain and independent of maternal prepregnancy weight status. This cohort study analyzes data from mother-daughter dyad participants in the Nurses’ Health Study II to assess whether a woman’s weight before or during pregnancy is associated with attention-deficit/hyperactivity disorder (ADHD) in grandchildren. Peripregnancy maternal weight can affect endocrine function21 and DNA methylation,22 both of which can affect the germline and are implicated in multigenerational effects of exposures.23 Animal studies have found evidence for such germline effects of weight-related variables around pregnancy,26 with possible implications for ADHD.28 Human studies have also found that maternal prepregnancy body mass index (BMI) and GWG may be associated with epigenetic effects in offspring.30Although heritability estimates for ADHD are 70% to 80%, the etiology of ADHD still appears partially attributable to nongenetic factors.31 The objective of the present study was to assess the possible multigenerational associations of prepregnancy BMI and GWG with ADHD by using the Nurses’ Health Study II (NHS-II), a large, well-characterized longitudinal study of nurses.Studies of grandmothers’ exposures and associated outcomes in grandchildren are inherently challenging, mainly owing to data collection over multiple generations.32 In 2001, 39 904 nurses’ mothers who were alive and free of cancer in 2000 were enrolled in the Nurses’ Mothers’ Cohort Study (NMCS) and provided additional information on the prenatal and childhood environment of the nurses.33 In the present study, we refer to the nurses as generation 1 (G1), their mothers as generation 0 (G0), and their children as generation 2 (G2). This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for cohort studies. Our study was approved by the institutional review board of Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health, Boston, Massachusetts. Returning completed questionnaire is considered evidence of informed consent in a manner consistent with the Common Rule requirements at enrollment in 1989, and participants have since completed biennial questionnaires. No one received compensation or was offered any incentive for participating in this study.The NHS-II is a prospective cohort study of 116 430 US registered female nurses who were born between 1946 and 1964.Our analytic population was first restricted to 23 898 G1 nurses who had children, returned the 2013 NHS-II questionnaire, were not themselves adopted, and whose mother participated in the NMCS. We then additionally excluded 1814 G1 nurses for whom data for G0 prepregnancy BMI or GWG were missing. We excluded G2 children with a sibling born in the same year to the same mother because if the mother reported a child with ADHD, we would not know which child it was. The final analytic sample included 19 835 G0-G1 dyads and 44 720 G2 offspring. Although the NMCS is a subset of the NHS-II, we found little difference in G2 ADHD prevalence by G0 NMCS membership status (7.9% of NMCS participants vs 7.2% of NMCS nonparticipants), suggesting that selection bias was unlikely.34 The G0 grandmothers also reported weight gain during the pregnancy with the G1 nurse, with categorical responses for GWG of less than 10 lb (<4.5 kg), 10 to 14 lb (4.5-6.4 kg), 15 to 19 lb (6.8-8.6 kg), 20 to 29 lb (9.1-13.2 kg), 30 to 40 lb (13.6-18.1 kg), more than 40 lb (>18.1 kg), or “don’t remember.”35 In our study, the majority (40%) of G0 grandmothers reported weight gain of 20 to 29 lb (9.1-13.2 kg) during pregnancy, which is consistent with the reported mean GWG of that era.40 We categorized G0 GWG as less than 20 lb (<9.1 kg), 20-29 lb (9.1-13.2 kg), more than 29 lb (>13.2 kg), or no response, which roughly reflected less than, equal to, and more than the recommended GWG in that era.40 We also considered the nurse’s self-reported BMI prior to the pregnancy with her G2 child as an exposure, with the same prepregnancy BMI categories as for G0.In 2001, the NMCS G0 participants reported their height and weight prior to the pregnancy with the NHS-II nurse (G1). From those data, we calculated BMI , which we categorized as underweight (<18.5), healthy/normal (18.5-24.9), overweight (25.0-29.9), and obese (≥30).42In 2013, the G1 nurses were asked “have any of your biological children been diagnosed with attention-deficit/hyperactivity disorder (ADHD)?” and if so, the year of birth of the child(ren). Our main analyses considered identified ADHD cases. The nurses were also asked about children with ADHD on the 2005 questionnaire, but not their year of birth; thus, the specific child with ADHD could not always be identified. In sensitivity analyses, we excluded any ADHD cases identified in 2013 by mothers who did not report a child with ADHD in 2005. Details of the validation study of ADHD diagnosis have been described.44 we included G1 birth year. We also considered G1 reporting of her mother’s (G0) lifetime history of major depression (yes or no) given its association with both overweight or obesity and ADHD.48 In additional analyses, we also considered G1 pregnancy-related complications  while pregnant with the G2 child50 and her child’s birth weight 51 as potential mediator variables.Potential confounders were identified by drawing directed acyclic graphs based on previous literature. We considered G0 grandmother self-reported race/ethnicity , grandmother educational level (high school or less vs college or more), smoking and alcohol use (separately) during pregnancy with the nurse (yes or no), and the grandfather’s educational level (same categories) and occupation . To account for increasing secular trends in obesity and ADHD prevalence,53 which was associated with ADHD in our data  and 95% CIs for G2 ADHD by G0 pregnancy-related weight characteristics, adjusting for covariates. Those equations weight by the inverse of the number of children for each nurse  to handle informative clustering. Generalized estimation equations simultaneously account for potential unknown correlations between outcomes among grandchildren born to the same nurse. A compound symmetric working covariance structure was applied, assuming constant correlation regardless of the order of children for each nurse. We adjusted for the described nonmediator covariates.Overweight or obesity may disrupt the reproductive functions of a woman, potentially affecting the number of G2 children for a given G1 nurse in the present study,56 which we verified by running models that included these interaction terms and evaluating their significance. Fifth, we carried out a quantitative bias analysis to assess the influence of possible exposure misclassification.57 Complete case analysis was conducted given the small amount of missing data . All analyses were performed from May 2018 to April 2021 using SAS (SAS Institute Inc) or the R package episensr, version 4.0.4 (R Development Core Team), for the quantitative bias analysis. A 2-sided value of P ≤ .05 was considered statistically significant.We conducted the following sensitivity analyses. First, we investigated whether the distribution of key G1-reported covariates differed according to the G0 NMCS membership status. Second, we excluded cases in which G1 mothers did not report a child with ADHD in 2005. Third, we considered differences by child sex by stratifying on G2 sex. Fourth, we examined how much of a potential association of our exposure with ADHD was accounted for by the described mediators by including those terms in the model. In addition, by having G0 prepregnancy BMI and GWG and G1 prepregnancy BMI in a model together, we were able to assess the extent to which G1 prepregnancy BMI mediated any association between G0 prepregnancy BMI or GWG and G2 ADHD, and the extent to which G0 GWG mediated any association with G0 prepregnancy BMI. Such checks on mediation are valid only if there is no interaction between the exposure of interest and the mediator,Of 19 835 G0 grandmothers, before pregnancy, 2113 (10.7%) had underweight and 1391 (7.0%) had overweight or obesity . During We found grandmaternal underweight to be associated with increased odds of ADHD in G2 , but no association with overweight  nor with GWG  . There . When boIn sensitivity analyses, no evidence of selection bias was found owing to loss to follow-up in our data, given the similar distribution of key G1-reported variables according to the NMCS membership. The results were essentially unchanged when we excluded cases in which mothers did not report a child with ADHD in 2005. Although there was little difference in results by G2 sex for the G0 exposures, G1 prepregnancy overweight or obesity was more strongly associated with risk of ADHD among girls than boys . By contrast, the association between G1 prepregnancy obesity and G2 risk of ADHD was slightly attenuated, suggesting that this association could partially operate via these potential mediators.58 indicating that mothers with overweight  or obesity  prior to pregnancy were at increased risk for ADHD. Potential underlying biological mechanisms include oxidative stress and inflammation or dysregulation of hormone signaling in the developing brain by maternal obesity prior to pregnancy.59 By contrast, there is a paucity of literature about maternal prepregnancy underweight in relation to ADHD in the offspring. A Danish National Birth Cohort study13 reported increased risk of autism spectrum disorder , but not of ADHD, among mothers who were underweight prior to pregnancy. Deardorff et al60 found prepregnancy underweight to be associated with higher total Behavior Problems Index and externalizing scores only among boys. Studies examining the association of GWG with the child’s (G2) risk of ADHD have shown limited and conflicting findings.61Previous research has found evidence for maternal prepregnancy obesity as a risk factor for ADHD, with a recent meta-analysis64 which may also be associated with ADHD.66 However, the association with G0 prepregnancy weight status remained robust to further adjustment for G2 birth weight and other G1 and G2 pregnancy factors, which suggests that these factors are not responsible for the association with G0 prepregnancy weight. Increased risk of ADHD in G2 from G0 with prepregnancy underweight may plausibly occur through G1 assortative mating  if G0 prepregnancy underweight leads to increased ADHD symptoms in G1. However, this explanation seems unlikely because we found lower—not higher—odds of ADHD in G2 for G1 with prepregnancy underweight . Unfortunately, no data were available regarding ADHD diagnosis or symptoms in G1 nurses.Several mechanisms may underlie the observed association between G0 prepregnancy underweight and G2 ADHD. Grandmothers who were underweight before pregnancy were more likely to have lower socioeconomic status, other factors of which could be associated with G2 ADHD. However, we adjusted for many G0-level socioeconomic status variables; furthermore, gaining more weight than recommended during pregnancy was also associated with lower socioeconomic status, yet no association was observed for that group. Similarly, grandmaternal prepregnancy underweight may be associated with G1 or G2 pregnancy factors, such as offspring weight and size,69 In humans, the precursors of eggs and sperm are primordial germ cells (PGCs), the fate of which is induced on embryo implantation in the uterine wall. During early embryogenesis, PGCs are formed and actively migrate to the gonadal ridge. This phase is followed by a second phase in which PGCs initiate controlled cell division directed by environmental cues.70 The PGCs lack protection from epigenetic dysregulation by environmental toxicants and thus remain vulnerable to damage during early development.71 When a grandmother (G0) experiences unhealthy weight around her pregnancy, both the G1 embryo and G2 germ cell precursors are directly exposed to the G0 pregnancy milieu and signals related to G0 weight status.72 Grandmother prepregnancy underweight could influence the epigenetics of the G2 germ cells, such as DNA methylation patterns,73 leading to neurodevelopmental deficits in that generation.Alternatively, a plausible underlying biological mechanism involves direct exposure of G2 germ cells to the G0 pregnancy milieu and epigenetic modifications.74 which was also suggested in our validation study.41 Second, exposure misclassification is also likely. The G0 prepregnancy weight, height, and total GWG data were self-reported. In a separate NHS validation study,75 maternal prepregnancy weight (r = 0.86) and height (r = 0.90) were found to be accurate compared with external data collected during their pregnancies, whereas recall of weight gained during pregnancy was only modestly correlated (r = 0.42). However, it is unlikely that grandmaternal BMI and GWG reporting differed according to grandchild ADHD diagnosis given that the data were collected from G0 many years before asking the nurses (G1) about ADHD in their children (G2) and there was not yet public awareness about potential multigenerational associations of grandmother weight around pregnancy with neurodevelopmental disorders in grandchildren. Third, obesity and ADHD may share common genetic variants.76 However, different patterns of association were found between G0 and G1 prepregnancy BMI in relation to G2 ADHD, which would have been similar if our findings were largely attributable to common genetic confounding between obesity and ADHD. Furthermore, the association between G0 prepregnancy BMI and ADHD risk in G2 was robust after adjusting for G1 BMI prior to pregnancy with G2, presumably partially blocking potentially unmeasured confounding factors due to common genetic susceptibility. Fourth, although our mediation analysis suggested a direct association between G0 prepregnancy BMI and G2 ADHD, our findings should be interpreted with caution given possible measurement error of the mediators.77 Fifth, as in all observational studies, the possibility of residual confounding remains, although we were able to adjust for a number of variables.This study has limitations. First, outcome misclassification is possible. The ADHD case ascertainment was based on nurses’ reports, rather than on medical records. However, maternal reports of ADHD diagnoses in their children have been found to be reliable,This study has several strengths. The large, prospective cohort of nurses provided a unique setting to investigate multigenerational associations by including information from 3 generations. A variety of covariates that were directly obtained from G0 gives us more confidence to address potential confounding factors compared with previous studies evaluating multigenerational associations, and data on the pregnancies with G2 enabled us to explore possible mediators of the G0 association.91 future research should assess risks to germline cells following different exposures.This cohort study provides novel evidence showing an association between grandmother weight status around pregnancy and increased risk of ADHD in their grandchildren. This study contributes important information to a growing body of literature on multigenerational associations in humans, suggesting that maternal weight around conception may be associated with neurodevelopment of the third generation. Our findings suggest a different pattern of associations with grandmother peripregnancy weight characteristics than with mother peripregnancy weight characteristics. Animal studies may help elucidate potential underlying biological pathways. Given emerging evidence of multigenerational associations with in utero exposure to various factors,


The use of surfactants in polymerization reactions is particularly important, mainly in emulsion polymerizations. Further, micelles from biocompatible surfactants find use in pharmaceutical dosage forms. This paper reviews recent developments in the synthesis of novel gemini and bicephalous surfactants, micelle formation, and their applications in polymer and nanoparticle synthesis, oil recovery, catalysis, corrosion, protein binding, and biomedical area, particularly in drug delivery. According to IUPAC definition, a micelle is a “Particle of colloidal dimensions that exists in equilibrium with the molecules or ions in solution from which it is formed” ,2. FurthSurfactants are amphiphilic molecules that have hydrophobic (head) and hydrophilic (tail) components, allowing their solubility in both organic solvents and water. At the air–water interface, the hydrophobic tail is in the air and the hydrophilic head in the water, causing a decrease of both, in the surface tension, which is defined as the force of attraction between the molecules at the air–water interface, and in the interfacial tension between two liquids ,5. UsualSurfactants are key compounds in many industrial processes  and a variety of useful products  have been vigorously developed in terms of functional variety and structural diversity in the last few years ,7,8.These compounds may be classified based on the chemical nature of their polar head . If the −), sulphonate (RSO3−), or sulphate (RO-SO3−) ..93].More recently, Asadov et al. synthesized and characterized the cationic gemini surfactant N,N′-bis-N,N′-bis (2-hydroxypropyl) ethylene diammonium dibromide with chain lengths of 9, 12, and 14 carbon atoms . They fo−3 mN/m, with surfactant concentrations less than 0.5 wt %  an an115] aOtherwise, the study of the interactions between proteins and surfactants is very important due to the numerous technical applications in the fields of pharmaceuticals, cosmetics, paints, coatings, etc. ,119,120.Recently, gemini surfactants were shown to be more efficient to interact with proteins by comparing them with single-chain surfactants ,126,127.Luo et al. focused on the investigations of the interactions between single-chain or gemini quaternary ammonium surfactants with hemoglobin.They observed that the interactions between the surfactants and hemoglobin were mainly caused by both electrostatic and hydrophobic interactions, and the hydrophobic chain length and linking group length of the surfactants had a significant influence on tuning the conformations of hemoglobin . For theMicellar catalysis is a process that consists of the accumulation of a catalyst in the internal part of a micelle . The micMicellar catalysis using gemini surfactants was shown to have high catalytic efficiency and accelerates processes reducing the generation of secondary reactions ,137. BunConventional surfactants are amphiphilic organic compounds that have a hydrophilic head and hydrophobic tail whose main functions are to reduce the interfacial tension in a colloidal system, forming an interface between the two immiscible phases . They arIn recent years, novel surfactants have been reported with different structural arrangements by comparing with the conventional ones. These new structures consist of two hydrophilic heads, a hydrophobic tail, and a spacer that prevents repulsion, which have been called “bicephalous” surfactants . There aBicephalous surfactants have been synthetically obtained by the 1,4 addition Michael reaction, in step 1, using as precursorspropanolamine (1) and tert-butyl acrylate (2) in methanol under constant stirring at room temperature, followed by a series of steps (2–5) that are represented in the route of synthesis for this surfactant type, as reported by Kalhapure et al.  146]..146].One year later, Ojewole et al.  made smaMore recently, Chaudhari et al. simplified the method of Kalhapure, using the same initial precursors and modifying the reaction media . They acOn the other hand, Hanssan et al. proposed a three-step synthesis to obtain a quaternary bicephalous cationic surfactant through the Michael reaction, using a different precursor to the previously reported synthetic methods . They usA select group of researchers has reported the synthesis of bicephalous surfactants in a relative short period. Among the most notable groups working along this line, it can be mentioned the group of Kalhapure and Ojewole, who have revealed four novel surfactants of this type. For their part, Bazylinska et al.  have proThe critical micellar concentration is an important parameter to characterize a surfactant since it describes the required concentration of this compound for the formation of stable micelles . Two-heaAn example of the decrease in the CMC is the bicephalous dianionic surfactant called disodium (Z)-3,30-((3-(oleoyloxy) propyl) azanediyl) dipropanoate, which reduces the CMC by almost 50% compared to the conventional sodium oleate surfactant This corroborates that the CMC can be reduced by increasing the amount of polar heads in the chemical structure of the surfactant .Micellar formation using a conventional surfactant is different from that resulting from a bicephalous surfactant. The main difference is that a greater amount of conventional surfactant is required to form a micelle a than thIn 2013, Kalhapure et al. synthesized a bicephalous dianionic surfactant, which was used to prepare solid lipid nanoparticles with ketoconazole, a drug with low solubility and high permeability. They achieved a decrease in CMC (<50%) compared to a formulation prepared with sodium oleate, increasing the solubility, stability, biocompatibility, and biosafety of the drug ,148. For their part, Ojewole et al. obtained three bicephalous dianionic surfactants derived from oleic acid to obtain gels for oral administration of the antiretroviral drug didanosine. These surfactants were the 9-octadecenolic acid(9Z)-,3-[bis[3--3-oxopropyl]amino] propyl ester, 9-octadecenolic acid(9Z)-,3-[bis(2-carboxyethyl) amino] propylester, and 9-Octadecenoic acid (9Z)-, 3-[bis(2-carboxyethyl) amino] propyl ester sodium salt. The results showed that the use of this type of surfactant increased the oral permeability of the drug compared to formulations where only oleic acid was used. It also was determined that an increase of the concentration of these surfactants caused an increase in oral drug permeation .12N(COONa)2 to formulate normal microemulsions with isopropyl myristate and/or oleic acid as the oil phase, achieving a thermodynamically stable microemulsion. This microemulsion was shown to have zero toxicity in the in vitro study with gingival fibroblast (HGF) and skin keratinocyte (HaCaT) cell lines, proving its potential application in the cosmetic industry for treatments that require cutaneous administration  amino] propyl ester, , whose precursor is a saturated fatty acid (linolenic acid), as an oil phase for the preparation of a nanoemulgel loaded with tenofovir, which is used in the treatment of human immunodeficiency virus. The permeation profiles were carried out and the results showed that the use of the bicephalous compound improved almost 40 times the permeation of the drug through the skin . On the Staphylococcus aureus. The toxic and hemolytic results demonstrated its biosafety and antimicrobial effect, which was improved up to eight times compared to a formulation without the use of the quaternary cationic bicephalic surfactant. In addition, sustained drug release was achieved by an intelligent system sensitive to pH 7.4 [More recently, Hassan et al. synthesized ammonia-based quaternary bicephalic cationic surfactants to formulate quatsomes with a broad-spectrum antimicrobial drug (vancomycin) to be tested against methicillin-resistant o pH 7.4 .As can be seen above, there are no reports about the use of bicephalous surfactants in the polymer area, including synthesis and characterization; therefore, many areas of opportunity and research can be opened in this field in the future.This review presents the synthesis, micelle formation, and applications status of two classes of non-conventional surfactants: gemini (dimeric) and bicephalous. The former is, by far, the most investigated of the two. Even though gemini surfactants have been known since 1935, commercial applications are scarce and conventional amphiphiles continue to dominate the market. Research has demonstrated that dimeric surfactants have improved properties over the conventional ones, which could lead to important savings in selected applications. The lower CMC indicates the potential to be advantageously applied, mainly in the cosmetic, pharmaceutical, oil recovery, catalysis, and polymer synthesis (emulsion polymerization) fields. Bicephalous surfactants represent a new generation of surface-active compounds that are starting to call to attention the need for future developments in applications dominated by conventional surfactants. Simplified synthetic routes for both gemini and bicephalous surfactants should be emphasized to open applications and markets in the near future.


Bartonella. Several studies have been conducted on the prevalence of Bartonella infections from animals and humans, including reports from wild and domestic ruminants. However, there has been only one report of Bartonella infection in water buffaloes from the northeastern part of Thailand. Moreover, the seroprevalence of Bartonella spp. in water buffaloes still remains unknown. This study was conducted to explore the prevalence of Bartonella spp. among water buffaloes from South Thailand using molecular and serological techniques.Bartonellosis is an emerging worldwide zoonosis caused by bacteria belonging to the genus Bartonella spp. using polymerase chain reaction and indirect immunofluorescence assay.A total of 312 samples (156 blood and 156 sera) of 156 water buffaloes from 29 farms in Phatthalung Province, South Thailand, were collected from January to March 2021. All samples were screened for Bartonella spp. was 16.03% , and among 25 water buffaloes with seroprevalence, 56%, 20%, and 24% were positive for antibodies against Bartonellahenselae, Bartonellavinsonii subspp. berkhoffii, and Bartonellatamiae, respectively. No significant difference was detected among seroprevalence, gender, age, and ectoparasite infestation.The seroprevalence of antibodies against three B. henselae, B. vinsonii subspp. berkhoffii, and B. tamiae in water buffaloes from South Thailand. Further studies are required on the epidemiology of Bartonella infection among water buffaloes, related personnel, and ectoparasites.This is the first report of the seroprevalence of antibodies against  Bartonella is the major causative agent of this disease. Bartonella spp. are Gram-negative fastidious oval bacteria that infect mammalian erythrocytes and endothelial cells of the host. It is well known that some Bartonella spp. are the cause of a wide spectrum of human illnesses such as cat scratch disease, chronic bacteremia, fever, and endocarditis [Bartonellahenselae, Bartonellabacilliformis, and Bartonellaquintana [Bartonella bovis, B. henselae, and Bartonellaclarridgeiae, have been detected from several animals, including buffalo, dog, and cat [Bartonellosis, an emerging zoonosis, is responsible for causing a variety of clinical syndromes in humans and animals and is associated with significant morbidity and mortality in humans worldwide ,2. A micquintana . Further and cat ,5. MammaBubalus bubalis) are one type of cattle belonging to the family Bovidae and subfamily Bovinae [Bartonella spp. The major transmission route of this bacterium is through biting from the arthropod vectors such as ticks, mites, lice, fleas, and flies to animals and humans [Bartonella spp. in buffaloes and the detection of bacterial species from buffalo samples [Bartonella infection in water buffaloes from the northeastern and middle parts of Thailand [Bartonella spp. in water buffaloes in Thailand still remains unknown.Water buffaloes  and indirect immunofluorescence assay (IFA).Therefore, the aim of the study was to explore the seroprevalence of The study was approved by the Institutional Animal Care and Use Committee of the National Institute of Health (NIH), Thailand.The study was conducted from January to March 2021. The samples were collected from Phatthalung providence of Thailand. The samples were processed at Department of Medical Sciences, NIH Laboratory, Ministry of Public Health, Nonthaburi, Thailand.We calculated the sample size based on 4124 water buffaloes using the Epitools program (www.epitool.net) with 95% confidence interval (CI), 0.05 precision, and 0.1 prevalence [A total of 156 water buffaloes were investigated in this study, comprising 106 females (67.9%) and 50 males (32.1%). These buffaloes were divided into two age groups as follows: 63 water buffaloes (40.4%) were aged ≤2 years and the remaining 93 buffaloes (59.6%) were aged >2 years. Furthermore, 109 (69.87%) buffaloes were infested with ectoparasites (lice).Bartonella was done according to a previously described method [2 for 2-4 weeks. This showed the presence of Gram-negative coccobacilli growing in small, rough, and grayish colonies that required long culture periods, which were tentatively considered as Bartonella organisms. The bacteria were subcultured on the culture media for further characterization.Isolation of d method  with modBartonella DNA from the samples was identified by the detection of genomic DNA using specific primers by PCR as described previously by Pangjai et al. [gltA) [i et al. . The geni et al.  (primer . [gltA)  , were fixed on the slides. Next, 10 μL of diluted serum (diluted in PBS containing 5% skim milk) was placed into the test holes, and the slides were incubated at 37°C for 1 h in a humid chamber. Then, the slides were washed twice with PBS for 15 min. Fluorescein-conjugated goat anti-bovine IgG  was diluted 1:800 in PBS with 0.001% Evans blue, and 10 μL of the mixture was applied into each hole. The slides were incubated for 1 h at 37°C, washed twice with PBS for 15 min, washed again with double-distilled water for 10 min, and then dried before examination under a fluorescence microscope. The intensity of the bacillus-specific fluorescence was scored subjectively from +1 to +4, and the fluorescence score of +2 at a dilution of 1:16 was considered to be positive. Serum samples were screened at 1:16 dilution, and the positive sample (at 1:16 dilution) was titrated in a series of 2-fold dilutions up to 1:1024. Positive and negative controls were included in the study.Buffalo sera were screened for antibodies against ai et al.. BrieflyThe odds ratio was calculated using “epiR” package embedded in R program, version 4.02. The obsBartonella antigens at the cutoff titer of 1:16. Bartonella antigens, wherein among the 25 positive buffaloes, 56% (14/25) were positive for B. henselae antigen only, 20% (5/25) were positive for B. vinsonii subspp. berkhoffii antigen only, and 24% (6/25) were positive for B. tamiae antigen only. Interestingly, 1 (4%) of the 25 buffaloes was found to be positive for B. vinsonii subspp. berkhoffii at the cutoff titer of 1:64. The comparisons of Bartonella spp. seroprevalence assessed by IFA among gender, age, and ectoparasite infestation using univariate analysis are summarized in Bartonella spp. Especially in farm 2, there was one buffalo harboring two antibodies against B. henselae and B. vinsonii subspp. berkhoffii. However, PCR analysis of any sample did not reveal Bartonella DNA.Overall, 16.03%  of the water buffalo sera samples were positive for three Bartonella spp. in water buffaloes using IFA. Our result showed that 16%  of the water buffaloes harbored antibodies against three Bartonella spp. We suspected that those buffaloes had been infected with Bartonella in the past. Some authors had reported that higher antibody titers were associated with bacteremia in animals and lower antibody titers might indicate slight bacteremia [Bartonella DNA was not detected using PCR. Due to the limitation of information on the seroprevalence of Bartonella antibodies from buffaloes in Thailand, we could not compare our results with other studies.This is the first report of the detection of cteremia . HoweverB. bovis, Bartonellachomellii, Bartonellaschoenbuchensis, Bartonellacapreoli, and Bartonellamelophagi infections in ruminants [B. bovis infection was reported in 6.8% of water buffaloes in Thailand [Syncerus caffer) in Mozambique, Africa [B. henselae, B. vinsonii subspp. berkhoffii, and B. tamiae, and not against B. bovis, in the water buffaloes.Recently, studies have reported the high prevalence of uminants -18. B. bThailand  and in 4, Africa , using mB. henselae infection has been reported in dogs and cats as well. Not only B. henselae but also B. vinsonii subspp. berkhoffii was detected in Thai cats [B. tamiae isolated from patients with febrile illness in Thailand who had possible rodent contact and were either dog or cat owners [Bartonella spp. isolated from ectoparasites (chigger mites) and ticks of rodents in Thailand [A high prevalence of hai cats -21. Our t owners ; we alsoThailand .Bartonella spp. to humans, such as B. henselae from cat fleas [B. quintana from human body lice [Bartonella spp. from cattle lice [Bartonella pathogens in those lice. Therefore, the mechanism through which the buffaloes acquired antibodies against Bartonella spp. remains unknown. It is necessary to clarify the knowledge gaps concerning the distribution of this bacterium, the genetic diversity, and the transmission mode among water buffaloes, related personnel, and ectoparasites.There are also some reports showing that infected arthropods could transmit at fleas ,24, B. qody lice , and Bartle lice . UnfortuB. henselae, B. vinsonii subspp. berkhoffii, and B. tamiae in water buffaloes from South Thailand. Further epidemiological studies should be conducted, including extensive surveys and additional studies of ectoparasites covering more areas and large samples sizes, to understand the risk factors for this disease among water buffaloes, related personnel, and ectoparasites.This is the first seroprevalence report of antibodies against KK, CS, WM, and IW: Collected the samples. DP: Provided technical help during the experiments. PS: Did the statistical analysis. SB: Designed the experiments and drafted and revised the manuscript. All authors read and approved the final manuscript.


This study examined the development of muscular fitness and coordination in children and adolescents with and without attention deficit hyperactivity disorder (ADHD) over a period of 11 years. Data was collected in three measurement waves as part of the longitudinal, representative Motorik-Modul (MoMo) study in Germany . The overall sample comprised 2988 participants . Structural equation modeling was conducted, and the estimated models had a good fit. No differences in muscular fitness were observed between participants with and without ADHD. Participants with ADHD had a lower coordinative performance at first measurement than those without ADHD. The difference in coordinative performance persisted throughout the study period. In the second wave of the nationally representative German National Health Examination and Interview Survey for children and adolescents KiGGS4, the prevalence of parent-reported ADHD diagnosed by a physician or psychologist at some point in the children’s or adolescents’ lives is somewhat lower, i.e., 4.4% in 3- to 17-year-old individuals5. Significantly more boys (6.5%) than girls (2.3%) in Germany are affected by ADHD, which is largely consistent with international findings even though diagnosis rates have recently risen more sharply in girls than in boys7.Attention deficit hyperactivity disorder (ADHD) is among the most commonly diagnosed mental disorders in children and adolescents. ADHD has a prevalence rate of approximately 5.3% worldwide which has remained stable in recent years8. ADHD is usually diagnosed once children enter school. In school, they are required to sit and maintain focus and concentration for extended periods of time, and are expected to only speak when called upon to do so by the teacher. This can be particularly challenging for children with ADHD who often present with motor restlessness or verbal disturbances in the classroom setting. Furthermore, they often cannot sufficiently profit from learning conditions and methods anchored in school and club structures9. In addition, children and adolescents with ADHD are particularly affected by the adverse consequences of the changing environmental situation, such as increasing digitalization and a related increase in sitting hours, a reduction in opportunities for free movement and play, and increasingly built-up open spaces. To this end, Roth9 points out that children and adolescents with ADHD require far more physical activity time and spaces than their peers without ADHD. Also, a recent research derived from three waves of the KiGGS study showed that physical activity was partly associated with lower specific symptoms of hyperactivity and inattention, as was general mental health10. Having a parent-reported ADHD diagnosis, however, did not interact with physical activity with respect to general mental health. Therefore, the authors concluded that the association between physical activity and mental health problems may not be influenced by the diagnosis of ADHD10.ADHD describes a cross-situational developmental disorder that usually has its onset in childhood. Inattention, impulsivity (deficit in inhibitory control), and/ or motor agitation are considered core symptoms of ADHD. A diagnosis of ADHD is complex and requires the occurrence of symptoms over a period of at least 6 months in several areas of life . Although the diagnostic criteria have been updated several times, the clinical description of the core symptoms of ADHD has remained essentially unchanged over several decades11, and about 75% of affected children and adolescents develop a comorbid disorder8. Mahone12 thus emphasizes the relevance of assessing motor developmental status as an important component in the diagnosis of ADHD. Indeed, research has shown that children and adolescents with ADHD have motor deficits13, particularly with regard to coordination and balance performance . Albeit few studies reported no difference in motor performance between children with versus without ADHD20 or reported that ADHD subjects had better motor performance than non-ADHD subjects21.About 60 to 80% of individuals with ADHD have comorbidities in psychological, physical, social as well as motor domains22. It is therefore critical to also examine potential long-term motor deficits and changes in motor performance. This is preferably done by conducting longitudinal studies among children with ADHD compared to those without23. To date, there is a lack of longitudinal, representative studies that examined motor performance trajectories among children and adolescents with ADHD, and that also compared motor performance among children and adolescents with versus without ADHD. In addition, available longitudinal research has mainly focused on cardiorespiratory fitness, e.g. a study derived from the European Youth Heart Study found that children with low cardiorespiratory fitness at the age of 9 years had a more than two-fold increased risk of increased ADHD symptoms at 6 years follow-up24.However, most previous studies are limited by cross-sectional designs, and are only comparable to a limited extent due to differences in methodological approaches, including large variety in sample sizes or lack of comparison or control groups. ADHD does not necessarily remit during adolescence but persists into adulthood in approximately 30% of affected persons, indicating the chronic nature of this disorder4, whether there are differences in motor performance, i.e. muscular fitness and coordination, between children and adolescents with and without parent-reported ADHD at baseline, and to compare the longitudinal development of motor performance between those with and without ADHD over a period of 11 years.To address this gap, our aim was to investigate based on the longitudinal, representative Motorik-Modul (MoMo) study, which is the in-depth motor module of the long-term, nationally representative German Health Interview and Examination Survey (KiGGS study)25. Overall, there is a trend for better test performance in all motor tests and across all three measurement points for non-ADHD as compared to ADHD subjects.A description of the characteristics of the study samples is given in Table Figures 2 = 56.742; df = 39; p = 0.033; CFI = 0.996; RMSEA = 0.017; muscular fitness: χ2 = 41.316; df = 27; p = 0.038; CFI = 0.997; RMSEA = 0.019). There were high stabilities over time for coordination, represented by high latent correlations between the three measurement points , varying between 0.64 and 0.95. For coordination, all latent correlations were slightly higher in the ADHD group than in the non-ADHD group. For muscular fitness, the latent correlations were slightly lower than for coordination and ranged from 0.45 to 0.83. Two of three latent correlations for muscular fitness between the three measurement points were slightly higher in the ADHD group than in the non-ADHD group. However, the correlation between T1 and T3 was lower in the ADHD than in the non-ADHD group.The model fit of both measurement models was excellent  and teachers . They observed coordination deficits in 34% of boys and 29% of girls with ADHD. The researchers also reported that both adolescents and younger children with ADHD were affected by coordination deficits, compared with healthy controls of the same age. In a follow-up study, Fliers et al.14 recruited 103 children  with a mean age of 10 years and objectively assessed motor performance using the Movement Assessment Battery for Children (MABC). In line with their previous findings, they observed a significantly poorer motor performance in ADHD participants compared to siblings and healthy controls , with significant effects for all three MABC subscales . Overall, 63% of children with ADHD had motor deficits and 34% of the ADHD-group were classified in the clinical DCD-range16. Cho et al.29 also assessed motor function in 58 school-aged children with ADHD with a mean age of 9.6 years, and 70 children in the control group with a mean age of 9.2 years using the Bruininks–Oseretsky test of motor proficiency (second edition). They observed significantly lower mean standard scores on fine motor control , manual coordination , body coordination , strength and agility , and total motor composite score  in children with ADHD than those in the control group. Similar findings were reported by Jeyanthi et al.30 who compared 16 Indian elementary school children aged 8–12 years with ADHD to 19 children without ADHD symptoms. Children with ADHD had significant deficits in motor performance and in gross and fine motor skills compared to those without ADHD symptoms. There was a significant difference in fine coordination (9-Hole Peg Test) between the two groups, i.e., children without ADHD symptoms completed the task faster  than children with ADHD , p < 0.001). In contrast, Colombo-Dougovito21 reported conflicting results based on a study of 51 elementary school-aged children  that underwent seven predominantly endurance and strength-based tasks from "The President's Challenge" fitness program. Surprisingly, children with ADHD performed better in number of push-ups and curl-ups than their peers without ADHD . The inconsistent results may be explained by different methods used in assessing motor performance, in diagnosing ADHD, or by differences in samples, e.g. with regard to representativeness. Some authors, such as Colombo-Dougovito21 and Jeyanthi et al.30 also note their small sample sizes and call for larger-scale future studies on the relationship between motor performance and ADHD.These results are in line with previous studies that reported motor deficits in children with ADHD, particularly with regard to coordination and balance performance21. However, these findings were primarily derived from cross-sectional studies comparing the motor performance of children with and without ADHD.Overall, previous studies showed that children and adolescents with ADHD have motor deficits, particularly in coordination performance, compared to children and adolescents without ADHD; albeit conflicting results for example with regard to muscular strength were reportedTherefore, our longitudinal study builds on previous research by examining developmental trajectories in motor performance of children and adolescents with and without ADHD. Also, we used objective motor performance tests rather than questionnaire data, and also carried out repeated assessments of coordinative performance and muscular fitness in our study participants. Of note, we observed that parent-reported ADHD may serve as a predictor of a decline in coordinative performance that was not only statistically significant but also potentially clinically relevant . Since we observed differences in the developmental trajectories between ADHD and non-ADHD subjects in coordination but not muscular fitness, this may have important implications for the design and conduct of intervention studies in children and adolescents with ADHD, particularly those aiming at increasing motor performance. Also, future studies should focus on different dimensions of motor performance and aim at investigating potential mechanisms that may underlie the observed differences in motor performance developmental trajectories between children and adolescents with and without ADHD. Furthermore, future research should consider physical activity patterns of participants, e.g. activity type, frequency and intensity, as well as physical activity history and setting in which physical activity or sports are carried out.22. Stability rates vary as a function of time and type of study  between 30 and 70%32. This corresponds well with the stability rates of the parent-reported diagnoses of child ADHD in the KiGGS study34. For example, the percentage of ADHD diagnoses that was consistently reported in the KiGGS cohort study 11 years after the baseline assessment was 37.3%34. An important assumption that we made to our analyses, however, is that ADHD is stable over the study period, which does not appear to be the case for all subjects. This may thus be considered a major limitation. However, in line with our hypothesis we observed that participants defined as ADHD subjects had poorer coordination performance over time than non-ADHD subjects although some of the former might have been misclassified. Given this, our results may need to be considered conservative. In addition, the observed ADHD prevalence of 9% in our study sample at baseline appears to be somewhat higher than what has previously been reported for the KiGGS study5. This is due to the fact that we have applied an extended ADHD definition, i.e., in our study, for case number reasons, we pooled participants with parent-reported ADHD diagnoses and the so-called “suspected cases”  according to the case definition of Schlack et al.5. A statistical limitation of the reported analysis is that, with respect to measurement invariance over time, only partial scalar invariance over time was found for the two constructs of coordination and muscular fitness. This suggests that some indicators were not fully invariant over time. Furthermore, the results are representative for German children and adolescents, but may not be generalizable to other populations or children and adolescents from other countries.The findings from our current study should be interpreted by considering its strengths and limitations. The major strengths of this longitudinal study are the population-based, representative sample, the long observation period from 2003 to 2017, and the objective and comprehensive measurement of coordination performance and muscular fitness. In addition, to address the research questions, this study estimated longitudinal SEMs that allow generalizing findings from single indicators or tasks to a higher construct level of coordination or muscular fitness. In addition, measurement invariance was explicitly tested as an important prerequisite for longitudinal analyses. One limitation of the study is that we did not consider physical activity behavior or sports participation in our analyses which may serve as moderators for the development of coordination and muscular fitness among children and adolescents with ADHD. Furthermore, as mentioned initially, ADHD does not necessarily remit over time36. Children and adolescents with ADHD may also benefit from the positive effects of sports and physical activity on the motor, cognitive, and social development38, and there is a growing body of intervention studies examining the effects of physical activity on motor performance in children and adolescents with ADHD40. More longitudinal studies are required in the future that confirm our observations and examine the development of motor performance in children and adolescents with ADHD. It is also important to investigate the potential effects of physical activity interventions in this population and how they may differ from those in individuals without ADHD symptoms. The results of such studies would make it possible to provide children and adolescents with ADHD with even more targeted support regarding the development of critical motor skills.In conclusion, our study provides additional evidence that motor skills of children and adolescents with ADHD develop over time, but remain below the performance levels of peers without ADHD. This underlines the need for increased promotion of physical activity in children and adolescents with ADHD. There is evidence that physical activity improves motor performance, social behavior and cognition, self-confidence, self-esteem, and social skills4, the first representative health monitoring for children and youth in Germany conducted by the Robert Koch-Institute, Berlin. The KiGGS study was set up in 2003 and included a core survey, with MoMo as one of five modular in-depth studies carried out in KiGGS subsamples. The samples were selected randomly from official population registries in 167 sample points across Germany41. The children and adolescents first participated in KiGGS and afterward in MoMo. Three MoMo measurement waves have been conducted so far . The MoMo participants and their parents were contacted individually and invited to test facilities nearby their homes in one of the 167 sample points, i.e., cities, municipalities43. In the test facilities, trained study staff accompanied and assisted the participants individually throughout the completion of physical tests and questionnaires. The MoMo study has a cohort sequence design with a cross-sectional and a longitudinal study arm. This article utilizes longitudinal data, i.e., participants from T1 were tested repeatedly at T2 and T3.The presented analyses are based on data from the long-term, representative Motorik-Modul (MoMo) study in Germany. The MoMo study is a longitudinal study assessing motor performance and physical activity levels of children and adolescents in Germany. MoMo is an in-depth study of the KiGGS studyThe longitudinal MoMo sample includes a total of 2988 individuals who participated in at least two out of three measurement waves. 1268 individuals participated at all three measurement waves. 941 individuals participated at T1 and T2 but not at T3; 167 individuals participated at T1 and T3 but not at T2. As it was possible to enter the study at T2, there are 612 individuals who participated at T2 and T3 but not at T1. Overall, the sample for our analysis comprised 2376 participants at T1 , 2821 participants at T2 , and 2047 participants at T3 . The mean age difference of the samples between T1 and T2 was 6.3 years, and that between T2 and T3 was 5.2 years. A description of the characteristics of the study samples is given in Table 33. For the present analyses, both categories were pooled and defined as ADHD for case number reasons5. According to this definition, 253  participants were classified as ADHD subjects and 2735  participants as non-ADHD subjects at baseline, i.e. their first measurement point. Of those participants with ADHD, 65% were males and 35% were females. Of non-ADHD subjects, 47% were males and 53% were females. There was no significant age difference between groups. Participants were aged between 4.0 and 17.9 years, the mean age of the ADHD group was 9.4 (± SD 3.3) years and that of the non-ADHD group was 9.0 (± SD 3.6) years.In the KiGGS study, participants were classified as ADHD cases if their parents reported a lifetime ADHD diagnosis made by a physician or psychologist. Additionally, participants were considered cases with “suspected ADHD” if they scored ≥ 7  in the hyperactivity/ inattention subscale of the Strengths and Difficulties Questionnaire (SDQ)46. The battery includes tests from validated test batteries that have been used in research studies in the past46. For the analysis presented in this manuscript, we considered six out of twelve tests that cover the motor dimensions coordination and muscular fitness. All tests have been documented in a comprehensive manual45.Motor performance was measured by a standardized test battery based on the systematization by Bös46.Coordination under time pressure was measured by the “jumping-sideways” test. In this test, the number of valid sideway jumps within 15 s carried out on a carpet mat  was assessed. Coordination under precision pressure was measured by the “balancing backwards” test. In this test, the participant is required to balance backwards on wooden beams of 3-m length and different widths: 3 cm, 4.5 cm, and 6 cm. A maximum score of eight steps on each beam without touching the floor is possible, and the sum of steps of two repetitions on each beam was recorded. Balance was measured by the “static stand” test which requires standing on the dominant leg on a wooden construction of 3-cm width and 7-cm height for one minute. The number of floor contacts of the contralateral leg within one minute during one test was recorded and there were no test repetitions46. In the ‘standing long jump’ test, the maximum jump distance in cm from an upright standing position was recorded out of two jumps. In the ‘push-ups’ test, the number of complete push-ups completed within 40 s was recorded and there was no test repetition. In the ‘sit-ups’ test, the number of valid sit-ups completed within 40 s was assessed with no test repetition.Muscular fitness was assessed by the “standing long jump”, “push-ups,” and “sit-ups” testsThe dataset contained missing data, which can be attributed to either the study design or particularities of the variables as described below. The coverage of all variables at all three measurement points was considered good, i.e., 57.3–88.5% at T1, 88.3–90.6% at T2, and 57.1–58% at T3. Due to developmental reasons, the push-up and sit-up tests were only conducted in participants aged 6 years and older. Sit-ups were introduced in T2, thus there are no sit-up data available from T1.25. According to this, a percentile value refers to the percentage of persons in the reference population with the same or lower performance. Thus, a percentile value of 1 represents the lowest possible performance, whereas a percentile value of 99 reflects the best possible performance25. We used percentile curves up to an age of 23 years for older participants. In cases where the age was above 23 years, the determined percentile in the last age range was assigned.Since age and sex differences were not the major interest of the current study, age- and sex-specific percentile curves were calculated for each of the six motor performance tests using the LMS transformation method47. The comparative fit index (CFI) and root mean square error of approximation (RMSEA) are reported as model fit indices, as well as χ2 values. According to Hu and Bentler48, RMSEA values ≤ 0.05 are considered as a good fit, and values between 0.05 and 0.08 are considered as an adequate fit. For the CFI, values of ≥ 0.90 reflect a satisfactory fit, whereas values ≥ 0.95 reflect an excellent fit. As a default option in Mplus, missing data were taken into account using the FIML approach.Descriptive and correlational analyses were conducted using IBM SPSS Statistics for Windows, version 24 . A significance level of 0.05 was set as a threshold to determine statistical significance. As our research questions focus on inter-individual differences, longitudinal analyses were performed by applying a structural equation modeling (SEM) approach in Mplus, version 8.049. For the evaluation of model fit in the invariance analysis, we considered the traditional likelihood-ratio test, which is based on the chi-squared distribution. However, for large sample sizes, the likelihood-ratio test tends to flag statistical significance even if the observed deviations are not practically meaningful. Therefore, we also considered the criterion of CFI difference (ΔCFI) suggested by Cheung and Rensvold50 as it is less vulnerable to sample size. The authors suggested that “a value smaller than or equal to − 0.01 indicates that the null hypothesis of invariance should not be rejected”50. A certain level of measurement invariance was interpreted as established if one of the criteria, i.e., only the likelihood-ratio test, only the CFI difference criterion, or both, applied to the results.As a requirement for the longitudinal analyses, we tested the measurement invariance for coordination and muscular fitness for both (a) ADHD vs. non-ADHD groups, and (b) the invariance over time. The results of the measurement invariance analysis are presented in Supplementary Tables In terms of invariance over groups (ADHD vs. non-ADHD group), full scalar invariance for the measurement models for coordination and muscular fitness could be established. Furthermore, with regard to the measurement invariance over time for both constructs, full metric invariance and partial scalar invariance over time could be established. For coordination and muscular fitness, we freed the intercept for the tasks “Balancing backwards” and “Push-ups within 40 s,” respectively. This indicates that these two single indicators were not completely invariant over time. The presented latent group comparisons are based on the multi-group models with completely fixed factor loadings and intercepts over both groups, as well as the restrictions over the measurement points according to the partial scalar measurement model over time.d effect size, i.e., a value of 0.2 indicates a group difference of 0.2 SD and represents a small effect; 0.5 indicates a medium effect, and d ≥ 0.8 indicates a large effect51. This statistical modeling approach allows us to model the covariance and the mean structure of the variables in both groups at the same time. It should be noted that high stability coefficients do not necessarily imply that there is no change in the latent means. In multi-group analysis, the regression coefficients refer to the rank order of persons in the sample. Thus, a regression coefficient of r = 1 between T1 and T2 indicates that the rank order of participants in the sample remains the same at T1 and T2. Nevertheless, it is still possible that all persons increase their abilities and show a higher latent mean at T2.In the latent multi-group model, the first measurement point in the non-ADHD group was used as a reference value and all other latent means were estimated in accordance with that reference. The differences between estimated latent means were interpreted similarly to the Cohen’s Supplementary Figure 1.Supplementary Figure 2.Supplementary Table 1.Supplementary Table 2.


N = 78, age 6–18 years) with ADHD were tested with the QbTest at baseline, visit 1 (1 month after baseline) and visit 5 (12 months after baseline). The ADHD-Rating scale was rated by investigator interview at the same visits. QbTest results and ADHD-RS ratings showed reductions in symptoms on all cardinal parameters of the QbTest and on all ADHD-RS subscales between baseline and 1 month and between baseline and 12 months. There was a weak but significant correlation between the total change scores on the two measures from baseline to 1 month. Eighteen participants dropped out of the study before visit 5; at baseline, these children showed significantly lower results on the inattention parameter of the QbTest, with faster reaction time and lower variation in reaction time, suggesting they suffered less problems with inattention. Both the QbTest and the ADHD-RS showed robust ADHD symptom improvements indicative of medication effect, and the QbTest results might also predict non-compliance of medication. Further research is warranted to increase knowledge about reliable monitoring of long-term medication and compliance.Documenting effectiveness of ADHD medication is essential throughout the course of treatment. A rating scale and a continuous performance test (CPT) with motion tracking were used to study the effect of ADHD medication including compliance during one year. Children ( Attention-deficit/hyperactivity disorder (ADHD) is characterized by a persistent pattern of symptoms of inattention and/or impulsivity/hyperactivity that clearly affects development and level of functioning in two or more life domains, such as in school, at home, with friends or in leisure activities . SymptomThe community prevalence of ADHD among school aged children is estimated to be between 2 and 7% with an average of about 5% worldwide , 4. SomeThe positive short-term effects of ADHD medication on core ADHD symptoms are well documented . One recContinuous performance tests (CPTs) are designed to capture inattention and impulsivity and are therefore often used in the assessment phase of ADHD. Several studies support that they reliably identify ADHD patients and differentiate them from normative controls . Howeverwww.qbtech.com), which is a CPT with an additional motion tracking system, has been used in assessment of ADHD and in treatment follow-up in children and adolescents [QbTest . The study included patients referred to the Child Neuropsychiatric Clinic (CNC) at Queen Silvia’s Children’s Hospital in Gothenburg, Sweden, from 2014 to 2020. The CNC is an outpatient clinic to which children up until age 18 years are referred for clinical evaluation and neuropsychiatric assessment.Patients between the ages of 6 and 18 years, with ADHD (any subtype/presentation) according to DSM-5 and with an intellectual ability in the normal range (IQ > 70), according to the Wechsler scales and clinical judgment, were invited to participate in the study. The study protocol includes screening, baseline, and 5 follow-up visits. Investigators in the trial were a pediatrician, a child psychiatrist, and two psychologists, with extensive experience in neuropsychiatric assessment and treatment.The screening assessment included a review of inclusion and exclusion criteria and was performed by a physician. The exclusion criteria were: (i) physical or psychological limitations making the QbTest unsuitable, (ii) cardiovascular disease, seizures or other unstable medical conditions that might increase the risk for the patient, (iii) bipolar disorder, conduct disorder, psychosis, severe autism or other severe psychiatric or medical conditions making participation unsuitable, (iv) other psychoactive medication, (v) substance use disorder. The screening assessment also included medical, neurodevelopmental and psychiatric evaluation including clinical evaluation of the ADHD diagnosis and subtype/presentation (according to the DSM-5). ADHD symptom severity was measured with the investigator-rated ADHD-Rating Scale-IV . Global Approximately 2–4 weeks after the screening visit, the baseline assessment was performed by a physician and a clinical psychologist. Inclusion and exclusion criteria were re-assessed and data regarding symptom severity, global functional impairment, vital signs and adverse events were collected by the physician. The overall cognitive ability and adaptive function of the participant was established by the psychologist, either consulting previous medical records or collecting new data through testing with the Wechsler Intelligence Scales (WISC) and The Vineland Adaptive Behavior Scales (VABS), when previous records were older than 2 years or unavailable. The QbTest measuring inattention, impulsivity and hyperactivity was performed. Medication for ADHD  was initiated or re-initiated on an individual basis regarding medication type and dose, tailored to each participant’s needs.One month after baseline, the first follow-up (visit 1) occurred. QbTest was completed for a second time. In addition, data on symptom severity, global functional impairment, treatment compliance, vital signs and adverse events were collected. This procedure was repeated at visits 2 to 5 . For some of the patients where the QbTest could not be completed at visit 1, efforts to complete the test at a later visit were made.In addition to the repeated procedure mentioned above, the QbTest and a follow-up assessment of intellectual ability and adaptive functioning were completed by the clinical psychologist at the twelve-month visit, and comorbidity status was assessed by the physician.The participants continued their medication at all post-baseline visits. Compliance (number of days with missed doses/number of days in period) was measured by parent and patient report, and was generally high .The present study includes data from baseline, visit 1 and 5. Data analysis focuses on the results on the QbTest and the ADHD-RS symptom ratings.n = 53 (68%) with combined presentation and n = 24 (31%) with inattentive presentation, and one participant (1%) with an unspecified ADHD diagnosis. Twenty participants (26%) also had an autism spectrum disorder (ASD) diagnosis. An additional 15 participants (19%) were described in their medical records to have autistic traits but without meeting criteria for a diagnosis. Other diagnosed neurodevelopmental “comorbid” disorders in the study group were Oppositional-Defiant Disorder , and subclinical ODD in an additional 37 participants (47%), i.e., with several symptoms but not meeting full diagnostic criteria), dyslexia (n = 7), developmental coordination disorder (n = 7), tic disorder (n = 5), and language disorder (n = 2). A large majority were treated with stimulants , a few with guanfacine  or atomoxetine (n = 1). The dropout rate after baseline was 18 , meaning that 60 of 78  and 38 boys (63%)) were followed from baseline to visit 5 (12 months after baseline). All but one of those who dropped out of the study were treated with stimulants, the single other patient with guanfacine. Fourteen of the 18 participants who dropped out of the study discontinued their ADHD medication - dropout reasons were lack of efficacy or adverse effects , wished to get by without medication , moved to other area (n = 2), other unrelated medical reason (n = 2). Four participants continued their medication - dropout reasons were move to other area (n = 3), lack of time (n = 1). Since they did not continue follow-up in the study, the duration of continued medication is unknown. Non-ADHD medication was limited to Melatonin (n = 8) or Hydroxizine (n = 2) for sleep, and Sertraline for anxiety/mood (n = 2). All patients also received the “standard” psychoeducation to the child, the parents, and the school about the child’s individual needs, which is given to all families after neuropsychiatric assessments at our center. No patients received Cognitive Behavioral Therapy (CBT) or other psychological interventions.The first consecutive 95 participants in the QbTest trial were selected for the present study. Fourteen (15%) of these participants declined participation in the trial after screening and three (3%) were excluded due to a diagnosed intellectual disability, leaving 78 participants to be included at baseline, 29 girls (37%) and 49 boys (63%). Mean age at baseline was 12.4 years (SD = 3.6) with an age range of 6–18.1 years. All participants had a confirmed diagnosis of ADHD, The QbTest , 21 is aThree cardinal parameters are presented in the test report: QbInattention, QbImpulsivity and QbActivity. These in turn are derived from a total of 10 parameters, four measuring activity , and six measuring impulsivity and inattention . The subject’s performance is compared to a normative data set of 1307 participants, matching age and gender. The results are presented as age- and gender-specific standardized scores  around a normative mean of 0 (standard deviation = 1). Positive scores (> 0) indicate more problems with inattention, impulsivity and hyperactivity, whereas negative scores indicate normal to extremely good performance .The Wechsler intelligence scales , 23 are The Vineland Adaptive Behavior Scales (VABS), Second Edition  is an inThe mean VABS total score at baseline in the total group was 79.4 , indicating borderline impaired adaptive functioning in the total group but with individual variation ranging from severely impaired to average. No significant score difference was found between those who were interviewed and those who completed the parent rating scale.The ADHD-RS-IV  is an 18The statistical software SPSS, version 25, was used for all data analysis. Non-parametric analyses were used due to the relatively small sample size and to reduce the impact of any outliers. The chi-square test for independence was used to calculate whether gender, ADHD subtype, comorbid ASD diagnoses and number of diagnoses were related to dropout. The Mann–Whitney U test was used to compare QbTest results, age, FSIQ and VABS between the drop-out group and remain group. Mann–Whitney U test was also used to check for gender differences in the QbTest results. The Friedman tests and post hoc Wilcoxon signed-rank tests with Bonferroni-adjusted pairwise comparisons were used to calculate the changes in QbTest and ADHD-RS results across the three different time points, before medication, at visit 1 and at visit 5. Finally, to examine individual differences in response to treatment, change (delta) scores were calculated by subtracting the baseline and the visit 1 scores on the QbTest and the ADHD-RS. Spearman correlation analyses were conducted between the change scores to examine whether these indices were associated.Fifty-one participants out of the 60 who remained in the study until visit 5 (85%) completed the QbTest across all three time points Table .Table 1QQ-values > 12, p values < 0.01). Bonferroni-adjusted pairwise comparisons showed consistently that there were significant reductions in symptoms from baseline compared with visit 1 and visit 5, whereas no significant change was observed between visit 1 and visit 5. No significant differences in the QbTest results related to gender were found.A series of non-parametric Friedman signed-rank tests were conducted to examine how scores from the QbTest and ADHD rating scale changed from baseline to visit 1 (after one month) and to visit 5 (after twelve months). Results were consistent in the sense that the effect of time point was significant for all variables  from baseline to 1-month follow-up, with improved performance on the QbTest associated with improved ratings on the ADHD rating scales. Regarding sub-scores on the ADHD-RS scale and QbTest, there were moderate significant positive correlations between the QbTest total change score and the ADHD-RS hyperactivity change score. Also, there were moderate significant correlations between the QbTest impulsivity parameter change score and the ADHD-RS total and hyperactivity change scores, respectively. The measures of inattention and of activity change scores on the QbTest did not correlate significantly to any of the other parameters  scores on the QbTest total and the change scores on ADHD-RS total , compared to the participants who remained , U = 632.5, z = 2.36, p = 0.019, r = 0.28. When extending the analysis to calculate the standard parameters comprising the cardinal parameter of inattention, separately, the parameters measuring reaction time and reaction time variation differed but not the measure of omission errors. The group of dropout participants had both faster reaction time  and lower reaction time variation  than the remain group  on both measures, respectively . No other cardinal parameters or test parameters on the QbTest differed significantly between the drop-out and remain groups , and not in all subdomains. Change scores from baseline to 1 month of the ADHD-RS hyperactivity subscale correlated with the QbTotal change score and with QbImpulsivity change score but not with QbActivity change score. We also found that ADHD-RS total change score correlated with QbImpulsivity change score. Bijlenga et al.  who simiSome previous studies have shown that the ability of QbTest to identify ADHD in a clinical sample was moderate, and the ability to differentiate between ADHD subtypes unsatisfactory , 15. HalOur study findings suggest that the reasoning of Toplak et al.  may alsoOur data support that all the parameters on the QbTest show significant improvement after 1 month and 12 months of medication. We also found some indications that information gained from the QbTest could predict adherence to ADHD treatment. The group who dropped out of the trial, of which a majority stopped medication, had on average less inattentive problems, faster reaction time and less variation in reaction time on the baseline QbTest than those who stayed on medication throughout the study. However, this difference was not seen in the activity parameters or in the impulsivity parameters, nor in the ADHD-RS. No other factors, such as FSIQ, age or gender, differed between the two groups. This might suggest that a CPT test could be a more sensitive indicator about who will continue medication and who will not, since this did not appear to be fully captured in the rating scales. However, to confirm this finding replication is required in a larger sample as well as analyzing subjective reasons for dropping out of the study in more depth.The strengths of the present trial are the relatively long follow-up period of 1 year, and the naturalistic study design in which medication was tailored to the needs of each participant, increasing the ecological and clinical validity of the trial. Furthermore, in many studies, cases with ASD are excluded. This was not an exclusion criterion in this trial, which may make the results more applicable to the group of patients with overlapping diagnoses, often seen in clinical practice. In the light of the previous study by Bijlenga et al.  in an adOur study also has important limitations. It is an open-label trial with no control group, which reduces the evidence strength since the longitudinal changes may rely on other factors than medication. Also, the sample size is relatively small. The significant difference found on the inattention parameter of the QbTest between those completing the study and those who dropped out merits further investigation in a larger sample. Another limitation is the heterogeneity of the drop-out group regarding medication status, since 14/18 participants stopped taking medication, whereas 4 continued medication but dropped out of the study.Both ADHD-RS and QbTest results appear to capture medication effect, but weak correlations between the two measures suggest that their role in medical follow-up might be complementary rather than interchangeable. Differences in the severity of inattention problems may affect the need and motivation for continued ADHD medication and/or participation in long-term follow-up. Further investigations of the reasons for non-compliance are needed. Results from this study suggest that looking at results from neuropsychological tests including CPT with motion tracking, may shed further light on the cognitive and behavioral profiles of those who benefit from medication and those who do not.