Crop rotation diversity has many benefits for integrated crop–livestock production systems. Cereal rye deserves consideration for use as a component of an integrated crop–livestock system as rye is a multi-use crop than can be grazed, harvested for forage, or harvested for grain and straw. Previous research from this research center has indicated that processed rye (processed to a processing index of 78.8%) is a suitable ingredient  for use in cattle-finishing diets. Processing grains prior to feeding is proven to enhance growth performance in feedlot cattle. A major impediment to the use of rye in finishing diets is that the rye should be processed prior to feeding, and processing rye requires different equipment settings than for processing dry-rolled corn. The objectives of the present study were to evaluate the impact of the complete substitution of dry-rolled corn with unprocessed rye in finishing diets fed to feedlot heifers on growth performance, dietary net energy use, and carcass traits.n = 7 heifers/pen and 4 pens/treatment). Treatments included a finishing diet that contained 60% grain (diet dry matter basis) as dry-rolled corn (DRC) or unprocessed rye grain (RYE). On study day 14, all heifers were consuming the final diet and were implanted with 200 mg of trenbolone acetate and 28 mg of estradiol benzoate . The RYE heifers had decreased (p ≤ 0.01) final body weight, average daily gain, and gain efficiency; however, they tended (p = 0.08) to have a greater dry matter intake compared to DRC heifers. RYE heifers had decreased (p ≤ 0.01) observed dietary NE and decreased (p ≤ 0.01) observed-to-expected dietary NE ratio for maintenance and gain compared to DRC heifers. The dressing percentage, 12th rib fat thickness, ribeye area, and the distribution of yield and quality grades were not altered (p ≥ 0.12) by dietary treatment. The hot carcass weight, calculated yield grade, estimated empty body fat (EBF), and body weight at 28% EBF decreased (p ≤ 0.02) and retail yield increased (p = 0.01) in RYE compared to DRC heifers. These data indicate that unprocessed rye is a palatable feed ingredient for inclusion in finishing diets for beef cattle and that rye inclusion only minimally influences the carcass quality grade. The feeding value of unprocessed rye is considerably less (21.4%) than that of dry-rolled corn using current standards and approximately 91% of the NE value of processed rye (processing index = 78.8%). Rye grain fed as processed or unprocessed grain has an NE value that is less than 90% of that of dry-rolled corn. Continental crossbred beef heifers were used in a randomized complete block design experiment to evaluate the effects of replacement of dry-rolled corn with unprocessed rye on the finishing-phase growth performance and efficiency of dietary net energy (NE) use. Fifty-six heifers (433 ± 34.0 kg) were transported 241 km from a sale barn in North Central South Dakota to the Ruminant Nutrition Center in Brookings, SD. Heifers were blocked by weight grouping and allotted to treatment pens ( Crop-rotation diversity has many benefits for integrated crop–livestock production systems. These include yield resiliency and crop yield increases compared to single- or two-crop rotation systems . When coCereal rye can be used as a component of an integrated crop–livestock system. Rye is a multi-use crop than can be grazed, harvested for forage, or harvested for grain and as straw. In addition, rye is harvested earlier in the season compared to other traditionally used row crops, allowing for greater flexibility related to manure management or use of shorter-growing-season forage crops to be fed to livestock if weather and market conditions are appropriate. Hybrid rye germplasms, which have recently become available to the United States from Europe, are of particular interest because of a greater yield potential and lesser ergot incidence compared to open-pollinated rye varieties .Previous research from this research center indicated that processed rye (processing index of 78.8%) is a suitable ingredient  for use in finishing diets [The objective of this experiment was to determine the effects of complete replacement of dry-rolled corn with unprocessed rye on intake, cattle growth performance, and feed conversion efficiency in finishing heifers. Our hypothesis was that unprocessed cereal rye has a lower NE value compared to dry-rolled corn and that rye can be substituted for dry-rolled corn in finishing diets, but would result in poorer growth performance and feed efficiency with no negative effects on carcass characteristics.The animal care and handling procedures used in this study were approved by the South Dakota State University Animal Care and Use Committee .3, and BRSV ) and clostridials , and administered pour-on moxidectin  according to label directions. On 1 September 2020 , all heifers were again individually weighed, and this body weight was used for allotment. The heifers were blocked by weight grouping and allotted to their study pens the following day (n = 7 heifers/pen and 4 pens/treatment), and test diets were initiated. Treatments included a finishing diet that contained either (1) dry-rolled corn (DRC) or (2) unprocessed hybrid rye (RYE) as the grain component. On study day 14, all heifers were consuming the final diet and were implanted with 200 mg of trenbolone acetate and 28 mg of estradiol benzoate ; an implant retention check was performed on day 42. The initial body weight (BW) was the BW captured on September 2, 2020. Following study initiation, the heifers were transitioned to a high-concentrate diet over the course of 14 d  and offered to heifers in equal amounts at each feeding. Orts were collected, weighed, and dried in a forced-air oven at 100 °C for 24 h to determine the DM content if carryover feed went out of condition or was present on weigh days. If carryover feed was present on weigh days, the residual feed was removed prior to BW measurements. The dry matter intake (DMI) of each pen was adjusted to reflect the total dry matter (DM) delivered to each pen after subtracting the quantity of dry orts for each interim period. The actual diet formulation was based upon weekly DM analyses (drying at 60 °C until no weight change) and corresponding feed-batching records. After weekly DM determination  were transported 241 km from a sale barn in North Central South Dakota to the Ruminant Nutrition Center (RNC) in Brookings, SD, on 24 August 2020. Upon arrival to the RNC, the heifers were housed in 7.62 m × 7.62 m concrete surface pens with 7.62 m of linear bunk space and provided ad libitum access to long-stem grass hay, and water. The following day heifers were offered a common 60% concentrate diet at 2% of body weight. On 27 August 2020 , all heifers were individually weighed , given a unique identification ear tag, vaccinated for viral respiratory pathogens  and carcass-adjusted final BW (FBW) from hot carcass weight (HCW) calculated as: HCW/0.625. The energetic assessment period was from day 14 to 77 using the BW from day 14, shrunk by 4%, and the FBW. The average daily gain (ADG) was calculated as the difference in the BW for the period of interest, divided by the days in that period, and feed efficiency was calculated as ADG/DMI.The heifers were harvested when they were visually appraised to have 1.27 cm of rib fat (RF). They were shipped the afternoon following the final BW determination and harvested the next day at Tyson Fresh Meats in Dakota City, NE, USA. The heifers were co-mingled at the time of study termination and remained as such until 7:00 a.m. the morning after shipping. The HCW was captured immediately following the harvest procedure. Video image data were obtained from the plant for the ribeye area, RF, and United States Department of Agriculture (USDA) marbling score. The yield grade was calculated according to the USDA regression equation . The dre1.119 × 0.0608W0.75, where W is the mean BW , a = −0.41EM, b = 0.877EM + 0.41DMI + EG, c = −0.877DMI, and NEg was determined as 0.877NEm − 0.41 [Observed dietary NE was calculated from the daily energy gain (EG) that was determined using the 1984 large-frame and compensating yearling heifer equation : EG = (Carcass-adjusted ADG from day 14 to 77)W in kg; ). The ma; [W0.75  of the dm − 0.41 ,17. The Growth performance, carcass traits, and efficiency of dietary NE use were analyzed as a randomized complete block design using the GLIMMIX procedure in SAS 9.4  with a pen as the experimental unit. The model included the fixed effect of dietary treatment, and a block  was included as a random variable. Least-squares means were generated using the LSMEANS statement of SAS, and treatment effects were analyzed using the PDIFF pairwise comparisons and the LINES option in SAS 9.4. The distribution of USDA yield and quality grade data was analyzed as binomial proportions in the GLIMMIX procedure of SAS 9.4 with fixed and random effects in the model, as described previously. An α-value of 0.05 or less determined significance, and tendencies were discussed between 0.05 and 0.10.p = 0.72). The final BW decreased by 6.8% in RYE compared to DRC heifers (p = 0.01); accordingly, the ADG decreased by 27.6% in RYE compared to DRC heifers (p = 0.01). It was demonstrated previously that complete replacement of dry-rolled corn with processed rye results in decreased growth performance in finishing steers [p = 0.08); as such, feed efficiency decreased by 32.3% in RYE heifers compared to DRC heifers. Studies indicated that as higher amounts of processed rye are included in finishing diets, the DMI and gain efficiency linearly decrease [Growth performance responses are given in g steers . The cumdecrease .p = 0.50). As previously mentioned, the final BW decreased by 6.8% in RYE heifers compared to DRC heifers. During the energetics assessment period, the ADG decreased by 29.0% (p = 0.01), and the DMI tended to be greater by 7.0% (p = 0.08) in RYE compared to DRC heifers. Complete replacement of dry-rolled corn with processed rye was shown to result in decreased intake and growth performance in finishing steers [p = 0.01). Observed dietary NEm increased by 34.7% (p = 0.01) and observed dietary NEg increased by 46.8% in DRC compared to RYE heifers (p = 0.01). The observed-to-expected NE ratio for maintenance and gain also increased in DRC compared to RYE heifers (p ≤ 0.01). The observed-to-expected NEm ratio for the DRC diet was 1.14, far from the expected ratio of 1.00. Thus, applying substitution, the corresponding NEm value for dry-rolled corn is 2.73 Mcal/kg. This NE value is much greater than what current standards indicate [Data from the energetics assessment period are provided in g steers . Alteratindicate ,17. Accoindicate ,19,20.p = 0.01). The dressing percentage was not influenced (p = 0.12) by dietary treatment. Reductions in HCW and dressing percentage were demonstrated when heifers were fed processed rye instead of dry-rolled corn [p ≥ 0.14) due to dietary treatment. The marbling score tended to be greater by 12.7% in DRC heifers compared to RYE heifers (p = 0.10). The calculated yield grade increased by 9.6% and the retail yield decreased by 1.0% in DRC heifers compared to RYE heifers (p ≤ 0.01). The estimated EBF was greater by 5.1% in DRC compared to RYE heifers (p = 0.02), and the final BW at 28% EBF decreased by 12 kg in RYE heifers compared to DRC heifers (p = 0.01). There was no influence (p ≥ 0.13) of dietary treatment on the distribution of the USDA yield or quality grade. Studies have indicated that partial or complete replacement of dry-rolled corn with rye has a minimal influence on the distribution of the USDA yield or quality grade [Carcass trait responses are given in led corn . The ribWe conclude that unprocessed rye is a palatable feed ingredient for inclusion in finishing diets for beef cattle, as indicated by a greater DMI compared to dry-rolled corn, and it only minimally influences the carcass quality grade distribution when considering the reduced carcass fatness of heifers fed unprocessed rye. The feeding value of unprocessed rye is considerably less (21.4%) than that of dry-rolled corn and approximately 91% of the net energy value of processed rye. Hence, gain and gain efficiency are correspondingly lower when heifers are fed unprocessed rye as a replacement for dry-rolled corn in feedlot finishing diets fed to cattle.


To estimate phenotypic and familial association between early-life injuries and attention deficit/hyperactivity disorder (ADHD) and the genetic contribution to the association using polygenic risk score for ADHD (PRS-ADHD) and genetic correlation analyses.Children born in Denmark 1995-2010  were followed from age five until a median age of 14 (IQR: 10–18 years). Using ICD-10 diagnoses, we estimated hazard ratios (HRs) and absolute risks of ADHD by number of hospital/emergency ward treated injuries by age five. In a subsample born 1995 to 2005 with genetic data available , we estimated incidence rate ratios (IRR) for the association between PRS-ADHD and number of injuries before age five and the genetic correlation between ADHD and any injury before age five.Injuries were associated with ADHD , in males  and females , with a dose-response relationship with number of injuries. The absolute ADHD risk by age 15 was 8.4% (3+ injuries) vs. 3.1% (no injuries).st- than 2nd-degree relatives. PRS-ADHD was marginally associated with the number of injuries in the general population , with a genetic correlation of 0.53 (0.21-0.85).ADHD was also associated with injuries in relatives, stronger in 1Early-life injuries in individuals and their relatives were associated with a diagnosis of ADHD. However, even in children with the most injuries, more than 90% were not diagnosed with ADHD by age 15. Despite a low positive predictive value and that the impact of unmeasured factors such as parental behaviour remains unclear, results indicate that the association is partly explained by genetics, suggesting that early-life injuries may represent or herald early behavioral manifestations of ADHD. In typically developing children, impulsive and risk-taking behaviour is more common at a younger age, as are injuries. The ability to plan ahead and foresee unsafe situations improves with age, as executive brain functions mature, and this cognitive maturation is paralleled by a decrease in the risk of injuries. Since the 1960s, studies of pre-school children have found injuries to be correlated with inattention, hyperactivity, and impulsivity. Meta-analyses provide strong evidence that individuals with ADHD have increased risk of emergency ward visits, and unintentional injuries. Traumatic brain injury (TBI) may be causally linked to ADHD, whereas literature on whether other injuries early in life tend to precede a diagnosis of ADHD is sparse.Attention deficit/hyperactivity disorder (ADHD) is more difficult to validly diagnose before age five, as the diagnostic criteria relates mainly to older children, and the clinical presentation of ADHD may differ in pre-school and school. Besides subthreshold symptoms of inattention, hyperactivity, and impulsivity, little is known on early-life manifestations of ADHD. with heritability estimates of around 74%. Independently derived ADHD polygenic risk score (ADHD-PRS) predicts ADHD-status, subthreshold ADHD-symptoms, and other neurodevelopmental traits in the general population. Genome-wide association studies (GWAS) recently identified several genome-wide significant loci, and estimated that common genetic variants explain 22% of the heritability of ADHD.ADHD is a neurodevelopmental disorder, and depression and ADHD are genetically correlated. A twin study suggest a small genetic contribution to injuries before age five, with most variance explained by child-specific environmental effects (including ADHD-symptoms) and familial effects, several of which are known risk factors for ADHD, such as low socioeconomic status (SES), and young and single parenthood. Similarly, a Finnish twin study found little indication of a genetic contribution to injuries in adults. However, Acar et al. found that fathers of children admitted to the emergency ward due to an injury had higher ADHD symptom scores themselves, compared with fathers of children acutely admitted to hospital due to a throat infection. The cross-generational association between ADHD and injuries may be explained by home-environment, parental behaviour, parent-child interactions or genetics.Less is known about the aetiology of early-life injuries. Recently, a Danish register-based study found maternal depression to be associated with injuries during early childhoodWe hypothesize that similar to early subthreshold symptoms of ADHD, injuries before age five are early manifestations of ADHD and may associate with ADHD genetic liability. Using a large Danish population-based cohort, our aims were to (1) estimate the association between the number of injuries before age five and a later diagnosis of ADHD, (2) estimate the association between early-life injuries in siblings and parents and the risk of ADHD, (3) estimate the association between ADHD-PRS and the number of early-life injuries in the general population as well as the genetic correlations between ADHD and early-life injuries. holds data on personal identification number, sex, date of birth, and continuously updated vital status of all persons living in Denmark, enabling accurate linkage of individual-level data across registers. The Danish National Patient Register (DNPR) and the Danish Psychiatric Central Research Register (DPCRR) holds data on all-inpatient admissions, and out-patient and emergency ward visits from 1995 onward and provided data on injuries and psychiatric disorders diagnosed in hospital departments. Diagnoses were registered according to the International Classification of Diseases, 8th revision (ICD-8) in 1969-93, and 10th revision (ICD-10) since 1994. The Danish Neonatal Screening Biobank provided dried bloodspots and The Integrative Psychiatric Research (iPSYCH) consortium processed these for genotyping.Since 1968 the Danish Civil Registration System DNA collection, genotyping and quality control has been described elsewhere. Included individuals fulfilled these criteria: Singletons, born between January 1, 1995 and December 31, 2005, alive and residing in Denmark at age 1, both parents born in Denmark and no diagnosis of TBI before age five. We further restricted to individuals of European ancestry and for genetic correlation analyses to unrelated individuals. See Population-based cohort study of individuals born in Denmark of Danish-born parents between January 1, 1995 and Dec 31, 2010, and living in Denmark at age five. Individuals were excluded if they fulfilled any of these criteria before age five: ADHD-diagnosis or filled prescriptions of ADHD-medication, TBI, or disease of the nervous system. Genetic analyses relied on the population-based iPSYCH2012 case-cohort, including all ADHD cases (N=18 726) and 2% random sample of the general population (sub-cohort) (N=30 000).We defined early-life injury in cohort members and their siblings as a hospital-treated injury before age five. This age cut-off ensured complete coverage of injuries and that injuries preceded the ADHD-diagnosis. We included diagnoses of injuries from all hospital contacts , excluding TBIs  and contParental injuries were based on ICD-8/10 codes  and defiUsing data from DPCRR and DNPR, we defined ADHD as the first hospital contact to a psychiatric, paediatric or neurological department with a diagnosis of ADHD  after the age of five. ADHD subtypes included combined (F90.0) and inattentive subtype ADHD (F98.8). Based on funding available at the time, sampling of ADHD-cases for the iPSYHC2012 case-cohort sample only included cases with ICD-10 code F90.0. and BOLT-LMM software and combined in a linear combination to obtain final PRS  were estimated by Cox regression for the association between early-life injuries and ADHD, using age as the underlying time-axis. Analyses were repeated for the two ADHD subtypes and in strata of parental education (completed high-school yes/no). Individuals were followed from their 5Second, we investigated familial co-aggregation of injuries and ADHD, estimating HRs for associations between injuries in mothers, fathers, full siblings, maternal and paternal half-siblings (exposures), and ADHD in the index child (outcome). All analyses were adjusted for sex, birth cohort , and the interaction between sex and birth cohort. Analyses of parental injuries were additionally adjusted for the parent’s birth cohort . Analyses of average number of injuries in siblings were additionally adjusted for the number of full/maternal half/paternal half siblings . IRRs correspond to a relative increase in the rate of early-life injuries for a one standard deviation increase in the PRS-ADHD. These analyses were adjusted for sex, age and calendar year at first ADHD diagnosis (both continuous), genotyping wave , and the first four principal components (PCs) to adjust for potential remaining population stratification. The PCA method for deriving the PCs used for ancestry outlier removal and adjustment is described in detail elsewhere. Follow-up started at birth and ended at age five, a diagnosis of disease of the nervous system (ICD-10 codes G00-G99), death or emigration from Denmark, whichever came first.Third, incidence rate ratios (IRRs) were estimated by negative binomial regression with the logarithm transformed time-at risk as an offset for associations between PRS-ADHD and the number of injuries before age five. Due to the iPSYCH2012 case-cohort sampling design, including all ADHD cases and a 2% random sub-cohort, inverse sampling probabilities were applied as weights.All analyses were repeated in males and females and applied a cluster-robust variance estimator with clusters defined as individuals having the same mother and father and for half-sibling cohorts defined as individuals having the same mother or father.2SNP) and genetic correlation (rg) between early-life injuries and ADHD were estimated using BOLT-REML software among the iPSYCH2012 ADHD cases and subcohort. SNPs were filtered and LD pruned according to BOLT-REML suggested guidelines .The main analyses were conducted using Stata 15.iPSYCH is approved by the Danish Data Protection Agency, the Danish Health Data Authority, the Danish Scientific Ethics Committee, and the Danish Neonatal Screening Biobank Steering Committee.The study population consisted of 786 543 children born in Denmark between 1995 and 2010, followed for a total of 6 910 193 person-years, until a median age of 13.9 years (IQR: 9.9 – 18.0). In total, 92 691 (11.8%) individuals sustained an injury before age five and 23 107 (2.9%) individuals were diagnosed with ADHD after five years of age and during follow-up. In 34 452 (4.4%) study participants, follow-up ended due to death (n=423), emigration from Denmark (n=11 704), or TBI (including concussion) after age five (n=22 053).Having sustained at least one injury before age five was associated with a subsequent diagnosis of ADHD . Furthermore, an increasing number of injuries was associated with an increased risk of ADHD, suggesting a dose-response relationship . ChildreWe found no significant interaction between injuries (yes/no) and sex (p = 0.455) and associations were similar in sex-specific analyses .We observed a dose-response relation with higher cumulative incidence of ADHD by increasing number of injuries before age five . By 15 yst-degree relatives, including mothers , fathers  and full siblings   and paternal half siblings . The number of injuries in full siblings also increased the HR of ADHD in the index child incrementally. An average number of >2 injuries in full siblings was associated with an increased risk of ADHD , when compared to the risk of ADHD in full siblings without injuries. Similar sex-specific trends were observed (ADHD was associated with injuries in 133-1.46) . The assobserved .g = 0.53 (95% CI 0.21-0.85) , a higher PRS-ADHD was associated with a higher number of injuries before age five , with similar-sized estimates were for males and females . In unre21-0.85) .In this population-based cohort study of almost 800 000 children, early-life injuries were associated with a subsequent clinical diagnosis of ADHD. Having sustained at least one injury before age five resulted in a 64% higher risk of ADHD, relative to those without injuries before age five. The association showed a dose-response pattern, as increasing number of injuries was associated with incremental increased risks of ADHD. Children with three or more injuries had a 2.5-fold increased risk of subsequently being diagnosed with ADHD after age five, when compared with children without early-life injuries. and that ADHD-medication reduces this risk. ADHD is associated with more collisions when riding a bicycle, more risk-taking behaviour, impulsive decision-making, and in adults higher rates of serious traffic accidents. The risk of fatal injuries is also increased in individuals with ADHD and accidents is their most common cause of death.Decades of research provide strong evidence that ADHD is associated with a 39-53% increased risk of injuries, Here, we show for the first time using an objective measure, that early-life injury is strongly associated with later ADHD risk and that increasing number of injures before age five may be a marker of ADHD liability already prior to diagnosis.Other than symptoms of inattention, hyperactivity and impulsivity, little is known about what characterises children with ADHD in their first years of life, prior to being diagnosed. Some retrospective studies have found that children attending emergency wards have higher rates of ADHD-symptoms, than other children.st-degree than 2nd-degree relatives, and in the general population, genetic liability for ADHD, as indexed by ADHD polygenic risk scores, was marginally associated with early-life injuries. However, we also found a genetic correlation of 0.53 between ADHD and early-life injuries; a higher correlation than ADHD shows with other neuropsychiatric disorders. This suggests that their underlying genetic liability is partly shared. However, the SNP-based heritability of injuries was low (6%), in keeping with findings from previous twin studies that have found little evidence for a genetic contribution to injuries. Other shared familial risks could be related the psychosocial environment in the family, which is associated with both injuries and ADHD . Finally, our observations could also be explained by gene-environment correlation whereby genetic liability to ADHD increases the risk for environmental stressors; a phenomenon that to date has only been observed for social stressors.We also found familial co-aggregation of injuries and ADHD, which suggests that the aetiology of the two phenotypes includes shared familial risks. The associations were stronger between 1 Based on that, the concept of ‘accident proneness’ was presented in 1926. During the 1940s and 50s, several publications supported this concept, linking it with different psychiatric symptoms, including distractibility, impulsivity, aggression, impatience, opposition, restlessness, and hyperactivity, and also documenting familial aggregation of accidents. Later, the concept of ‘accident proneness’ was criticised for placing the responsibility of sustaining an injury solely on the injured individuals themselves. While our data suggest shared liabilities for injuries and ADHD, and that individuals more prone to injuries are more likely to have ADHD, this still explains very little of the variance in early-life injuries.We find that the risk of injuries may be linked to individual and familial characteristics, and not merely caused by external factors, which supports a hypothesis suggested in 1919. yet the validity of this ICD-diagnosis in the Danish registers is not fully known. However, we expect this misclassification to be non-differential by groups of the injuries exposure, meaning our estimates of association might be underestimated. Fourth, while BOLT-REML seems to provide robust estimates for the genetic correlation in a setting with shared controls and case-control ascertainments, the estimated SNP-based heritabilities are likely underestimated and should be interpreted with caution. Finally, even in children with the most injuries, more than 90% were not diagnosed with ADHD. Hence, with such a low positive predictive value our data does not support obtaining information about early-life injuries should be recommended as part of the clinical assessment for ADHD, or that such information would improve diagnostic validity.Major strengths of our study include the longitudinally recorded individual-level data on a population-based national sample in a country with free universal health. Furthermore, we were able to link the individual information with information in parents and siblings and to link with genetic information on all with an ADHD diagnosis and randomly selected controls. The randomly selected cohort enables us to present valid estimates of absolute risk in the general population, something most studies cannot. Furthermore, our similar findings among individuals of high and low educated parents suggest that the association between injuries and ADHD is only minimally biased by differences in hospital-seeking behaviour due to SES. However, our study also has some important limitations. First, we did not estimate the potentially mediating or moderating effects of other mental disorders often comorbid to ADHD (e.g. conduct disorder). Second, with TBI excluded we did not hypothesize a direct causal relationship between early injuries and ADHD and hence, we decided not to adjust analyses for e.g. parental SES or mental disorders. However, the impact of home-environment, parental behaviour, or parent-child interactions was not assessed and hence we were not able to fully disentangle the genetic and non-genetic contributions to the observed association. Third, we relied on previously validated data from the national health registries on clinical diagnoses of ADHD from hospital departments, and persons diagnosed by child and adolescent psychiatrists in private practices were not included. According to Danish guidelines, inattention without hyperactivity should be coded as F98.8,To conclude, our study adds important new findings to the literature on what characterizes children with ADHD, years before they are diagnosed and the data suggest that early-life injuries may be an early manifestation of impairment and risks related to symptoms of ADHD. Furthermore, the observed association between early-life injuries and ADHD is partly due to shared familial and genetic risks.Supplement


Compton scatter photons are generally considered problematic in nuclear medicine imaging. Therefore, efforts are being made to minimize the involvement of these photons by employing some special strategies in daily practice. Basically, photons scattering at a small angle and traveling in the proper direction stand a chance of getting recorded and thereby participate in the image formation. These photons may create artifactual hot spots in the vicinity of a region with high concentration of radioactivity. The present study focuses on the negative impact of such photons during routine imaging in clinical setting, through an artifact detected in technetium-99m-IDA hepatobiliary scintigraphy, with the purpose of highlighting this issue to the nuclear medicine practitioners.


Attention deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder characterized by age-inappropriate symptoms of inattention, impulsivity, and hyperactivity that persist into adulthood in the majority of the diagnosed children. Despite several risk factors during childhood predicting the persistence of ADHD symptoms into adulthood, the genetic architecture underlying the trajectory of ADHD over time is still unclear. We set out to study the contribution of common genetic variants to the risk for ADHD across the lifespan by conducting meta-analyses of genome-wide association studies on persistent ADHD in adults and ADHD in childhood separately and jointly, and by comparing the genetic background between them in a total sample of 17,149 cases and 32,411 controls. Our results show nine new independent loci and support a shared contribution of common genetic variants to ADHD in children and adults. No subgroup heterogeneity was observed among children, while this group consists of future remitting and persistent individuals. We report similar patterns of genetic correlation of ADHD with other ADHD-related datasets and different traits and disorders among adults, children, and when combining both groups. These findings confirm that persistent ADHD in adults is a neurodevelopmental disorder and extend the existing hypothesis of a shared genetic architecture underlying ADHD and different traits to a lifespan perspective. Attention deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder that severely impairs the daily functioning of patients due to age-inappropriate levels of impulsivity and hyperactivity, and/or difficulties in focusing attention . ADHD haADHD is a multifactorial disorder with heritability averaging 76% throughout the lifespan –5. ThereThe presentation of ADHD symptoms changes from childhood to adulthood, with lower levels of hyperactivity in adulthood but a high risk for ongoing attention problems, disorganization, and emotional dysregulation , 14. As Several risk factors measured in childhood predict the persistence of ADHD symptoms into adulthood, such as the presence of comorbid disorders, the severity of ADHD symptoms, being exposed to psychosocial adversity, as well as having a high polygenic risk score (PRS) for childhood ADHD –28. TwinGiven this background, we set out to study the contribution of common genetic variants to the risk for ADHD from a lifespan perspective by conducting the largest GWAS-MAs performed so far on persistent ADHD in adults (diagnosed according to DSM-IV/ICD-10 criteria) and on ADHD in childhood (that may include remittent and persistent forms of the disorder) separately and jointly. For the first time, we estimated the genetic correlation between childhood and persistent ADHD, compared their patterns of genetic correlation with other traits and disorders, assessed the effect of childhood ADHD PRSs on persistent ADHD, and explored whether individuals in which ADHD symptoms may persist into adulthood could be distinguished already in childhood using genetic data.A total of 19 GWAS of ADHD comprising 49,560 individuals , provided by the Psychiatric Genomics Consortium (PGC), the Lundbeck Foundation Initiative for Integrative Psychiatric Research (iPSYCH), and the International Multi-centre persistent ADHD CollaboraTion (IMpACT), were analyzed. All participants were of European ancestry, had provided informed consent, and all sites had documented permission from local ethics committees.The meta-analysis on persistent ADHD was conducted in 22,406 individuals  using six datasets from the IMpACT consortium, two datasets from the PGC, and the adult subset from the iPSYCH cohort included in Demontis and Walters et al. .–43.httpsLINK 1.9 , assuminLINK 1.9  and resuNca+1Nco . The gensoftware  was usedDetails of downstream analyses for top signals identified are provided in the online supplement and include conditional analysis, Bayesian credible set analysis, and functional characterization of the significant variants.h2 was estimated by single-trait LD score regression using summary statistics, HapMap 3 LD-scores, considering default SNP QC filters (INFO > 0.9 and MAF > 0.01) and assuming population prevalence of 3.4, 5.5, and 5% for persistent ADHD, ADHD on childhood, and ADHD across the lifespan, respectively, [h2. Partitioning and enrichment of the heritability by functional categories was analyzed using the 24 main annotations  described by Finucane et al. [P < 2.08E−03).The SNP-ctively, . Data ofe et al. . Statisthttp://www.broadinstitute.org/gsea/msigdb), which integrates Kyoto Encyclopedia of Genes and Genomes (http://www.genome.jp/kegg/), BioCarta (http://www.biocarta.com/), Reactome (https://reactome.org/), and Gene Ontology (GO) (http://www.geneontology.org/) resources. Bonferroni correction  and 10,000 permutations were used for multiple testing correction in the gene-based and gene-set analyses, respectively.MAGMA software was undertaken for gene-based and gene-set association testing using summary data from our GWAS-MAs . Variantr2 = 0.1, kb = 10,000) with MAF > 0.05 from the GWAS-MA of persistent ADHD using two different P value thresholds of P < 5.00E−05 (62 SNPs) and P < 1.00E−03 (710 SNPs). Results were meta-analyzed using the standard weighted sum of z-score approach, where z-scores are weighted by the square root of the effective sample size. The statistical power was calculated using 1,000 simulations, considering the ADHD children meta-analysis sample size, the odds ratios and risk allele frequencies from the GWAS-MA of persistent ADHD, and assuming 65% of heterogeneity proportion (π).The Breaking Up Heterogeneous Mixture Based On cross(X)-locus correlations (BUHMBOX) analysis  was usedr2 = 0.05, kb = 500, p2 = 0.5) and different P value thresholds . The concordant direction of effect was evaluated using a one sample test of the proportion with Yates’ continuity correction against a null hypothesis of P = 0.50 with the “stats” R package.The direction of the effect of variants associated with ADHD in childhood was tested in persistent ADHD and vice versa, using strict clumping  to select independent variants  from the childhood GWAS-MA of ADHD and were then tested for association with persistent ADHD in each of the nine datasets, adjusting for the covariates included in the GWAS and using PRSice-2 (https://choishingwan.github.io/PRSice/). Best guess genotypes for nonambiguous strand variants present in all the persistent ADHD studies (missing rate < = 0.02) were included . Results from the nine PRS analyses at each P value threshold were combined using inverse-variance weighted meta-analysis.PRSs were constructed using different z-scores (observed heritability/observed standard error) >4 and with an observed heritability > 0.1 were considered (N = 139 out of 689 available traits). Statistical significance was set using Bonferroni correction (P < 3.60E−04). Pearson’s correlation coefficient (Pearson’s r) was calculated between the genetic correlations of persistent ADHD with the phenotypes from the LD-hub and the genetic correlations of ADHD in childhood with the phenotypes from the LD-hub.Cross-trait LD score regression with unconstrained intercept was used to calculate genetic correlations (rg) between pairs of traits, considering HapMap3 LD-scores, markers with INFO ≥ 0.90, and excluding the MHC region hg19:chr6:25-35M) . Other A:25-35M .h2) was 0.19 (SE = 0.024), with a nominally significant enrichment in the heritability of variants located in conserved genomic regions (P = 5.18E−03) and in the cell-specific histone mark H3K4me1 (P = 3.17E−02)  significantly associated with persistent ADHD, with ST3GAL3 being the most significant one (P = 8.72E−07) (Table P = 1.69E−07)  Fig. . The gen7) Table . The sin07) Fig. . Similarh2 for ADHD in childhood was 0.19 (SE = 0.021), with a significant enrichment in the heritability of variants located in conserved genomic regions after Bonferroni correction (P = 1.21E−06)  (Table P = 1.67E−07) , we conducted a GWAS-MA on children with ADHD in a total of 10,617 ADHD cases and 16,537 controls. We found no evidence of genomic inflation or population stratification  Fig. . The gen7) Table . No sing07) Fig. , and no P = 2.13E−21) and from 1 (P = 0.02). Sign test results provided evidence of a consistent direction of effect of genetic variants associated with ADHD in childhood in persistent ADHD and vice versa   explaining the most variance (r2 = 0.0041 and P = 1.20E−27) , significantly different from 0 (t) Table . In addi27) Fig. . The qui27) Fig. .Fig. 2PoP < 5.00E−05 and P < 1.00E−03, respectively  analysis. We found no evidence of subgroup genetic heterogeneity in children, supporting that the sharing of persistent ADHD-associated alleles between children and adults was driven by the whole group of children, with a statistical power of 98.4 and 100% for thresholds of ly Table .h2 for ADHD across the lifespan was 0.17 (SE = 0.013), and a significant enrichment in the heritability of variants located in conserved genomic regions was observed after Bonferroni correction (P = 1.53E−06)  significantly associated with ADHD across the lifespan (Table P = 1.57E−08), followed by loci on chromosome 7 (index variant rs3958046), chromosome 4 (index variant rs200721207), and chromosome 3 (index variant rs1920644) (Table P.adj = 0.021)  Fig. . We iden06) Fig.  and ninean Table . The mos4) Table , Fig. 3.One of the four loci identified in the single-variant analysis also reached genome-wide significance in the previous GWAS-MA on ADHD , and allr2 = 0.56), was associated with the expression of RSPH3 in blood and brain , and with the expression of VIL2 in blood (P.adj = 3.21E−03). The credible set for the second most associated locus on chromosome 7 included 24 variants. The index variant, rs3958046, and other variants in this set, were eQTLs for CADPS2 in brain (maximum P.adj = 2.91E−03). The credible set for the locus on chromosome 4 contained 50 variants, most of them located in or near PCDH7, but no eQTLs were identified. In the credible set for the locus on chromosome 3, which included 98 variants, the index variant, rs1920644, was associated with the expression of KPNA4, IFT80, and KRT8P12 in brain . Many other variants in this set were eQTLs for these genes and also for TRIM59, OTOL1, and/or C3orf80 in brain (P.adj < 0.05)  using available data in blood or brain , 55. We RMI1 expression in blood (PSMR = 5.36E−06) (Table PHEIDI was 0.47.In a summary-data-based Mendelian randomization (SMR) analysis, we used summary data from the GWAS-MA of ADHD across the lifespan and the eQTL data in blood and brain from Westra et al.  and Qi eN = 53,296) and persistent ADHD , ADHD in childhood , and ADHD across the lifespan  (Table N = 7086), a significant genetic correlation was also obtained between their sample and ours .We found significant genetic correlations of ADHD in children and adults from the previous GWAS-MA   Table . When reN = 17,666) and persistent ADHD , ADHD in childhood , and ADHD across the lifespan . Similarly, significant genetic correlations between GWAS of self-reported ADHD status from 23andMe  and persistent ADHD , ADHD in childhood , and ADHD across the lifespan  were observed   Fig. .Fig. 4GeIn the current study, we set out to explore the contribution of common genetic variants to the risk of ADHD across the lifespan by conducting GWAS-MAs separately for children and adults with persistent ADHD that meet DSM-IV/ICD-10 criteria. Using the largest GWAS datasets available from the PGC, the iPSYCH, and IMpACT consortia we found evidence for a common genetic basis for ADHD in childhood and persistent ADHD in adults and identified nine new loci associated with the disorder.h2 = 0.19), which is consistent with the SNP-h2 estimate reported in the recent GWAS-MA on ADHD [h2 = 0.22), that included children and adults, and is in line with multiple studies supporting the stability of ADHD’s heritability from childhood to adulthood [We found a highly similar proportion of the heritability of ADHD explained by common variants in children and in adults  and the GWAS-MA of childhood rating scales of ADHD symptoms in the general population [We also found strong and significant positive genetic correlations of ADHD ascertained in clinical populations of adults, children, or both with other ADHD-related measures from general population samples, including the largest GWAS of self-reported ADHD status from 23andMe participants N = 9,652 and Similar patterns of genetic correlation of ADHD with different somatic disorders and anthropometric, cognitive, and educational traits were identified for children and adults. These findings were highly similar to those observed in the recent GWAS-MA  and furtFEZF1, a candidate for autism spectrum disorder implicated in the formation of the diencephalon [RSPH3, which participates in neuronal migration in embryonic brain [CADPS2, which has been associated with psychiatric conditions due to its role in monoamine and neurotrophin neurotransmission [AMN, which is involved in the uptake of vitamin B12 [FBXL17, which has previously been related to intelligence [We report 13 loci in gene- and SNP-based analyses for childhood ADHD, adult ADHD, and/or ADHD across the lifespan. Four ADHD-associated loci were previously identified by Demontis et al. , which wcephalon , 59, RSPic brain , CADPS2,smission –64, AMN,amin B12 , 66, essamin B12 , and FBXlligence .The main limitation of this study is the sample overlap (85.7%) between the present GWAS-MAs and the previous one by Demontis et al. , which hIn summary, the present cross-sectional analyses identify new genetic loci associated with ADHD and, more importantly, support the hypothesis that persistent ADHD in adults is a neurodevelopmental disorder that shows a high and significant genetic overlap with ADHD in children. Future longitudinal studies will be required to disentangle the role of common genetic variants on ADHD remittance and/or persistence.www.adhdinadults.com. JK has given talks at educational events sponsored by Medice; all funds are received by King’s College London and used for studies of ADHD. HL has served as a speaker for Evolan Pharma and Shire and has received research grants from Shire; all outside the submitted work. K-PL served as a speaker for Eli Lilly and received research support from Medice, and travel support from Shire, all outside the submitted work. LAR reported receiving honoraria, serving on the speakers’ bureau/advisory board, and/or acting as a consultant for Eli Lilly, Janssen-Cilag, Novartis, and Shire in the last 3 years; receiving authorship royalties from Oxford Press and ArtMed; and receiving travel awards from Shire for his participation in the 2015 WFADHD meetings and from Novartis to take part of the 2016 AACAP meeting. The ADHD and juvenile bipolar disorder outpatient programs chaired by him received unrestricted educational and research support from the following pharmaceutical companies in the last 3 years: Janssen-Cilag, Novartis, and Shire. MC has received travel grants and research support from Eli Lilly and Co., Janssen-Cilag, Shire, and Lundbeck and served as consultant for Eli Lilly and Co., Janssen-Cilag, Shire, and Lundbeck. BF has received educational speaking fees from Medice and Shire. JAR-Q was on the speakers’ bureau and/or acted as consultant for Eli Lilly, Janssen-Cilag, Novartis, Shire, Lundbeck, Almirall, Braingaze, Sincrolab, Medice, and Rubió in the last 5 years. He also received travel awards (air tickets + hotel) for taking part in psychiatric meetings from Janssen-Cilag, Rubió, Shire, Medice, and Eli- Lilly. The Department of Psychiatry chaired by him received unrestricted educational and research support from the following companies in the last 5 years: Eli Lilly, Lundbeck, Janssen- Cilag, Actelion, Shire, Ferrer, Oryzon, Roche, Psious, and Rubió. The other authors have nothing to disclose. All members of the 23andMe Research Team are current or former employees of 23andMe, Inc. and hold stock or stock options in 23andMe. Authors of the ADHD Working group of the PGC that participated in this study: Catharina A. Hartman, Ziarih Hawi, Jennifer Crosbie, Sandra Loo, Josephine Elia, Russell Schachar, Christie Burton, Ted Reichborn-Kjennerud, Aribert Rothenberger, Søren Dalsgaard, Irwin Waldman, Mark Bellgrove, Hakon Hakonarson, Johannes Hebebrand, Anke Hinney, and Robert Oades have nothing to disclose. Joseph Biederman 2019–2020: he received research support from Genentech, Headspace Inc., Lundbeck AS, Neurocentria Inc, Pfizer Pharmaceuticals, Roche TCRC Inc., Shire Pharmaceuticals Inc., Sunovion Pharmaceuticals Inc., and Tris. He was a consultant for Akili, Avekshan, Jazz Pharma, and Shire/Takeda. Through MGH CTNI, he participated in a scientific advisory board for Supernus. Dr Biederman’s program has received departmental royalties from a copyrighted rating scale used for ADHD diagnoses, paid by Bracket Global, Ingenix, Prophase, Shire, Sunovion, and Theravance; these royalties were paid to the Department of Psychiatry at MGH. Tobias Banaschewski served in an advisory or consultancy role for Lundbeck, Medice, Neurim Pharmaceuticals, Oberberg GmbH, Shire, and Infectopharm. He received conference support or speaker’s fee by Lilly, Medice, and Shire. He received royalties from Hogrefe, Kohlhammer, CIP Medien, and Oxford University Press. James McGough is an expert testimony for Eli Lilly; DSMB for Sunovion. Benjamin Neale is a member of the scientific advisory board at Deep Genomics and consultant for Camp4 Therapeutics, Takeda Pharmaceutical, and Biogen. Ole Andreas Andreassen received speaker’s honorarium from Lundbeck, and is a consultant for HealthLytix.VR has served on the speakers for Eli Lilly, Rubio, and Shire in the last 5 years. She has received travel awards from Eli Lilly and Co. and Shire for participating in psychiatric meetings. The ADHD Program has received unrestricted educational and research support from Eli Lilly and Co., Janssen-Cilag, Shire, Rovi, Psious, and Laboratorios Rubió in the past 2 years. MC received travel awards for taking part in psychiatric meetings from Shire. CF received travel awards for taking part in psychiatric meetings from Shire and Lundbeck. GEM received travel awards for taking part in psychiatric meetings from Shire. EJSS-B received speaker fees, consultancy, research funding, and conference support from Shire Pharma. Consultancy from Neurotech Solutions, Copenhagen University and Berhanderling, Skolerne & KU Leuven. Book royalties from OUP and Jessica Kingsley. Financial support—Arhus University and Ghent University for visiting Professorship. Editor-in-Chief JCPP—supported by a buy-out of time to University of Southampton and personal Honorarium. King’s College London received payments for work conducted by PA: consultancy for Shire, Eli Lilly, Novartis, and Lundbeck; educational and/or research awards from Shire, Eli Lilly, Novartis, Vifor Pharma, GW Pharma, and QbTech; speaker at events sponsored by Shire, Eli Lilly, Janssen-Cilag, and Novartis. JKB has been in the past 3 years a consultant to/member of advisory board of and/or speaker for Shire, Roche, Medice, and Servier. He is not an employee of any of these companies, and not a stock shareholder of any of these companies. He has no other financial or material support, including expert testimony, patents, royalties. In the past year, SVF received income, potential income, travel expenses continuing education support and/or research support from Tris, Otsuka, Arbor, Ironshore, Shire, Akili Interactive Labs, Enzymotec, Sunovion, Supernus, and Genomind. With his institution, he has US patent US20130217707 A1 for the use of sodium–hydrogen exchange inhibitors in the treatment of ADHD. He also receives royalties from books published by Guilford Press: Straight Talk about Your Child’s Mental Health, Oxford University Press: Schizophrenia: The Facts and Elsevier: ADHD: Non-Pharmacologic Interventions. He is principal investigator of https://www.ecnp.eu/research-innovation/ECNP-networks/List-ECNP-Networks/ADHD.aspx). Over the course of this investigation, PR is a recipient of a pre-doctoral fellowship from the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR), Generalitat de Catalunya, Spain (2016FI_B 00899). The iPSYCH project is funded by the Lundbeck Foundation (grant nos. R102-A9118 and R155-2014-1724) and the universities, and university hospitals of Aarhus and Copenhagen. ADB and NRM’s work is also supported by the EU’s Horizon 2020 programme . Data handling and analysis was supported by NIMH (1U01MH109514-01 to Michael O’Donovan and ADB). CSM is a recipient of a Sara Borrell contract from the Instituto de Salud Carlos III, Ministerio de Economía, Industria y Competitividad, Spain (CD15/00199). K-PL and his team are supported by the Deutsche Forschungsgemeinschaft , ERA-Net NEURON/RESPOND, no. 01EW1602B, ERA-Net NEURON/DECODE, no. 01EW1902, and 5-100 Russian Academic Excellence Project. JH thanks Stiftelsen K.G. Jebsen, University of Bergen, the Western Norwegian Health Authorities (Helse Vest). HL thanks the Swedish research council. BC received financial support from the Spanish “Ministerio de Economía y Competitividad” (SAF2015-68341-R) and AGAUR (2017SGR738). MSA is a recipient of a contract from the Biomedical Network Research Center on Mental Health (CIBERSAM), Madrid, Spain. MR is a recipient of a Miguel de Servet contract from the Instituto de Salud Carlos III, Spain (CP09/00119 and CPII15/00023). The research leading to these results has received funding from the Instituto de Salud Carlos III , and cofinanced by the European Regional Development Fund (ERDF), from the Pla estratègic de recerca i innovació en salut (PERIS); Generalitat de Catalunya , from “la Marató de TV3” (092330/31) and from the Agència de Gestió d’Ajuts Universitaris i de Recerca-AGAUR, Generalitat de Catalunya (2017SGR1461). ES’s work is supported by a personal Hypatia grant from the Radboud University Medical Center. MH received a Veni grant from of the Netherlands Organization for Scientific Research . The NeuroIMAGE study was supported by NIH Grant R01MH62873 (to SVF), NWO Large Investment Grant 1750102007010 (to JKB), ZonMW grant 60-60600-97-193, NWO grants 056-13-015 and 433-09-242, and matching grants from Radboud University Nijmegen Medical Center, University Medical Center Groningen and Accare, and Vrije Universiteit Amsterdam. Organization for Scientific Research . BF and MK’s work is supported by the Dutch National Science Agenda (NWA) for the NeurolabNL project (grant 400-17-602). This paper represents independent research part funded by the National Institute for Health Research (NIHR) Biomedical Research Centre at South London and Maudsley NHS Foundation Trust and King’s College London. BF received additional funding from a personal Vici grant of the Dutch. The authors declare no competing interestsThe research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007–2013) under grant agreement n° 602805 (Aggressotype) as well as from the European Union H2020 Programme (H2020/2014–2020) under grant agreements n° 643051 (MiND), n° 667302 (CoCA), n° 728018 (Eat2beNICE), and n° 278948 (TACTICS). The work was also supported by the ECNP Network “ADHD across the Lifespan” (Supplementary materialSupplementary tables


Streptomyces, frequently occurring in an aquatic environment. To understand the potential role of maduramicin in crayfish consumption related Haff disease, a mouse model was established in this study. Two exposure routes of maduramicin in the abdominal muscle and the hepatopancreas tissue homogenates of crayfish were given intragastrically to mice in different doses for seven days. Action changes, clinical symptoms, feed consumption, body weight, blood biochemistry, and histopathology examination of mice were observed and analyzed. In the natural exposure group, relatively low concentration of maduramicin in crayfish muscle and hepatopancreas had no obvious effects on mental state, body weight, blood biochemical indexes, or histologic appearance. However, in the artificial exposure group, with increasing concentrations, maduramicin in crayfish muscle and hepatopancreas homogenates both induced mental sluggishness and weight loss of mice. Blood biochemical examination showed that 3.5 mg·kg−1 and 7 mg·kg−1 maduramicin in crayfish tissue homogenates significantly increased levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), lactate dehydrogenase (LDH), and creatine kinase (CK). Additionally, histopathological examination showed that multiple organs were damaged by maduramicin, including degeneration of liver cells, shedding of renal epithelial cells, and disturbance and partial lysis of myocardial and skeletal muscle filaments in the mice. In summary, maduramicin may not cause Haff disease through contamination of the aquatic environment under normal conditions. Maduramicin can be used as a potential toxin tool to establish a rhabdomyolysis disease animal model for drug development.Maduramicin is a toxic ionophore antibiotic that is isolated from  Procambarus clarkii), buffalo fish (Ictiobus cyprinellus), freshwater pomfret (Colossoma brachypomus), Atlantic salmon , pike (Esox species), and boxfish (Ostraciontidae spp.), which were probably contaminated by a heat-stable toxin [L.lota), pike (Esox spp.), and freshwater eel (A. anguilla) [Mylossoma spp.), tambaqui black finned colossoma (Colossoma macropomum), or pirapitinga freshwater pompano (Piaractus brachypomus) [Haff disease is a rare syndrome of unexplained myalgia and rhabdomyolysis in a person who has ingested seafood from fresh or brackish water within the previous 24 h. It was first reported in 1924 near the Königsberger Haff shores along the Baltic coast in East Prussia [le toxin ,4. Sincenguilla) ,6. The fnguilla) , and as nguilla) . In 2008hypomus) . In Chinhypomus) . Subsequhypomus) ,12. A crhypomus) . Four huhypomus) . IncreasStreptomyces, is extensively used in the poultry industry [As an important anticoccidiosis drug for the control of broiler chicken coccidiosis and growth promotion, maduramicin, one of the polyether ionophore antibiotics that were isolated from industry . Due to industry ,19,20,21industry . Humans industry , which aindustry . Drugs iindustry ,25,26. T−1 of maduramicin has been detected [Maduramicin was approved as an additive for control coccidiosis and growth promotion of broiler chickens at one day old during the whole period of growth. During the summer and autumn seasons, a large amount of maduramicin is widely and heavily used in the poultry production industry . As madudetected . Considedetected ,29. In Cdetected . Similardetected . Occurredetected . In addidetected . ConsumpProcambarus clarkia) consumption is very popular in central and south China during July and August. This correlates with maduramicin application in poultry production and the peak of crayfish-related Haff disease cases during the year. In Nanjing, people consume 60–80 tons of crayfish per day from June to September each year [Crayfish  was obtained from Zhejiang Esigma Biotechnology Co., Ltd.  Phosphate buffer saline , giemsa staining solution , heparin sodium , and ethylene diamine tetra acetic acid disodium salt  were purchased from Solarbio Science & Technology Company . Other analytical grade reagents used in the present study were obtained from Sinopharm Chemical Reagent Co., Ltd. .−1, pH at 7.0, total ammonia at <1 mg/L, and water hardness (as CaCO3) at 116–121 mg·L−1. Crayfish were fed with drug-free commercial pellets (1% body weight each day), and excrement and dead crayfish bodies were removed in time. During the whole acclimation period, cultured water was renewed every two days, and the cumulative mortality of the crayfish did not exceed 5%.To safeguard animal welfare, all of the methods performed in animals were carried out in strict accordance with the National Institutes of Health standards established in the Guidelines for the Care and Use of Experimental Animals. All experimental protocols in animals were approved by the Ethics Committee on Animal Experimentation of Nanjing Agricultural University. Adult male crayfish  were purchased from Freshwater Fisheries Research Institute of Jiangsu Province . Crayfish were acclimated in a plastic tank containing fully aerated, dechlorinated tap water (21 ± 2 °C) for 14 d in advance. Water quality characteristics were measured and recorded daily. The concentration of dissolved oxygen was higher than 6.0 mg·LAdult Institute of Cancer Research (ICR) mice  were purchased from Nanjing Qinglongshan Animal Breeding Farm . Mice were housed in stainless steel cages in an air-conditioned room maintained at 24 ± 2 °C and 55 ± 5% humidity with a 12 h dark/light cycle. All mice were acclimated for 7 d prior to crayfish tissue exposure, and they were allowed free access to a normal diet and water.50 of maduramicin on crayfish  were used in exposure treatment to obtain maduramicin contaminated crayfish tissues that could be used for subsequent mice intragastric administration. Maduramicin  was dissolved in 1.5 mL of ethanol and diluted in 10 L of fully aerated dechlorinated tap water. As a vehicle control, 1.5 mL of ethanol was dissolved in 10 L of cultured water in the inert plastic tanks. Twenty crayfish per tank were used in the whole trial. All of the crayfish were exposed to maduramicin and solvent control for 72 h based on a preliminary study (unpublished data). After 72 h of exposure, the concentrations of maduramicin in the hepatopancreas of different treatment groups were 89.5, 406.5, and 756.7 μg·kg−1 and in abdominal muscles were 12.3, 83.5, and 135.5 μg·kg−1, respectively (unpublished data). Feeding conditions in the whole exposure study were in accordance with the acclimation process of crayfish.To simulate the natural exposure pathway of maduramicin to crayfish, 1/100, 1/20, and 1/10 LC−1 in muscles and 0.00179, 0.00813, and 0.01513 mg·kg−1 in hepatopancreas. In the artificial exposure group, the other 60 negative hepatopancreas and muscles were separately homogenized with maduramicin solution in a ratio of 1:1 to prepare homogenates, making the gavage doses of maduramicin in the homogenates 0.7 mg·kg−1, 3.5 mg·kg−1, and 7 mg·kg−1 that corresponded to 1/50, 1/10, and 1/5 LD50 of maduramicin on mice. The hepatopancreas and abdominal muscles of the remaining crayfish were used as a vehicle control and were homogenized with normal saline at a 1:1 ratio. All homogenates were prepared and stored at 4 °C prior to mice gavage on the same day.After maduramicin exposure, the hepatopancreas and abdominal muscles (without the intestine cord) of all crayfish were collected and stored at −80 °C. The obtained crayfish tissues were divided into the natural exposure group and artificial exposure group. To prepare tissue homogenates in the natural exposure group, in brief, hepatopancreas and muscles of crayfish with different concentrations of maduramicin were homogenized with physiological saline in a ratio of 1:1. The concentrations of maduramicin in different tissues were converted to the doses for mice gavage, 0.00025, 0.00167, and 0.00271 mg·kg−1), and the other 30 mice were administered crayfish hepatopancreas with different doses of maduramicin . Artificial administration group: 30 mice were divided into three groups and orally administered the homogenates of crayfish abdominal muscle with maduramicin , and the other 30 mice were administered the homogenates of crayfish hepatopancreas with different doses of maduramicin . All mice of the fifteen groups were orally administered normal saline, negative crayfish homogenates, or maduramicin contaminated crayfish tissue homogenates (0.2 mL·10 g−1 bw·d−1) once a day for seven consecutive days.One hundred and fifty ICR mice were randomly divided into fifteen groups. Three groups were the normal saline group, negative homogenate of crayfish hepatopancreas, and muscle group. Natural exposure group: 30 mice were divided into three groups and were administered crayfish muscle homogenate with different doses of maduramicin , dissected, and fixed in neutral buffered formalin solution  for further histopathology analysis.During the whole animal experiment period, the general clinical observation of mice was performed once a day at eight in the morning. The weight of each mouse and the food consumption of each group were recorded every day. At day seven, mice were fasted for 4 h before sample collection. Blood samples were collected via orbit venous sinus into centrifuge tubes and centrifuged at 3500 r·minBiochemical parameters lactate dehydrogenase (LDH), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatine kinase (CK) were determined using commercial assay kits provided by Nanjing Jiancheng Bioengineering Institute . The detailed protocols were carried out according to the manufacturer’s instructions.The fixed tissue samples were embedded in paraffin wax, sectioned into two slides of 5 μm thickness, subsequently dehydrated in a series of graded ethanol, and stained with hematoxylin and eosin (H&E). After that, each tissue section was observed and photographed under a light microscope  with a digital camera for histopathology analysis.p < 0.05 was considered as statistically significant.Results were presented as mean ± SD (standard deviation). Statistical analysis of the data was performed using one-way ANOVA, followed by an LSD (least significant difference) multiple comparison test by SPSS . The value of −1) showed low activity, listlessness, limb weakness, thinness, and reduced spontaneous activity. Several mice in the artificial exposure group that were administered with a medium dose of maduramicin (3.5 mg·kg−1) exhibited toxic symptoms, such as mental depression, slow response, and thinness. The mice administered with the low dose of maduramicin (0.7 mg·kg−1) appeared to have normal activity and mentality compared to the negative control group.To understand the effects of maduramicin on clinical symptoms of mice, we observed the general performance of all tested mice. Compared to the negative control group, the mice in the natural exposure group exhibited good activity and smooth fur. However, the mice in the artificial exposure group that were administered with a high dose of maduramicin (7 mg·kgp < 0.05). As shown in p < 0.05).As shown in −1), the feed intake of the mice was gradually reduced . When the concentration of maduramicin was elevated to 3.5 and 7 mg·kg−1, LDH, ALT, AST, BUN, and CK of mice were increased significantly (p < 0.05) compared to the negative control group.As shown in p > 0.05). However, when the dose of maduramicin was increased to 0.7 mg·kg−1 in the artificial exposure group, the AST activity of mice was elevated significantly (p < 0.05). In addition, ALT, AST, BUN, and CK of 3.5 mg·kg−1 and 7 mg·kg−1 maduramicin treated mice were significantly higher than that in the blank control (p < 0.05).As shown in −1) did not show pathological changes. On the contrary, in the artificial exposure group, crayfish muscle contaminated with the higher dose of maduramicin (7 mg·kg−1) caused severe liver injury in mice, exhibiting a deteriorated hepatic lobule, vacuolar degeneration (black arrow), and nuclear condensation (yellow arrow), as shown in −1), and contamination showed intact tubular epithelial cells, normal glomerular size, and no degeneration (−1) fortified in crayfish muscle homogenates induced severe degeneration of renal tubular epithelial cells, epithelial exfoliation (black arrow), nuclear condensation (yellow arrow), and nuclear fragmentation (green arrow) (−1) and the negative control, showing arranged muscle fibers and no inflammatory infiltration. A high dose of maduramicin (7 mg·kg−1) in crayfish muscle homogenates caused myocardial interstitial edema (yellow arrow) and fibrin exudation (black arrow) (−1) in crayfish muscle homogenates did not show a change in muscle fiber arrangements or morphology of skeletal muscle cells (−1) induced severe edema of muscle silk (yellow arrow) and rhabdomyolysis  F. As fork arrow) F. Comparle cells A,B,D. Ho−1) had no effects on mice liver morphology (−1) in crayfish hepatopancreas homogenates induced severe vacuolar degeneration (black arrow) and nuclear condensation (yellow arrow) of mice livers (−1), compared to the blank control (−1) in crayfish hepatopancreas homogenates in the artificial exposure group caused epithelial exfoliation (black arrow), nuclear condensation (yellow arrow), and nuclear fragmentation (green arrow) (−1) in crayfish hepatopancreas homogenates showed no abnormal changes in mice hearts compared to those of the blank control group and the negative crayfish hepatopancreas group (−1) in crayfish hepatopancreas homogenates induced serious pathological lesions, such as disordered and broken myofilaments of mice (−1) showed no observed differences with normal muscle tissue (−1) in crayfish hepatopancreas homogenates caused serious damage of skeletal muscle, showing rhabdomyolysis  G. Furtheas group A,C,E. In of mice G. Similae tissue A,C,E. InSince the outbreak of Haff disease in early 1965, studies of the risk factors have been conducted consistently. For the first time, Leschchenko et al. in the Soviet Union experimentally induced a condition mimicking Haff disease in mice and cats by feeding them fresh fish implicated in a Haff disease outbreak , proposiActinomadura madurae, was suspected due to its characteristics of heat-stable hydrophobicity. Maduramicin is the most toxic ionophore antibiotic with a very narrow safety range, and often causes severe rhabdomyolysis in target/non-target animals and humans who consumed it by accident [As neglected in previous studies, a potential rhabdomyolysis inducer, maduramicin, which is produced by accident ,19,27. Iaccident . Other daccident ,25,26. Iaccident ,33. Prevaccident ,40,41,42accident . We hypo50 of maduramicin in mice were used as the exposure dose to crayfish, and the final doses of maduramicin were 0.00025, 0.00167, and 0.00271 mg·kg−1 in muscles and 0.00179, 0.00813, and 0.01513 mg·kg−1 in hepatopancreas of crayfish, respectively. After administration of the crayfish tissue homogenate containing a low concentration of maduramicin by gavage for seven days, there were no significant changes in body weight, food intake, or blood biochemical indexes. Pathological observation showed no obvious histological changes compared to the negative control. Based on these findings, we first demonstrated that maduramicin residue in crayfish will not cause symptoms of rhabdomyolysis in mice under normal exposure conditions, which is consistent with the opinions of the European Food Safety Authority (EFSA) that ionophores have no harmful effects on humans, animal health, or the environment [−1). Laboratory examination of blood biochemical indicators are crucial to the determination of Haff disease [−1 and 7 mg·kg−1 of maduramicin were significantly increased in a dose-dependent manner. ALT and AST activities were increased significantly by maduramicin treatment, presumably due to damage to the liver and myocardial tissues, which allowed the release of transaminases into the blood. Elevated LDH levels also suggested skeletal muscle or myocardial damage by maduramicin exposure. As the most important indicator to diagnose Haff disease, CK often elevated significantly along with rhabdomyolysis in most Haff disease cases [−1 of maduramicin induced CK elevation about two times higher than the normal level, indicating that the laboratory diagnostic index of Haff disease is still unable to be reached just by maduramicin treatment in mice. However, it cannot be ruled out that in a complex aquatic environment, maduramicin may have a synergistic effect with other unknown exogenous substances. A rhabdomyolysis animal model has been established in previous studies by coadministration of statin and fibrate [To mimic natural exposure of maduramicin-contaminated crayfish to mice, 1/10, 1/20, and 1/100 LDironment . Further disease . In gene disease . ALT, AS disease . In seve disease . In thisse cases ,35,36. I fibrate , and cip fibrate , both wi fibrate . AlthougIn conclusion, as an environmental pollutant, maduramicin will not induce crayfish-related Haff disease in a mouse model under normal circumstances due to its low exposure concentration. In some extreme conditions, maduramicin residue with a high concentration in edible tissues of crayfish will cause significant damage of skeletal muscle, myocardial muscle, livers, and kidneys of mice, but will not reached the criterion of Haff disease. Base on maduramicin-contaminated crayfish consumption in the mice model, it is concluded that maduramicin is unlikely to induce Haff disease by a crayfish-transmitted food chain.


The purpose of this paper is to evaluate the efficacy and safety of acupuncture in the treatment of childhood attention deficit hyperactivity disorder (ADHD).We will electronically search PubMed, Medline, Embase, Web of Science, the Cochrane Central Register of Controlled Trial, China National Knowledge Infrastructure, China Biomedical Literature Database, China Science Journal Database, and Wan-fang Database from their inception. Also, we will manually retrieve other resources, including reference lists of identified publications, conference articles, and grey literature. The clinical randomized controlled trials or quasi-randomized controlled trials related to acupuncture treating pediatric ADHD will be included in the study. The language is limited to Chinese and English. Research selection, data extraction, and research quality assessment will be independently completed by 2 researchers. Data were synthesized by using a fixed effect model or random effect model depend on the heterogeneity test. The scores of Revised Conners’ Parent Rating Scale (CPRS-R), Conners Teacher Rating Scale (CTRS-R), and Child Behavior Checklist (CBCL) will be the primary outcomes. Besides, the scores of the Conners Continuous Performance Test, Internal Restlessness Scale, and Behavior Assessment System for Children (BASC), and the possible adverse events will also be assessed as secondary outcomes. RevMan V.5.3 statistical software will be used for meta-analysis, and the level of evidence will be assessed by Grading of Recommendations Assessment, Development, and Evaluation (GRADE). Continuous data will be expressed in the form of weighted mean difference or standardized mean difference with 95% confidence intervals (CIs), while dichotomous data will be expressed in the form of relative risk with 95% CIs.The protocol of this systematic review (SR) does not require ethical approval because it does not involve humans. We will publish this article in peer-reviewed journals and presented at relevant conferences.https://osf.io/xvyp9)OSF Registries, DOI: 10.17605/OSF.IO/XVYP9 ( Among children with ADHD, approximately one-third persists to adulthood.Attention deficit hyperactivity disorder (ADHD) mainly manifests as inattention, and/or hyperactivity and impulsivity but failed to improve mood, self-esteem, and social relationship; besides, ADHD patients showed low medicine compliance after 1 year. Nonpharmacological treatments, including behavior management approaches , cognitive training, motivational enhancement, academic, organizational, social skills training, and neurofeedback are also considered as valid options.–12In the treatment of ADHD, pharmacological therapies, including methylphenidate, amphetamine, atomoxetine, and guanfacine, were reported to be effective for some features,–15 As one of the most common form of complementary and alternative medicine, acupuncture is widely applied to treat various of conditions, it is reported to improve attention and school performances on children,,18 and it is popular in treating ADHD in Asian countries. Till today, it is still uncertain if acupuncture could improve the core symptoms in ADHD; thus, to access the safety and efficacy of acupuncture in the treatment of ADHD, we plan to conduct this systematic review and meta-analysis, hoping to provide further reference for future clinical practice.Meanwhile, a wide range of complementary and alternative medicines are used; a study showed that 12% to 64% of ADHD patients received complementary and alternative medicine approaches, including nutritional intervention, herbal supplements, acupuncture, massage, and yoga.2https://osf.io/ha97r). The protocol follows the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) 2015 statement guidelines. We will report the changes in the full review if necessary.The protocol has been registered on OSF as Registration DOI: 10.17605/OSF.IO/XVYP9  of acupuncture for ADHD children, whether using blind method or allocation concealment method, including those using a quasi-random method such as alternate allocation or allocation by birth date. We included both parallel and crossover studies. The language of the trials to be included should be English or Chinese.2.1.2Following studies would be excluded: case reports and reviews, literature not in English or Chinese language, and clinical research studies that compared different kinds of acupuncture.2.2We would include patients with ADHD under the age of 18 years, regardless of gender or race, diagnosed by standard criteria such as the Diagnostic and Statistical Manual of Mental Disorders (DSM) or the International Classification of Diseases (ICD).2.3 involving the needle selection, acupoints selection, manipulations, course, etc. Controlled interventions include no intervention, placebo acupuncture or sham acupuncture; standard treatments ; or other non-acupuncture interventions.Acupuncture for treating ADHD include traditional acupuncture, electro-acupuncture, laser acupuncture, auricular acupuncture, etc. These interventions can be used alone or in combination. The details of acupuncture treatment should be clearly illustrated according to STRICTRA,2.42.4.1We select the score of Revised Conners’ Parent Rating Scale (CPRS-R), Conners Teacher Rating Scale (CTRS-R), and Child Behavior Checklist (CBCL) as primary outcomes.2.4.2We also care about the scores of the Conners Continuous Performance Test, Internal Restlessness Scale, and Behavior Assessment System for Children (BASC); meanwhile, the possible adverse effects are also taken into consideration, including fainting, stuck needle, bent needle, broken needle, and hematoma.33.1Following databases will be searched: PubMed, Web of Science, the Cochrane Central Register of Controlled Trials, AMED, MEDLINE, EMBASE, Cochrane Library, China National Knowledge Infrastructure (CNKI), Wanfang data, Chinese Scientific Journals Database (VIP), and China biomedical literature database (CBM). We will select the eligible studies published up to October 31, 2020. The search terms used in the systematic review are as follows: acupuncture, attention deficit hyperactivity disorder.We will not apply any language, population or national restrictions. The specific search strategy will be (taking PubMed as an example) listed on Table We will identify relevant RCTs and the selected studies will be analyzed according to the Cochrane Handbook.3.2We also retrieve manual related documents, such as replacing and supplementing some reference documents, medical textbooks, clinical laboratory manuals, and the World Health Organization International Registry of Clinical Trials (ICTRP). At the same time, we will contact experts and authors in this field to obtain important information that cannot be found in the search. The research flow chart is shown in Figure 44.1Two independent researchers (LY and JH) will assess the full-text articles from the search results independently against the inclusion and exclusion criteria. Discrepancies will be discussed and resolved by consensus with a third author (HB).4.2The following information will be extracted from each study: research number, data extractor, date of data extraction, general situation of the study, research methodology, research population, baseline comparability, interventions, main outcome indicators, secondary outcome indicators, combined drug use, adverse reactions or complications, etc. For those with questions or incomplete information, we will try to contact the author to obtain information before deciding whether to include it.4.3Two of the authors (LY and JH) individually assessed the risk of bias using assessments included in the study were evaluated in the Cochrane System Evaluator's Manual for RCT quality evaluation criteria. Assessing the risk of bias: random sequence generation; allocation concealment; blinding of participants and personnel; blinding of outcome assessment; incomplete outcome data; selective outcome reporting; other bias. Every domain was classified as high risk of bias, low risk of bias, or unclear risk of bias. Any arising difference was resolved by discussion.4.4We will measure continuous data with mean difference (MD) or standard MD (SMD) for the therapeutic effect with 95% CIs. For dichotomous data, risk ratios (RRs) with 95% CIs will be calculated.4.5To obtain the missing data, we will contact the corresponding author. If no response will be obtained, we will analyze only the available data and describe the reason and impact of this exclusion in the paper.4.6Publication bias will be explored through funnel plot analysis. GRADE profiler 3.6 is used to evaluate the quality of evidence. The specific contents include limitations of research, inconsistency of research results, indirect evidence, inadequate accuracy, publication bias. Finally, the quality of evidence is divided into 4 levels: high-level evidence, intermediate evidence, low-level evidence, and very low-level evidence.4.7I2 value of the Chi-squared test (a = 0.1) to determine the heterogeneity. When I2 ≤50%, it is considered acceptable. When I2 > 50%, subgroup analysis should be performed to identify potential causes and record them.All literature will use 4.8P and I2 represent the size of heterogeneity among multiple studies. When P is greater than .1 and I2 is less than 50%, it suggests heterogeneity is small; on the contrary, it suggests heterogeneity is large. Heterogeneity is mainly handled by subgroup analysis. Sensitivity analysis is used to test the reliability of the overall effect.RevMan 5.3 software was used for statistical analysis of data. RR was used for binary variables and MD was used for continuous variables. Heterogeneity analysis will be conducted by heterogeneity test; 4.9When the heterogeneity test results are heterogeneous, we will conduct subgroup analysis to explore the possible causes of heterogeneity. The effects of different types of acupuncture therapy including design scheme, severity of illness, age, sex, and mild or severe AP were analyzed. We will also delete low-quality and/or medium-quality studies to check the robustness of the results.4.10Sensitivity analysis will be used to test the quality of the research contained in the sampled documents. The stability of the conclusions can be tested by reanalyzing the conclusions by inputting missing data and changing the type of research.4.11The results of the system review will be published in peer-reviewed journals, disseminated at relevant meetings, or disseminated in peer-reviewed publications, and we use aggregated published data to exclude individual patient data, so ethical approval, and informed consent are not required.5 In children, ADHD mainly presents executive impairments in inhibitory and working memory; pragmatics; emotional dysregulation, including emotional inflexibility, slow return to emotional baseline, and low threshold for emotional excitability; sensory integration deficits, including visual motor integration and time perception deficits; and motor performance issues.ADHD is a multidimensional chronic neurodevelopmental condition that affects 8.4% of U.S. children between 2 and 17-years-old, when untreated, it may become a long-term condition into the adulthood. Along with medication and behavior therapy, complementary and alternative medicine (CAM) is widely applied in the treatment of ADHD, between 12% and 64% ADHD children received CAM, including nutritional interventions, electroencephalographic biofeedback, herbal and natural health products, massage and yoga, etc. As a major form of CAM, acupuncture is reported benefit for ADHD children and being prevalent in the management of this condition.On children with ADHD, the goals of treatment are symptom reduction, social and cognitive function improvement.,29 Recent studies offered more refences on this topic. A statistically significant greater reduction in attention deficit and hyperactivity symptoms was observed on children who received auricular therapy on acupoints, comparing with those who received sham acupoints. Besides, a pilot study of RCT suggested a trend of greater improvement on ADHD scores on children received acupuncture, and no reserve event was reported. Furthermore, the combination of scalp acupuncture and EEG biofeedback achieves the superior efficacy on children with ADHD as compared with the EEG biofeedback therapy alone. Moreover, on ADHD children, a complementary and alternative medicine combination, including acupuncture, applied kinesiology, and respiratory exercises improved sleeping conditions rapidly, as well as hand writing. Another study showed that combining with behavior therapy, acupuncture had a positive effect on reducing ADHD symptoms on preschool children. Comparing to routine western medicine, acupuncture showed a better effect on improving mild brain dysfunction on children.A decade ago, due to the limited RCTs evidence, former systematic reviews failed to conclude the efficacy and safety of acupuncture in the treatment of ADHD.As a result, to further investigate the safety and efficacy of acupuncture in the treatment of childhood ADHD, we would like to conduct this systematic review and meta-analysis, this work might contribute to the further evaluation of acupuncture treatment.Conceptualization: Yong Lin, Hongjiao Jin.Data curation: Ning Zhao, Zhu Li.Formal analysis: Hongjiao Jin, Ning Zhao.Funding acquisition: Bo Huang.Methodology: Hongjiao Jin, Jie Xu.Project administration: Bo Huang, Yong Lin.Software: Jiao Mao, Changda Chen.Supervision: Bo Huang.Validation: Jie Xu, Jun Zhang.Visualization: Jun Zhang, Biqin Shuai.Writing – original draft: Yong Lin, Hongjiao Jin.Writing – review & editing: BO Huang, Biqin Shuai.


To explore the relationship between adult Attention Deficit/ Hyperactivity Disorder (ADHD), antistreptococcal titers, ABGA, and recurrent infections during early childhood.Childhood history of recurrent infections and a blood sample were collected in a sample of DSM-IV adult outpatients with ADHD. The anti-streptolysin O (ASO), anti-deoxyribonuclease B (anti-DNase B), and anti-basal ganglia antibodies (ABGA) titers were determined in patient plasma by enzyme-linked immunosorbent assay (ELISA). Titers positivity was evaluated following manufacturer’s specifications. Absolute titers were also collected as continuous variables.Fourteen out of 22 (63.6%) have had recurrent infections in childhood . Eighteen patients (81.9%) were positive for anti-DNase B, five (22.7%) for ASO, and 4 (18.2%) were positive for both of them. Five participants (22.7%) were ABGA positive, whereas only two (9.1%) were positive for all three antibodies.patients with ADHD might be more prone to infections during childhood and subclinical streptococcal infections during adulthood. Moreover, they seem to have an increased risk for basal ganglia autoimmunity in adulthood. Both infections and the ensuing acquired autoimmunity could influence the neurodevelopmental process, by contributing, at least in part, to the ADHD pathogenesis. ADHD is one of the most prevalent neurodevelopmental disorders and it is characterized by a persistent pattern of inattention and/or hyperactivity and/or impulsivity, leading to functional impairment  and seveAlthough the etiopathogenesis of ADHD is still unclear, a gene by environment model has been proposed to explain the high family history of the disorder , 10, 11.Over the last 20 years, the recognition of the role of some infections  in determining acute, somewhat mixed, movement  and psychiatric disorders , have increased the attention to infective agents as possible causes of childhood neurodevelopmental disorders, involving basal ganglia –18. A moSince Kiessling and colleagues  have repADHD appears to play an important role in post-streptococcal basal ganglia disorder at least as much as obsessive-compulsive and tic disorders even when considered alone: higher titers of GAS infection markers were found in children with ADHD than in those with obsessive-compulsive or tic disorder; ADHD and even more hyperactivity symptoms significantly predicted anti-DNase B titer; and ASO titers were correlated with basal ganglia volume in children with ADHD .More recently, three case-control studies have focused on GAS infection and ABGA titers of children with ADHD and without comorbid obsessive-compulsive/tic disorders , 27, 28.To the best of our knowledge, no studies have assessed GAS antibodies and ABGA titers in adult patients with ADHD in an attempt to confirm the lifespan vulnerability of this population to GAS infection, which could also, in some cases, involve basal ganglia autoimmunity.The aim of the present preliminary study was to explore ASO, anti-DNase B, and ABGA titers in a sample of adult outpatients with ADHD.This preliminary study was conducted at the adult ADHD outpatient center of the San Luigi Gonzaga University Hospital , between July 1, 2019 and September 1, 2019, on a consecutive sample of patients newly diagnosed with adult ADHD according to DSM-IV-TR criteria (age > 18 years). According to the outpatient service routine, all patients had been screened for adult ADHD using the Adult ADHD Self Reporting Scale (ASRS-1.1) screener, and then the DSM-IV ADHD diagnosis was confirmed through the Diagnostic Interview for Adult ADHD (DIVA 2.0). The severity of ADHD symptoms was rated by Adult ADHD Investigation Rating Scale (AISRS).All patients were asked to participate in the study signing a written informed consent and a unique identification code was assigned to each patient, in order to maintain data anonymity and patient confidentiality. The research protocol of the present observational study was approved by the Research Ethics Committee of the San Luigi Gonzaga University Hospital , therefore the study was conducted in accordance with the Helsinki Declaration.During the assessment process, information about the history of both infections and psychiatric disorders were collected and patients were also assessed for other psychiatric disorders through an in-depth psychiatric interview. Recurrent infections in childhood  were defined as at least three symptomatic episodes of infection with fever  per year for at least 1 year.Blood samples from patients were collected in potassium ethylenediaminetetraacetic acid (EDTA) - containing tubes  at the end of each recruitment. Plasma was separated by centrifugation at 24,104 x g at 4 °C for 10 min and immediately stored at − 80 °C ready for analyses.Commercial, ready-to-use, microwell ELISA kits were used for the quantitative determination of plasma concentration of ASO , anti-DNase B , and ABGA . Sample absorbance values were detected in a 96-multiwell plate reader  using dual-wavelength recording modes at 450 nm and 655 nm (the latter used as reference). ELISA protocols instructions, and threshold values for adults provided by the manufacturer were adopted. . Thereby, the following values defined patient positivity: ASO > 200 U/ml; anti-DNase B > 86 ng/ml; ABGA > 1.18  but absolute titers values were also collected as continuous variables.The ASRS 1.1  is a sixThe DIVA 2.0  is a valThe AISRS is a validated  18-item All analyses and calculations were performed using RStudio for MAC OS .p-value of 0.05 was used to designate statistical significance. Mean and standard deviation (SD) were used to report normally distributed variables, whereas median (Mdn) and interquartile range (IQR) were used for nonnormally distributed ones.The positivity rate for every titer was calculated. The distributions of continuous variables were tested by Shapiro-Wilk’s test. A p < .001). Most of the patients have reached a middle or high school diploma, but more than a fifth of them have got a degree. Although the majority of the sample referred from GPs, psychiatrists, or child neuropsychiatrists, less than half accessed the adult ADHD center on their own .p < .001; anti-DNase B, Median = 95, IQR = 17, Shapiro-Wilk’s test W = .915, p = .049; ASO, Median = .54, IQR = .17, Shapiro-Wilks W test = .661, p < .001). Within sample subgroups comparison could not be performed due to low numerosity.As regards antistreptococcal antibodies, 18 patients (81.9%) had positive anti-DNase B titer, five (22.7%) had positive ASO titer, four (18.2%) had them both positive. Five (22.7%) patients were ABGA positive, whereas only two (9.1%) resulted positive for all three titers. All titers were nonnormally distributed in the examined population , more than a quarter of the sample still showed ASO titer positivity (index of recent GAS infection) and the same amount presents ABGA positivity (index of acquired autoimmunity). The comparison with previous studies conducted on children with ADHD is challenging because of methods heterogeneity, albeit they all excluded patients with tics, obsessive-compulsive disorder, and Sydenham’s chorea. On average, the positivity rate of ASO titers reported by previous studies  doun = 7) of the sample had a history of GAS infections [All patients were recruited and tested for titers during summer, the season having the fewest GAS outbursts . On the fections . All parfections , 4), we Even more emerged about basal ganglia autoimmunity as over a fifth of the sample had positive ABGA titers, consistently with a previous report regarding Italian children with ADHD 25%, ). This r%, . ThisThe main aim of the present study was to provide preliminary findings on the usefulness of ASO, anti-DNase B, and ABGA titers measurement in adult patients with ADHD. In this respect, we used easily available, standard, ready-to-use ELISA kit tests for the quantitative determination of blood titers that increased the reproducibility of our research and findings.n = 20, [n = 22, [Nevertheless, different limitations should be taken into account. First of all, the sample size of the present study was small, although it was not so far from those of some clinical groups of previous case-control studies conducted on children , which Further studies including appropriate controls should be conducted on wider samples to confirm our preliminary findings, providing more solid evidence to support the already suggested vulnerability of patients with ADHD to some type of infections in childhood and perhaps also in adulthood , 28. NevAccording to the present preliminary study, patients with ADHD might be prone to infections during childhood, subclinical streptococcal infections in adulthood, and they seem to have a high risk for basal ganglia autoimmunity as adults. Both infections and the ensuing acquired autoimmunity may influence the neurodevelopmental process, by contributing, at least in part, to the ADHD pathogenesis. ASO, anti-DNase B and ABGA titers seem to be easy to determine by using specific ELISA kits. It may be possible that a childhood predisposition to infection, which persists into adulthood as antistreptococcal titer positivity, might also contribute to the pathogenesis of ADHD and even to its persistence. Future controlled studies should confirm on wider samples our findings on streptococcal infections, autoimmunity, and ADHD. As for some other neuropsychiatric disorders, ADHD may be related to streptococcal infections or basal ganglia autoimmunity and this could have implications on pharmacological treatment.