Background: Attention-deficit/hyperactivity disorder (ADHD) is a common pediatric condition with significant developmental, social, educational, and safety implications. The American Academy of Pediatrics has developed guidelines to support quality care of children with ADHD, but studies demonstrate that the guidelines are variably followed.Methods: This review highlights patterns of diagnosis and treatment of children with ADHD nationally and in Louisiana and provides examples of system- and practice-level opportunities to improve adherence to quality standards.Results: Possible contributors to the higher prevalence of ADHD and medication use in Louisiana compared to the nation are specialty workforce shortages, factors in the educational system, and factors associated with race and geography. Innovative system approaches have been developed to address workforce shortages and training limitations. Practice-level innovations include improving the use of validated measures, offering adequate scheduling, and identifying relevant resources and sharing the information with families.Conclusion: Despite the availability of evidence-based recommendations and resources, significant opportunities exist to provide enhanced ADHD care at the primary care level, especially in Louisiana where the high prevalence of some risk factors for ADHD and the high rates of ADHD and medication prescriptions have been noted nationally and at the state level. Attention to these factors can potentially help address these disproportionalities. Additionally, innovative models of training and collaboration in pediatrics are imperative. Pediatric clinicians, mental health providers, and families can work together to increase awareness about the needs of children and families affected by ADHD in medical, educational, and policy arenas and move the system forward for children. Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5)  is one of the most common psychiatric conditions in children and adolescents and contributes to significant academic and functional impairment. Diagnosis is primarily clinical, involving confirmation of symptoms and differentiation from other causes of hyperactivity and inattention based on the criteria in the  (DSM-5) .1 ADHD c2 and 20163 suggest that 9.4% to 11% of children in the United States had received an ADHD diagnosis, with a 42% increase in the rates of diagnosis between 2004 and 2011.2 Additionally, rates reported in the United States are much higher than those reported in other developed countries4 and higher than the worldwide prevalence.5 Results from a large epidemiologic study from the United States suggest a prevalence (8.6% to 10%) similar to those reported by parent surveys.6 However, the study identified that a substantial proportion of children taking ADHD medications did not meet the criteria for ADHD, and only about half of children who met the criteria for ADHD had been previously identified.6 These differences highlight the importance of attending to patterns of identification, diagnosis, and treatment. Discussion of ADHD in Louisiana is important because Louisiana leads the nation in rates of pediatric ADHD medication use.7Parent-reported surveys from 2011This review describes trends in ADHD diagnosis and management nationally and in Louisiana and describes opportunities for adherence to best-practice guidelines.8 The guidelines include age-specific treatment recommendations for preschool-age children (4 to 5 years), elementary school–age children (6 to 11 years), and adolescents (12 to 18 years).8 Familiarity with these guidelines is important for primary care clinicians caring for children who have any behavioral concerns.In 2011, the American Academy of Pediatrics (AAP) published an update to the best-practice guidelines for the evaluation and management of ADHD in children and adolescents with a focus on developmentally specific recommendations .8 The gu7 established in 2014, have emphasized the importance of using standardized tools such as ADHD rating scales as part of the diagnostic process and of tracking the response to treatment.8 Validated rating scales are particularly valuable for eliciting information from teachers to demonstrate the presence of symptoms and impairment in more than one setting.9 Rating scales allow for comparisons across children, across reporters, and across time and assist in laying a foundation for managing ADHD using a chronic care and medical home model as recommended by the AAP ADHD guidelines.8The AAP and the Louisiana Task Force on ADHD,8 In this age group, the AAP and other practice guidelines recommend consideration of medication if symptoms persist after an adequate trial of behavioral therapy or if behavioral therapy is not available.10 For children 6 to 11 years, the AAP recommends prescribing stimulant and/or behavioral therapy, and for adolescents 12 to 18 years, the recommendation is stimulant with or without concurrent behavioral therapy.8For preschool-age children diagnosed with ADHD, the AAP recommends behavioral therapy as the first-line treatment.In young children, behavioral therapy focuses on parent management training strategies. In older children, behavioral therapy includes a combination of family-focused strategies to support a child's organization and impulse control and the development of self-regulation skills and organizational skills in the child.11 as recommended by the guidelines. However, duration from starting a stimulant to first follow-up averaged 72 days,11 substantially longer than the recommended 30-day follow-up.12 Delayed follow-up can result in undertreatment with ineffective doses and prolonged exposure to side effects.Despite the 2011 AAP guidelines, significant variability in ADHD care persists. In a 2014 study of pediatricians across 50 practices, approximately 70% reported applying DSM-IV criteria when diagnosing children with ADHD, and approximately half used an adult-report measure 11 Further, while 93% of the children were receiving medications, only 13% received behavioral therapy. These findings suggest that patients in the 6- to 11-year-old age group—when stimulant and/or behavioral therapy is recommended—overwhelmingly received stimulants instead of behavioral therapy.Despite recommendations to use validated measures, the Epstein et al 2014 study showed that only 10.8% of pediatricians used a parent follow-up ADHD rating scale to monitor treatment outcomes, and only 7.5% obtained teacher rating scales to monitor treatment outcomes.13 However, the proportion of preschoolers with ADHD who were treated with medication did not change.Information about ADHD treatment in preschool patients is limited, but after the publication of the AAP guidelines in 2011, rates of diagnosis of ADHD in preschoolers, which had been increasing, slowed.14 used data from the National Survey of Children's Health2 to show that 15.8% of children in Louisiana had been diagnosed with ADHD , and 10.4% of children in Louisiana were taking medications for ADHD.14 Louisiana children covered by Medicaid receive more prescriptions for ADHD medications (12.9%) than the state average (10.4%), costing Louisiana Medicaid approximately $30 million.7 Furthermore, the rate of ADHD diagnosis for Louisiana boys is substantially higher than the national rate (19% vs 12%), with an even higher rate among boys covered by Medicaid (22%).7 Interestingly, the rate of ADHD diagnosis for girls in Louisiana does not differ substantially from the national rates. In Louisiana, white boys receive ADHD prescriptions at the highest rate of all youth which is comparable to national trends l trends .716 Beginning at age 6 in Louisiana, children with birthdays in September (the youngest children in the class) are 26% more likely to have an ADHD prescription than children born in October,7 suggesting that classroom expectations influence treatment patterns.Another identified factor in prescribing patterns is age compared to classroom peers, which has been demonstrated in Louisiana and internationally.7 Hypothesized factors, including school districts, demographics in the region, insurance status (public vs private), and density of mental health providers, did not explain the variability, raising questions about the contribution to differences in trends from national data of other nonclinical factors such as variability in the diagnostic process.Geographic factors also seem to play a role in treatment decisions, with significant variability across the state, although the specific factors remain somewhat elusive. Generally, but with notable variability, the southern part of the state along the I-10/I-12 corridor (with the exception of New Orleans) showed higher rates of ADHD prescriptions than northern Louisiana .7 Hypoth8 and are seen in Louisiana at rates higher than the national rates, specifically, the incidence of low birth weight.17 Postnatal environmental risk factors for ADHD include adversity, stress, and exposure to toxins such as lead. Rates of exposure to adversities such as natural disasters, murder, and adverse childhood experiences occur at high rates in Louisiana.20 Louisiana's overall rate of exposure to elevated lead levels (1.3%) is similar to the national rate.21Louisiana's patterns of diagnosis and prescribing raise an important question about why Louisiana has high rates of ADHD identification and, relatedly, high rates of ADHD prescriptions. Children in Louisiana may possibly be at higher risk for ADHD than children in other states. Consistent risk factors and correlates of ADHD in the literature can be categorized as genetic, prenatal, and environmental. The high inheritability of ADHD may result in increased rates of ADHD among populations that tend to stay in a geographic area. Prenatal exposures to substances including tobacco, alcohol, and illicit substances; slow fetal growth; and maternal stress are associated with developing ADHD7 Additionally, the Louisiana Medicaid managed care organizations provided evidence-based treatment training for Louisiana clinicians and collaborated with the Louisiana Chapter of the AAP to purchase licenses for the AAP ADHD toolkit for all Louisiana providers.22 The toolkit provides evidence-based screening tools to help clinicians assess ADHD in children aged 4 to 18 years and specific screening tests to help clinicians identify comorbid conditions. The toolkit includes handouts and educational materials for both clinicians and parents that address the treatment of ADHD, monitoring, and follow-up and also includes tools designed to help families better understand ADHD  estimates that ≥47 child and adolescent psychiatrists (CAPs) are needed for every 100,000 children.23 No counties in the United States achieve this goal, and most of the country is defined as a “severe shortage” area. Louisiana has approximately one-seventh of the required CAPs for the pediatric population at 7.17 CAPs per 100,000 children, and the CAPs are generally clustered in large population centers.23 Other specialties have workforce shortages as well. For example, according to the AAP membership directory, Louisiana has 5 developmental and behavioral pediatricians, principally in urban centers.24 Similarly, as of 2013, Louisiana ranked lowest for per capita clinical and school psychologists and also had a severe shortage of social workers.25 Overall, these shortages mean extensive wait times; limited access; and a burden on pediatric primary care, clinicians, and families.Although noncomorbid mild to moderate ADHD can be managed in the primary care setting, children with complex presentations or more severe ADHD benefit from specialty evaluations and treatment to assess for comorbid conditions and/or to identify other causes for the clinical presentation.26 However, pediatric residency training rarely prepares trainees sufficiently for this role. In a 2010 survey, fewer than half of graduating pediatric residents reported they had good to excellent competence in dealing with children's mental health.27 Further, 65% of practicing pediatricians reported in 2013 that they lacked training in children's mental health problems.28 One contributing factor may be that the faculty members who are training most pediatric residents have limited training or confidence in addressing mental health concerns themselves. To date, pediatric training does not require any clinical training exposure to child psychiatry, despite the expectation to learn to assess and to manage many psychiatric disorders.29 The required developmental and behavioral pediatrics rotation, which was mandated in 2000,30 may provide some training, but limited access to developmental and behavioral pediatricians means that the training remains variable.31Both the AAP and the AACAP recommend that primary care clinicians play the primary role in addressing common mental health issues such as ADHD and depression.After residency training, continuing medical education opportunities are available to keep physicians up to date on trends and guidelines. These opportunities are generally designed as short didactic courses rather than interactive educational experiences.32 and the Post Pediatric Portal Program33 are two innovative residency programs developed as alternative pathways to train physicians in child and adolescent psychiatry. The Triple Board Program, started in 1986, is a 5-year program that trains physicians who are eligible for board certification in pediatrics, psychiatry, and child and adolescent psychiatry. The Post Pediatric Portal Program is a 3-year program to train board-eligible pediatricians in general and child and adolescent psychiatry. In Louisiana, the Triple Board Program at Tulane University has trained 15 residents since 2010, more than half of whom practice pediatric psychiatry along the Gulf Coast, principally in the Greater New Orleans area. While these innovative training programs contribute valuable and well-trained specialists, the shortage remains substantial. Several states, including Louisiana, have offered loan repayment programs for healthcare providers trained in psychiatry, with the goal of addressing local workforce shortages.34Some efforts have been made to increase the workforce of specialists who can meet the needs of children with ADHD and related mental health concerns. The Triple Board Programwww.AAP.org). The availability of these training modules allows all residency programs, including those with limited access to specialists, to provide quality training on these topics.Efforts also address the training of pediatric residents. The AAP has developed training modules about several mental health topics, motivational interviewing, and the ADHD toolkit, most of which are publicly available on the AAP website  Institute in which primary care clinicians participate in an intensive course on child mental health, followed by ongoing longitudinal group case discussions.39 Unlike one-on-one direct services, telehealth consultation can influence the entire population cared for by a primary care clinician, not just the child being discussed, because the clinician can apply the new knowledge to similar clinical situations. Telehealth consultation offers a dynamic learning opportunity for primary care clinicians that potentially can help them manage patients with ADHD or other mental health concerns. Although the pediatric data are limited, long-term cost analyses in adults have shown significant cost savings: $1 spent on collaborative care saves $6.50 in direct healthcare costs.40 One study in adolescents with depression showed an overall net decrease in cost and improvement in the quality of life.41 Because consultation codes are not reimbursable in most states, most consultation programs are funded at the state level through Medicaid, state sources, federal grants, or philanthropic grants.At least 19 states have statewide telehealth child psychiatry consultation programs.40 and high rates of satisfaction. In Louisiana, collaborative models have limited scope but have been well received and show promise. The Tulane Early Childhood Collaborative (TECC) in the Greater New Orleans area and Project LAUNCH , an Office of Public Health/Office of Behavioral Health project, offer interdisciplinary direct consultation at the pediatric office and telehealth consultation for young children by primary care clinicians.43 TECC has developed handouts and training about ADHD and is specifically focused on enhancing ADHD care through educational tools and quality improvement activities. Clini-cians at Louisiana State University provided collaborative treatment to patients in Louisiana parishes affected by the Deepwater Horizon oil spill and reported a significant decrease in psychiatric symptoms among the adults who received treatment.44 In addition, the primary care clinical staff reported an increase in their confidence and competence in evaluating and managing patients’ mental health needs in the future. While this study did not include treatment for children, a similar approach could be used to provide mental health care to children in areas that lack access to mental health care.Data demonstrate that participation in the consultation is associated with increased use of mental health skills discussed in consultation45 Other studies have shown that using case managers who are trained to provide psychoeducation to parents and their children with ADHD leads to reduced family stress, improved provider diagnostic and management skills, and improved ADHD symptoms.47Other models involve the addition of lay care coordinators to pediatric practices to improve mental health outcomes in children. A 2010 study showed that the addition of case managers to pediatric teams facilitated communication between primary care clinicians and specialty services treating children with ADHD, resulting in improved clinical outcomes and parental satisfaction.To date, no data support that one approach may be better than the other. Future research can focus on whether the most successful models are those that are flexible, utilize local expertise, address community-specific needs, and work within existing healthcare systems.At the practice level, the use of validated measures, development of comprehensive assessment recommendations, scheduling changes, and adequate access to resources can influence the quality of ADHD care.11 Several barriers may influence the limited use of these measures in pediatrics. Pediatricians’ concerns about using checklists include time, resources, and the perception that checklists are too impersonal for use in practice.48 However, a 2017 study showed that using the Early Childhood Screening Assessment49 did not add to appointment time, and many providers felt that their practice was more efficient when using written measures.50Caregiver-report measures to diagnose ADHD in children are used in less than half of evaluations.12 This schedule may be a useful guide for when providers can use measures to follow up on symptoms and consider titration of medication doses.Pediatric clinicians can consider obtaining follow-up measures at regular intervals  or in association with medication titration or symptom changes. The Healthcare Effectiveness Data and Information Set, which defines quality indicators for most insurers, sets the standard of 3 follow-up visits for children in the 10 months after initial diagnosis.Primary care clinicians may not want to rely solely on parents and children with ADHD to be the liaison between providers and the children's schools. To encourage the collection of information from teachers, one strategy is to include the clinician's office phone and/or fax number on the scale. Providing this information can lead to direct conversation if the teacher desires. School fax numbers may be available on websites, and keeping a list of these numbers for easy access can be a useful practice for primary care clini-cians. The AAP ADHD toolkit includes a letter for clinicians to send to teachers that explains why the teacher's input is valuable in guiding treatment plans; this information may increase some teachers’ motivation to complete and return the measure.51 Primary care clinicians have limited time during pediatric visits, so procedures that support efficient data collection are useful. First, knowing a family history informs risk status. Children with a family history of ADHD, mood disorders, and substance use disorders should be considered at higher risk for developing ADHD or other disorders than children without such a family history. Identifying these disorders routinely during an initial visit with a family increases efficiency and may facilitate the early identification of disorders in children. Considering developmental status and learning problems is critical when a child presents with school or behavioral concerns. Conducting validated developmental screens routinely as recommended by the AAP can be helpful for the early identification of children at risk of learning problems. Additionally, every child presenting with emotional and behavioral concerns such as ADHD should be verbally screened for depression, anxiety, and PTSD. While the Vanderbilt Assessment Scale is well validated for identifying ADHD, its 37% sensitivity in identifying anxiety or mood problems is inadequate, and clinicians must use an additional measure or explicitly review symptoms of mood and anxiety as part of an ADHD assessment.52 For children older than 8 years, the Pediatric Symptom Checklist-17 can be used to explore a broad range of symptoms, including mood and anxiety.54 Other measures such as the Patient Health Questionnaire-9A56 or the Screen for Child Anxiety Related Emotional Disorders58 can be used in cases of suspected depression or anxiety, respectively. To explore environmental stressors, primary care clinicians can consider using tools such as the Safe Environment for Every Kid (SEEK),59 Well-Child Care Visit, Evaluation, Community Resources, Advocacy, Referral, Education (WE CARE),60 or Center for Youth Wellness Adverse Childhood Experiences Questionnaire.61 The Tulane Early Childhood Collaborative uses a version of the SEEK that is modified to include locally common traumatic events.62 Using such measures routinely elicits information efficiently and establishes a framework for biopsychosocial assessment that can be consolidated in future visits and further evaluations. The Screening Technical Assistance Resource (STAR) center is an initiative from AAP that provides healthcare professionals with evidence-informed technical assistance and resources to assist in implementing effective screening, referral, and follow-up recommendation.63 The website also includes an online tool finder to assist providers in finding and utilizing an evidence-based screening tool.64Approximately two-thirds of children with ADHD have a comorbid condition.65 In this study, a group of 579 children, aged 7 to 10 years, with ADHD combined type were assigned to 14 months of medication management (titration followed by monthly visits); intensive behavioral treatment ; the two combined; or standard community care (treatment by community providers). More frequent visits (8.8 vs 2.3 in a year) and longer appointments (30 minutes vs 18 minutes) were key differences between the medication management group and standard community care group, despite both groups receiving medication treatment.65 The standard community care group had the worst outcomes and received lower doses of stimulants, suggesting that the scheduling differences may have resulted in insufficient time to assess and effectively treat the symptoms.The landmark multimodal treatment of ADHD study demonstrated that scheduling likely plays an important role in adequate treatment.Adequate visit schedules can be achieved by developing a patient registry for children with ADHD. A patient registry offers a way to track patients with special healthcare needs who should be followed at shorter intervals than the children who are healthy and developing typically. A patient registry, ideally embedded in an electronic medical record, can provide flags if a patient has missed or not scheduled appointments and may include reminders to contact the patient. Registries can also include ticklers to remind primary care clinicians or the team to send a symptom checklist to the child's school.66While many practices do not have a case manager on site, every practice can have a list of community and electronic resources related to ADHD management. A printed list of resources can be easily shared with families, and clinicians can circle relevant resources or print selected electronic resources. Phone numbers and URLs for developmental services, the school system's point of entry for special education evaluations, organizations that support families with educational advocacy, and preferred local mental health providers for children and adults should be included. One resource that families or providers can use to identify support for basic needs including housing, food, transportation, and legal resources is called Aunt Bertha, available at auntbertha.com.Point of care educational resources for families are also valuable to reinforce the verbal guidance provided during a visit .Despite the availability of evidence-based recommendations and resources, significant opportunities exist to provide enhanced ADHD care at the primary care level, especially in Louisiana where the high prevalence of some risk factors for ADHD and the high rates of ADHD and medication prescriptions have been noted nationally and at the state level. Specialty workforce shortages, factors in the educational system, and factors associated with race and geography seem to play important roles. Attention to these factors can potentially help address these disproportionalities effectively. When the youngest children in classrooms are predictably being diagnosed with ADHD more than their older peers, questioning whether the existing classroom structure can address the needs of the entire developmental spectrum is reasonable. Mental health consultation in classrooms may be useful in supporting teachers to adapt to the wide developmental range in early elementary school. Additionally, examining if high-stakes testing and teacher evaluations based on student performance influence these patterns may be worth considering.Specialty workforce shortages in child and adolescent psychiatry are unlikely to be fully remedied in a short time span, so innovative models of training and collaboration in pediatrics are imperative. Specifically, required training in residency, ongoing case-based learning after residency, and consultation and collaborative care are promising approaches to improving the quality of care. At the practice level, the AAP ADHD guidelines, the AAP ADHD toolkit, and scheduling recommendations offer practical approaches that can enhance the care children receive. Statewide initiatives to support innovative healthcare delivery approaches, such as consultation, could bring improved care to Louisiana's most underserved children with ADHD. Pediatric clinicians, mental health providers, and families can work together to increase awareness about the needs of children and families affected by ADHD in medical, educational, and policy arenas and move the system forward for children.


Plasmodium parasite, however, the underlying mechanism needs to be further elucidated.Malaria remains to be one of the deadliest infectious diseases and imposes substantial financial and social costs in the world. Mosquitoes rely on the immune system to control parasite infection. Peptidoglycan recognition proteins (PGRPs), a family of pattern-recognition receptors (PRR), are responsible for initiating and regulating immune signaling pathways. PGRP-LA is involved in the regulation of immune defense against the pgrp-la in Anopheles stephensi were analyzed by qPCR. The function of PGRP-LA was examined using a dsRNA-based RNA interference strategy. Western blot and periodic acid schiff (PAS) staining were used to assess the structural integrity of peritrophic matrix (PM).The spatial and temporal expression patterns of pgrp-la in An. stephensi was induced in the midgut in response to the rapid proliferating gut microbiota post-blood meal. Knocking down of pgrp-la led to the downregulation of immune effectors that control gut microbiota growth. The decreased expression of these immune genes also facilitated P. berghei infection. However, such dsLA treatment did not influence the structural integrity of PM. When gut microbiota was removed by antibiotic treatment, the regulation of PGRP-LA on immune effectors was abolished and the knock down of pgrp-la failed to increase susceptibility of mosquitoes to parasite infection.The expression of Plasmodium parasites in An. stephensi.PGRP-LA regulates the immune responses by sensing the dynamics of gut microbiota. A mutual interaction between gut microbiota and PGRP-LA contributes to the immune defense against  Plasmodium. It remains to be a high concern of the World Health Organization due to the continued emergence and spread of drug-resistant parasites and insecticide-resistant mosquitoes  were quantified by qPCR using specific primers . The recPer1 was purified through Ni-NTA Superflow resin by the AKTA Explorer system  and was used to generate the antibody commercially . For PM structure analysis, the abdomen of An. stephensi was fixed at 45 h post-infectious blood meal and sectioned at 5 μm using the paraffin sectioning method and stained with Periodic Acid Schiff (PAS) (Sigma-Aldrich) as previously described [For measurement of mRNA levels, 3 PM synthetic genes [escribed . The sliAll statistical analyses were performed using GraphPad Prism version 6 . Statistics of gene expression were tested for using the Student’s t-test. The Mann–Whitney test was used to determine the significance of infection rate and microbiota level of dsRNA-treated mosquitoes.pgrp-la in the midgut and the remaining carcass of An. stephensi 24 h prior to (− 24 h) and 24, 48 and 72 h post-infectious blood meal. It was expressed abundantly in the carcass before blood-feeding. However, pgrp-la was upregulated significantly in the midgut within 48 h after the mosquito took an infectious blood meal  mosquitoes. As expected, once the gut microbiota was removed by antibiotic treatment, the expression of pgrp-la in the midgut significantly decreased, compared to control mosquitoes  , 1 negative regulator of IMD signaling pathway  and 4 proteins related to cellular and epithelial immune responses . As expected, the expressions of four genes, attacin, tep1, nos and defensin were significantly decreased in dsLA-treated mosquitoes comparing to those in dsGFP  was injected intrathoracically into An. coluzzii, we next investigated the influence of PGRP-LA on parasite defense in An. stephensi [pgrp-la (dsLA) resulted in a significant increase in the infection rate of P. berghei from 9 oocysts in dsGFP to 46 oocysts in dsLA mosquitoes  and two PM digesting chitinases [peritrophin1 (per1) and chitinase A which were differentially regulated 24 h post-infection in dsLA mosquitoes, the expression of most of the genes was comparable to dsGFP mosquitoes , thereby inhibiting nfection , 39, 40.itinases . Except pgrp-la is correlated with the abundance of gut microbiota, we next investigated whether the regulation of PGRP-LA on immune response could rely on the presence of gut microbiota. The dsRNAs were introduced into An. stephensi of which gut microbiota was removed by antibiotic treatment. Surprisingly, once the gut microbiota was cleared, knock down of pgrp-la had no impact on the expression of the immune effectors, especially attacin, tep1, nos and defensin , which is a negative regulator of Drosophila innate immune signaling. Such interaction helps control the homeostasis of commensal bacteria [Wolbachia, which in turn induces the expression of PGRP-LE in the carcass of A. aegypti [The interactions between insect immune system and gut microbiota play vital roles in determining infection outcomes. Here we show that the regulation of immune responses by PGRP-LA relies on the presence and abundance of gut microbiota. In agreement with our findings, other PGRP family members are also involved in maintaining the homeostasis of gut microbiota. For example, PGRP-SC2 contributes to the gut immune homeostasis through limiting age-related gene, osophila . PGRP-LCbacteria . PGRP-LE aegypti . Additio aegypti –49.pgrp-la, which in turn controls the homeostasis of gut microbiota. Such finely tuned balance is key in regulating the defense against pathogen infection. Our findings will pave the way for further understanding the tripartite interactions between the mosquito, its gut microbiota and the Plasmodium parasite.In this study, we show that gut microbiota regulates the expression of Additional file 1: Table S1. Primers used in this study.Additional file 2: Figure S1. a PGRP-LA silencing efficiency in control, dsGFP and dsLA mosquitoes. b The weight of dsRNA-treated mosquitoes at 0 h and 24 h post-infectious blood meal.Additional file 3: Text S1. Significant results from statistical analyses in this study.


N = 462; 16–17 years), we assessed the level of ADHD symptoms using the Strengths and Difficulties Questionnaire, social behavior using the Social Responsiveness Scale, and the allelic distribution of the dopaminergic catechol-O-methyltransferase (COMT) Val158Met polymorphism. We found a significant association between COMT and social impairment, insofar as Met-allele carriers showed increased levels of social impairment. Moreover, social impairment significantly determined an association between COMT and ADHD (explained variance: 19.09%). This effect did not significantly differ between males and females. COMT and social impairment might interactively affect ADHD symptomatology, and could thus represent significant gene-phenotypic risk factors for ADHD symptomatology. This might have interesting implications for prevention and intervention strategies with a focus on social behavior in genetically at-risk individuals.The dopaminergic system has been shown to have substantial effects on the etiology of attention-deficit hyperactivity disorder (ADHD). However, while some studies found a significant direct effect, others did not. In this context, social behavior might play an important role as a factor that is related both to the dopaminergic system and ADHD. In a large epidemiological sample of adolescents ( Attention-deficit hyperactivity disorder , charactO-methyltransferase gene (COMT)  . The COM 22q11.2  and code 22q11.2 . Previoul allele , which wl allele  or to bel allele . Others l allele . The disCOMT inhibition was found to be associated with increased egalitarian tendencies  at the age of 16–17 years. Data from the sample assessed in France were not analyzed (N = 96), because no French validation was available for one of our measures, the Social Responsiveness Scale (SRS) (see below for details) .The subjects of the present study were part of the European Imaging Genetics (IMAGEN) study , a studyExclusion criteria for participation in the study were: any mental disorder as defined by the Development and Well-Being Assessment (DAWBA) , IQ < 80Deoxyribonucleic acid was extracted from venous blood samples. To ensure high quality and sufficient quantity, DNA extraction was performed by a semi-automated process. All samples were part of a genome-wide genotyping of ∼600,000 autosomal SNPs within the IMAGEN study using the Illumina Quad 610 chips .COMT Val158Met in the sample were as follows: Val/Val: n = 93, Val/Met: n = 243, and Met/Met: n = 126. The genotype distribution did not differ according to sex  or site .The genotype frequencies of www.sdqinfo.org). For the present analyses, we only used the hyperactivity/inattention problem scale, with the total hyperactivity score as dimensional variable [sum of the five hyperactivity items (self-ratings)] (see Tables 2).To assess ADHD symptom strength, we used the hyperactivity scale from the Strengths and Difficulties Questionnaire (SDQ) , a screeTo measure social behavior/impairment, we used the SRS , a widelTo assess the two questionnaires, we used the Psytools software  via its Internet-based platform.All analyses were conducted using the Predictive Analytic Software  for Windows, version 24.COMT Val158Met polymorphism on ADHD symptoms and social impairment, we conducted a univariate analysis of covariance (ANCOVA), with ADHD symptoms or social impairment as dependent variables and COMT as between-subject factor with three levels , adjusting for sex and conduct problems as potential confounding variables. Data were also corrected for multiple comparisons.Effects of COMT on ADHD symptoms and social behavior: To test the direct effect of the COMT, social behavior, and ADHD symptoms: In a subsequent moderator analysis  and ADHD symptoms as dependent variable (outcome)  . Sex andp-values < 0.05 are reported.For all analyses, COMT on ADHD (F(2.460) = 1.537; p = 0.216; partial eta-squared = 0.007; covariates: sex: F(1.460) = 16.248; p < 0.001; partial eta-squared = 0.034; conduct problems: F(1.460) = 94.844; p < 0.001; partial eta-squared = 0.172). However, COMT did have a significant impact on the level of social impairment (F(2.460) = 4.376; p = 0.013; partial eta-squared = 0.019; covariates: sex: F(1.460) = 0.026; p = 0.873; partial eta-squared = 0.000; conduct problems: F(1.460) = 22.941; p < 0.001; partial eta-squared = 0.048): Homozygote Met-allele carriers showed increased levels of social impairment compared to Val-allele carriers .We found no significant effect of COMT as predictor, ADHD symptoms as dependent variable, and social impairment as moderator was significant . There was a direct effect of COMT on social impairment , and of social impairment on ADHD symptom strength , but no effect of COMT on ADHD symptoms  . However, we found an indirect effect of COMT on ADHD moderated by social impairment , and significant effects of the two covariates .The regression model with COMT and ADHD, and thus represent a significant moderator driving the influence of the dopaminergic system on ADHD symptomatology. In the present study, we found no significant association between the dopaminergic COMT polymorphism and ADHD symptoms, although this might depend on social impairment: The level of social impairment served as a moderator of the association between COMT and the levels of ADHD symptoms. Moreover, COMT was further directly significantly associated with social impairment.The dopaminergic system has substantial effects on ADHD etiology. However, so far, studies have yielded inconsistent results, with some finding a significant association and others failing to do so. These discrepant findings may be attributable to a significant effect of further factors such as social behavior, which might modulate the direct association between COMT Val158Met on ADHD and related symptoms or traits only in boys, while others observed the opposite results. In the present study, we did not observe any significant difference between male and female participants. This indicates that social impairment is an important general facet of ADHD that is strongly triggered by the catecholaminergic system, but not so much by sex-related biological or social constraints.While previous studies found positive associations of both the Val and the Met allele with ADHD symptomatology ; severalCOMT and its associated functional consequences may thus serve as critical neurobiological determinants for ADHD-related risk and problem behavior. By contrast, compared to the Met genotype, the Val genotype was associated with positive emotionality and extraversion  is rather small for a genetic association study; thus, we were not able to further subdivide the sample to perform a replication or sex-related analyses. It could be speculated that a direct effect between COMT and ADHD – against conclusion from three meta-analyses (COMT on antisocial behavior in ADHD patients (COMT effects in this context. However, it would also be of interest to include other dopaminergic genes or genes that have been identified by GWAS in ADHD samples in further investigations using larger clinical or non-clinical samples. Moreover, finding has to be treated with caution due to a possible overestimation of the genetic effect (COMT, ADHD symptoms, and social impairment in an epidemiological sample of adolescents was observed. This indicates significant gene–environment risk factors for ADHD symptomatology also at subthreshold levels, and could inform strategies to prevent or manage social problems in daily life in genetically at-risk individuals.As one limitation of the present study we have to mention that our sample sizes (analyses  – could patients  and posspatients  and thusc effect  based onAB, CBü, HG, PG, AH, J-LM, TP, MR, MS, HW, RW, GS, TB, and HF study design. SM, FN, SHo, SHe, CBa, SV-K, UB, EQ, JUF, and HL data assessment and recruitment. SM, FN, JOF, SD, VF, BI, M-LM, DP, LP, and TB data management and analyses. SM, FN, SHo, and TB paper writing.TB has served as an advisor or consultant to Bristol-Myers Squibb, Desitin Arzneimittel, Eli Lilly, Medice, Novartis, Pfizer, Shire, UCB, and Vifor Pharma; he has received conference attendance support, conference support, or speaker’s fees from Eli Lilly, Janssen McNeil, Medice, Novartis, Shire, and UCB; and he is involved in clinical trials conducted by Eli Lilly, Novartis, and Shire; the present work is unrelated to these relationships. SM received conference attendance support from Shire. SHo, SHe, SV-K, AB, UB, CB, EQ, SD, HF, VF, HG, PG, AH, BI, J-LM, M-LM, DP, TP, LP, JF, MS, HW, RW, GS, and FN report no financial relationships with commercial interests.


Tricin  is a flavone that has been found to be incorporated in grass lignin polymers via 4′–O–β coupling. Herein, we investigated the tricin-lignin structure using nuclear magnetic resonance (NMR) methods by comparing the 1H–13C heteronuclear correlation (HSQC) NMR spectra of the isolated lignin with a series of dimeric and trimeric tricin-4′–O–β-ether model compounds. Results showed that the tricin moiety significantly affects the chemical shift of the Cβ/Hβ of 4′–O–β unit, producing peaks at around δC/δH 82.5–83.5/4.15–4.45, that differ from the Cβ/Hβ correlations from normal 4–O–β units formed solely by monolignols, and that have to date been unassigned. Lignin, a phenylpropanoid polymer, is one of the major components of plant cell walls. Many aspects of lignin structure and biosynthesis remain elusive . SeveralHSQC is the most frequently applied 2D nuclear magnetic resonance (NMR) technique for lignin characterization because it provides comprehensive information on the types of units and their characteristic interunit linkages in the polymer. Signal assignments in a HSQC spectrum of lignin are mostly based on the assignments of low molecular weight model compounds, particularly dimers and trimers, which provide the key NMR data for structural authentication. In this short communication, we report on work in which we synthesized several 4′–O–β-coupling products of tricin with coniferyl and sinapyl alcohol, as well as a trimer that resulted from further cross-coupling with another monolignol. By comparing the NMR data of the model compounds with those of an isolated lignin sample, we have elucidated the tricin-lignin structure  in polymeric lignin and reveal characteristic new correlations in the HSQC spectrum.2, 1 mm, normal phase, Analtech.  were used for raw product fractionation and purification using hexane/ethyl acetate or methanol/dichloromethane as eluent. Flash chromatography was performed using Biotage SNAP silica cartridges on an Isolera One instrument  using a hexane/ethyl acetate (EtOAc) gradient as eluent.All chemicals and solvents used in this study were purchased from commercial sources  and used without further purification. The wheat sample was the same as that used in a previous publication ; chalcon1 Tricin-(4′–O–β)-coniferyl alcohol, compound 2 tricin-(4′–O–β)-syringyl alcohol, and compound 4 tricin-(4′–O–β)-syringyl alcohol-(4–O–β″)-coniferyl alcohol were all synthesized according to a previous study [3 tricin-(4′–O–β)-coniferyl alcohol (4-O-methylated) was synthesized using the same method as other tricin-containing products, but using 3,4-dimethoxyacetophenone instead of 3-methoxy-4-hydroxyacetophenone as starting materials. All of the synthetic compounds were characterized by NMR and the data match those in a previous publication [Compound us study . Compounlication .3 and then transferred to an NMR tube for characterization.The model compound (10 mg) or enzyme lignin (25 mg) was dissolved in 0.25 mL pyridine and 0.1 mL acetic anhydride (1 mL pyridine and 0.5 mL acetic anhydride for lignin) and stirred for 2 h (12 h for lignin) at room temperature. When the reaction was completed, the solvent was evaporated under reduced pressure at 45 °C. Ethanol was added as co-solvent and repeatedly evaporated to completely eliminate the residue of pyridine and acetic anhydride. The acetylated samples can be readily dissolved in CDClv/v, and acetylated samples in CDCl3) and the solution was then transferred to an NMR tube for NMR acquisition. NMR spectra were recorded on a Bruker Biospin AVANCE 500 or 700 MHz spectrometer  fitted with a cryogenically cooled 5-mm TCI (500 MHz) or quadruple-resonance 1H/31P/13C/15N QCI gradient cryoprobe (700 MHz) gradient probe with inverse geometry (proton coil closest to the sample). Bruker’s Topspin 3.1 (Mac) software was used to process spectra. The central solvent peaks were used as internal references .The model compound or lignin sample (10–25 mg) was dissolved in 0.7 mL of deuterated solvent -sinapyl alcohol-(4–O–β″)-coniferyl alcohol. By comparing the lignin HSQC spectra with those of the model compounds, we were able to resolve the tricin-lignin structure from these Cβ/Hβ correlations. The aromatic region of the tricin HSQC spectrum has been well studied previously ,6, showiBrachypodium (unpublished data in our lab), and oat (unpublished data in our lab) all clearly showed the correlations in the range of δC/δH 82.5–83.5/4.15–4.45, but researchers were not able to assign this correlation at the time because of the lack of data from model compounds. The lignin from coconut coir fibers contains tricin as well, but the content was too low to show the characteristic tricin correlations in the HSQC spectrum [The HSQC spectra of the lignin preparations from monocot species such as wheat ,10,11, bp-coumaroyl-CoA toward flavonoid biosynthesis. The tricin moiety was completely depleted in the CHS mutant and, therefore, the corresponding HSQC spectrum did not show the characteristic correlations from tricin in the aromatic region [To further confirm our hypothesis, we characterized the lignin isolated from the CHS-deficient maize and its wild-type control. CHS is the main enzyme that controls the carbon flux from the common pathway intermediate c region . As was In summary, the data from model compounds, together with the study of the lignin structure of different species, as well as the lignin from CHS mutant maize leaf and its corresponding wild-type control, unambiguously support our hypothesis that the tricin moiety affects the Cβ/Hβ 4′–O–β unit chemical shifts such that correlations in the region of δC/δH 82.5–83.5/4.15–4.45 in the HSQC spectrum are diagnostically assigned to these tricin-lignin unit Cβ/Hβ entities. As such, these correlations provide another “marker” for tricin in lignins, this time from the viewpoint of the lignin rather than the tricin moiety.


M = 21.23, SD = 8.04) took part in the study. Virtual reality (VR) variables such as the number of correct answers, omission and commission errors, among others, were used to predict current and retrospective self-reported symptoms of ADHD using multiple regression models. Correct answers and omission errors in the VR test significantly predicted both current and retrospective ADHD symptoms. However, only the number of perseveration errors and gender were able to significantly predict retrospective ADHD symptoms. These findings suggest that inattention problems tend to remain after adolescence, while perseveration errors (which have been related to impulsive behavior) and gender differences tend to diminish.Despite the persistence of attention deficit hyperactivity disorder (ADHD) into adulthood and adolescence, there are few objective, reliable instruments (based on patient performance) that have been shown to be able to predict current and retrospective ADHD symptoms. The present study aimed to explore whether a validated VR test called Nesplora Aquarium is able to predict ADHD symptoms in adults and adolescents, based on both current and retrospective self-reports. A non-clinical sample of 156 adults and adolescents (70 women and 86 men) between 16 and 54 years of age ( Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by persistent attentional problems, difficulty in controlling impulses, and hyperactivity . It affePrevious neuroimaging studies suggested that people with ADHD show reduced activation, compared to controls, in right lateral prefrontal cortex regions when performing go/no go tasks. This suggests that a deficit in executive function (EF) gives rise to the behavioral symptoms of ADHD . EF inclCorrect responses: the sum of the participant’s correct responses, considered as a measure of working memory due to the high cognitive load involved in responding simultaneously in the two sensory modalities with varying instructions.Omission errors: the sum of the participant´s errors, failing to press the button when they were supposed to press it, considered an indicator of attentional arousal in situations of high cognitive load for working memory.Commission errors: the sum of the participant´s errors, pressing the button when they were not supposed to press it, considered an indicator of impulsivity.Reaction time: the average time the participant took to press the button once the target stimuli was presented, considered an indicator of processing speed.Variability of reaction time: the difference between the fastest reaction time and slowest reaction time registered in the test. This measure is indicative of changes in sustained attention or fatigability during the task.Conversely, in the case of the adult population, various studies have noted the existence of underdiagnoses of ADHD. The main reason is due to the absence of CPTs or other objective tools (based on patient performance) which allow assessment of possible ADHD cases in adolescence and more especially in adulthood. Both late adolescence and adulthood require more complex testing and assessment protocols because of the presence of comorbid psychiatric disorders.Considering the complexity of diagnosing ADHD in adulthood or late adolescence, it is important to have adequate objective tests. Most current tests demonstrate poor ecological validity and are administered in unrealistic conditions that might generate extraneous variance depending on the many ways subjects adapt to these conditions . AnotherIt is important to develop reliable, ecologically valid measures for measuring ADHD symptoms and the incorporation of virtual reality (VR) is a promising approach . While mThis study aims to explore whether a validated VR test called Nesplora Aquarium  is able Each of these tasks is composed of 140 evaluation items and 20 training items, but only the evaluation items of the final two tasks are considered in the analysis. The objective of the first task is to produce fatigue and ensure learning of the stimuli. The whole test takes about 20 min. Once the tasks are finished, the new VR test not only provides the aforementioned variables , but also provides data on two new important variables: (1) switching: the difference between the participant´s correct responses in the last part of a task and the beginning of the next task, considered an indicator of the capacity to shift attention; and (2) perseveration errors: the errors registered in the dual execution tasks, when subjects respond to the task by following the instruction of the previous task. This variable is interpreted as deficit in cognitive flexibility.Given the potential benefits of using VR tools that provide different variables related to attention, impulsivity, and hyperactivity problems, it is likely that VR variables will be significant predictors of current and retrospective ADHD symptoms.This objective would have significant practical implications in the diagnosis of ADHD in adolescence and adulthood. If a VR test was able to predict a high percentage of retrospective patient symptoms, VR variables could be used as a good indicator for determining whether ADHD symptomatology had been present throughout a patient’s life.M = 21.23, SD = 8.04). The sample was recruited by a convenience method, which means that all participants were previously informed about the objectives of the present research. The participants came from different high schools, universities, and companies in Spain. Participants reported a medium-high socio-economic level. Regarding education, 13.4% had studied up to secondary school level and 86.6% had university qualifications. From the complete sample, 53.3% were in mid and late adolescence , while the remaining 46.8% were adults . None of the participants reported having been diagnosed with ADHD or other psychiatric or neurological diseases.A non-clinical sample of 156 adults took part in the study: 70 women (44.9%) and 86 men (55.1%), between 16 and 54 years of age , and all procedures were performed in compliance with relevant laws and institutional guidelines. After providing informed consent, participants completed two self-report measures (current ADHD symptoms and retrospective ADHD symptoms) and completed the Nesplora Aquarium VR test. The evaluations were conducted in a laboratory and lasted for 1 h. A member of the research group was responsible for contacting the participants and supervising the assessment process.The Spanish adaptation  of the AA cut-off score ≥ 12 suggests that symptoms are consistent with ADHD . Ramos-Quiroga et al.  reportedTo measure retrospective ADHD symptoms, the ADHD subscale from the Wender Utah Rating Scale (WURS)  was admiFinally, the VR EF Test called Nesplora Aquarium   were calculated .p < 0.05.Then, two hierarchical regression analyses were conducted : one anaSimilarly, with the retrospective symptoms, model 1, with four significant predictors, explained 88.80% of the variance in childhood ADHD symptoms, and the introduction of other variables in models 2 and 3 also showed no significant increases in the variance explained.This study supports the utility of VR EF measures for predicting current and retrospective ADHD symptoms in adolescents and adults. Specifically, the number of correct responses in the test and the number of omission errors were associated with current and retrospective ADHD symptoms, while gender and the number of perseveration errors were only related to retrospective ADHD symptomatology. These findings suggest that the number of omissions and correct answers are the only variables able to predict both current and retrospective ADHD problems. The findings suggest that inattention problems tend to continue after adolescence, while perseveration errors (which have been related to impulsive behavior) and gender differences tend to diminish ,13,14,16The associations found between the VR measures and ADHD symptoms were higher than those found when traditional EF performance measures were used . In partIn summary, Nesplora Aquarium has been shown to provide significant predictors of current and retrospective ADHD symptoms in adults and adolescents, combining independence from respondent subjectivity and ecological validity. However, continued work on that explanation is crucial to determine how the symptoms can be either accentuated or masked in adulthood, their real implications in daily life, and the design of appropriate interventions. It is especially important if we consider the potential high prevalence of ADHD symptoms in adulthood .


Low birth weight  is an adverse perinatal risk that may reflect a poor intrauterine environment. While LBW has been a well-known predictor of physical, neurological, cognitive and psychological deficits later in life, minimal research has been done on small head circumference and low 5 minute Apgar scores, and their association with subsequent developmental abnormalities.The current study aims to demonstrate that small head circumference and low 5-minute Apgar scores are predictors for developmental abnormalities throughout childhood and later.Using a longitudinal design, 2,151 individuals' physical, neurological, and cognitive functioning in childhood, as well as psychological functioning in adulthood, was assessed as a function of three perinatal risk factors: LBW, small head circumference and low Apgar scores.Similar to findings with LBW, small head circumference or a low Apgar score were associated with increased number of hospital visits (p<0.0001 and p=0.005 respectively) and neurological abnormalities (p<0.0001 and p=0.001 respectively) at age 1. Intelligence quotient (IQ) scores at ages 4 and 7 were significantly lower for those born with small head circumference (p<0.0001) or low Apgar scores (p=0.002). Finally, the incidence of anxiety in adulthood was significantly higher for those born with small head circumference (p=0.03) or low Apgar scores (p=0.004) compared to their counterpart.Small head circumference and low a Apgar score are predictors of later physical, neurological, cognitive and psychological abnormalities, and can complement LBW, a more frequently used perinatal risk factor, and thus be used to screen for future developmental deficits, together with LBW. Low birth weight (LBW), defined as less than 2500 g, has been implicated as a predictor for subtle, but adverse, childhood neuro-cognitive functioning, including cognitive, neurological and psychological challenges -3. IndivWhile not extensive, prior researchers have investigated the effects of small head circumference and low Apgar scores on various functioning of the offspring. For example, small head circumference at birth has been associated with increased vulnerability to the immature brain  lower veThe current study will examine the prevalence of abnormality in neurological development, cognitive functioning and academic achievement, medical problems, and language, hearing and speaking in childhood by the three perinatal risk factors, i.e, LBW, small head circumference, and low Apgar scores, simultaneously. LBW would serve as a reference, i.e, the established surrogate measure, in order to have a base of prediction with which we could compare the head circumference and Apgar scores predictability. Considering multiple indicators of poor intrauterine environment may also help identify infants at greater risk for neurobehavioral development and psychological symptoms in adulthood.Data come from part of the Johns Hopkins Collaborative Perinatal Study. Pregnant women receiving prenatal care at the perinatal clinic delivered their babies at Johns Hopkins Hospital during 1960-1964. Their children were followed until they were 8 years of age. Twenty-five years later, those offspring were re-contacted (mean age 31). Hardy et al.  for a fund generation subjects (G2) eligible for follow-up between 1992 and 1994, 2,344 (79.8%) G2 had information on the three perinatal problems  and an initial neurological examination at 4 months old. Between those who are included in the analysis  and those excluded (n=543), there is no major demographic differences except for the sex of the participants, where a greater proportion of male than female offspring was excluded. The frequency of missing data on neurobehavioral and cognitive function measures ascertained between ages 0 to 8 for dependent variables vary, ranging from 0% to 2.9%. 7.6 to 9.1% of speech, language, and hearing function at age 3, and 10.5% of the health care utility between 4 and 8 years old, and are missing.Of the 2,694 2Of those 2,151 cohorts, 1,540 were located 25 years later. Approximately 91% of the located sample (n=1403) provided their general psychological status. The frequency of missing data for controlling variables was negligible (less than 0.2%).Birth weight, head circumference, and 5 minute Apgar scores were recorded by a nurse observer in the delivery room at the birth of the infant. Mean (sd) of these three perinatal indicators are 3,024 g (561 g), 33.6 cm (4.0 cm), and 8.8 (1.2) respectively. These risk factors were dichotomized at the cut-off points, to create indicator variables for LBW , small head circumference (≤ 32 cm), and low Apgar score (<7), which are conventionally used at clinical setting.The number of medical visits and the number of hospitalizations at ages 1, 4, and 8 year were reported based on caretakers' self-reports.Neurological abnormalities at ages 4 months, 1, and 7 years were evaluated through physical examination conducted by a pediatrician, with special training in neurology, or a pediatric neurologist. Neurological abnormalities included skull size and shape, spinal abnormalities, primary muscle disease, mental retardation, emotional and psychiatric disorders and detected squint. Non-neurological abnormalities included all conditions except neurological abnormities and minor acute upper respiratory infection. Based on observation of 116 items the child's neurological status was characterized as none , suspect, or definite. Finally, the impression of the child's non-neurological status was characterized as none, minor, or questionable/definite.Bayley Scales of Mental and Motor development  was usedSpeech, language, and hearing problems were assessed by a speech pathologist and audiologist, using The Three-Year Speech, Language, and Hearing Examination . The exaDevelopmental profiles were assessed by child psychologists in four areas: fine motor development, gross motor development, concept formation, and behavior. Each area was scored separately and the child's performance was indicated as “normal” or “suspect or abnormal.” Normal was coded as 0 and suspect or abnormal was coded as 1.Fine motor development was measured by Wallin pegboard, coping forms, stringing beads, and porteus maze. Gross motor development was measured by line walk, hopping, and ball catch. Each developmental task was scored pass or fail and established the presence or absence of motor defects first. Concept formation was measured by Graham-Ernhart block sort test, which provided a significant discrimination between brain damaged and non-brain damaged preschoolers. The test consists of sorting materials based on size, shape and color.Cognitive functioning was assessed using the Stanford-Binet IQ  and admiChild psychologists measured intelligence, visual motor coordination, and academic performance. Intelligence was measured using Wechsler Intelligence Scale for Children (WISC) at age 7 . StandarAdult psychiatric status was measured using the General Health Questionnaire-28 (GHQ-28) ,27. DeprSocio-demographic confounders include mother's race and poverty level at delivery and child's sex. Poverty level represents the ratio of the mother's annualized income to the poverty level based on the Social Security Bulletin Annual Statistical Supplement . GestatiFirst, to examine group differences by LBW, small head circumference, and low Apgar scores for rates of offspring problems at different times in infancy, childhood, and adulthood, univariate analyses were conducted using X2 tests for dichotomous outcomes and analysis of variance (ANOVA) for continuous outcomes. The univariate analyses were followed by multivariate analyses to adjust for potential confounders: logistic regression analysis was used for dichotomous outcomes and analysis of co-variance (ANCOVA) was used for continuous outcomes. Potential confounders were considered a priori and were included in the model as covariates for statistical adjustment. All reported p values are two-tailed.Among 2,151 offspring, 82.4% were Black, 17.94% were White, and 49.9% were female. Approximately a quarter of the mothers completed at least high school and approximately 30% had less than or equal to an 8th grade education. With regard to mothers' demographics, the mean (SD) number of progeny, age and individual income during the 1st trimester pregnancy were 3 (2.4), 24.9 (7.1) and $1,022 (515) respectively.Research pediatricians and pediatric neurologists who were blind to perinatal risk status, assessed development of the offspring in three categories  at ages 4 months, 1 year, and 7 years. LBW was associated with a 2-fold increased risk for definite non-neurological abnormality at 4 months and 1 year, but not with minor non-neurological abnormality, and the differences diminished by 7 years of age. There was a significant increased risk for questionable/definite non-neurological abnormality among offspring with small head circumference at 4 months. However, unlike with LBW, the risk for non-neurological abnormality diminished by 1 year of age. There was no notable increased risk of non-neurological abnormality (neither minor nor definite) with low Apgar scores.At the 4 year neurobehavioral development assessment, with an exception of the association between low Apgar scores and concept formation, all of the three perinatal risk factors were associated with a smaller, but significant, increased risk for fine motor , gross motor , intelligence , and concept formation abnormalities The current study evaluated the short- and long-term neuropsychological developmental consequences of LBW, small head circumference and low Apgar scores simultaneously, capitalizing on a longitudinal design. Our findings are generally consistent with previous literature and extend its scope by highlighting four main findings. First, the number of hospital admissions increased for all three perinatal risk factors-LBW, small head circumference, and low Apgar scores-during the first year of life (0-12 months) and the significant difference by LBW persisted until age 8. Second, each of the three perinatal factors was an independent predictor of neurological, non-neurological, cognitive and language difficulties later on in childhood (ages 4 months-8 years). Third, the difference on various indicators of neurobehavioral impairment between offspring with and without a perinatal factor appear to dissipate on its own over the course of the first few years, however, in adulthood there were modest but significant differences in psychological functioning. Lastly, with the exception of a brief period of time in preschool years, small head circumference and low 5 minute Apgar scores are significant independent predictors for suboptimal functioning, even after controlling for the effect of LBW.Our findings on the increased risk of hospital admissions in relation to birth weight status were consistent with previously demonstrated links between LBW and hospital admission ,33. An aThere is a large difference between groups for the number of hospitalizations from ages 0 to 12 months because the severity of the same illnesses is intensified for children with LBW. During early infancy, children born with LBW appeared to be more vulnerable, such as by the presence of compromised immune systems, than their normal BW counterparts thus contributing to a substantial difference between groups (p<0.0001). However, for ages 13 to 48 months, those born with LBW who have survived the first 2 years of life might have caught up with their deficits, including their immune functioning. By age 8 the possibly compromised immune systems of children born with LBW might have been highlighted once again when diseases from school exposure may be more likely.In our study, when the effects of potential confounders, such as sex, race and mother's poverty level during pregnancy, were controlled for, LBW was no longer associated with minor non-neurological abnormalities at 4 months or 1 years of age. Those findings, especially in non-neurological abnormalities among offspring born with LBW, can be explained by malformation in the central nervous system  and loweWe found that neurological development at 8 months and 4 years was significantly affected by all of the three perinatal risk factors we examined. Such distinctions between groups persisted in IQ at ages 4 and 7 as well as in achievement scores at age 8. Readers, however, need to be cautious when interpreting the greater magnitude of effects on development as measured by the Bayley Scales. 22 Recent meta-analysis shows that the Bayley Scales on development of very preterm and very LBW children are limited . AdditioFinally, it is important to be reminded that our findings provide evidence that small head circumference was a significant and even stronger predictor of psychological functioning, such as depression, social dysfunction, and somatic symptoms, and low Apgar scores were associated with an increased risk for anxiety. Although growing evidence show that LBW is associated with adult psychiatric disorders . LBW on Our study has some methodological strength. First, the second generation cohort  was prospectively and systematically followed from birth and studied longitudinally for over 30 years. Our sample came from predominately unprivileged backgrounds of low socioeconomic status with little to no education and a high proportion of ethnic minorities (82.1%). However, they were randomly selected regional  representatives who sought prenatal care at Johns Hopkins Hospital rather than a clinical sample with either serious psychopathology or medical illness. Nevertheless, we acknowledge that generalizability may be limited. Second, birth weight, Apgar scores, and head circumference were recorded by a research nurse at the time of delivery rather than based on mother's retrospective report. We also acknowledge potential limitations. First, we need to interpret our findings in light of the level of obstetric care in the1960's. Our sample was born in the pre-NICU era and the mortality rate for those born very early and with very LBW was higher than it is currently. Second, although we have adult psychological function data, the participants included in this analysis in adulthood is relatively low. While 75% of adult participants were identified and 67% of the original participants were followed, we were unable to include participants who could have survived had they been born in the modern times, who maybe more likely to have various and more serious impairment. Finally, the concept of special or remedial education, and a realization of the need for early intervention, has also advanced considerably since the late 1960s. It may be that with early intervention and special education programs provided at the preschool level, that some of these results may have ameliorated or disappeared. However, there are also many children who did not survive in the 1960s who would today. They may be more involved and thus more likely to demonstrate long term negative sequellae.Despite these limitations, the study contributes to the existing literature by providing evidence that 1) examining different perinatal risks, can improve our ability to predict different signs of neuro-cognitive impairments at different times in a life cycle, 2) that small head circumference which does not resolve by age 2 is a risk factor for additional neuro-cognitive impairments, and 3) that low 5 minutes' Apgar scores is a long term predictor of neurological, cognitive, and psychological development. There is ample evidence that early intervention education services can have positive long-term effects most shown for neurological abnormalities and less so for genetic ones ,42. Gene


Shannon = 0.036) and that the composition (beta diversity) differed significantly between patients and controls . In detail, the bacterial family Prevotellacae was associated with controls, while patients with ADHD showed elevated levels of Bacteroidaceae, and both Neisseriaceae and Neisseria spec. were found as possible biomarkers for juvenile ADHD. Our results point to a possible link of certain microbiota with ADHD, with Neisseria spec. being a very promising ADHD-associated candidate. This finding provides the basis for a systematic, longitudinal assessment of the role of the gut microbiome in ADHD, yielding promising potential for both prevention and therapeutic intervention.ADHD is a psychiatric disorder which is characterized by hyperactivity, impulsivity and attention problems. Due to recent findings of microbial involvement in other psychiatric disorders like autism and depression, a role of the gut microbiota in ADHD pathogenesis is assumed but has not yet been investigated. In this study, the gut microbiota of 14 male ADHD patients (mean age: 11.9 yrs.) and 17 male controls (mean age: 13.1 yrs.) was examined via next generation sequencing of 16S rDNA and analyzed for diversity and biomarkers. We found that the microbial diversity  was significantly decreased in ADHD patients compared to controls (p With a worldwide prevalence of 3–5% , 2, ADHDgut-brain axis. Recent studies underline the importance of the human gut microbiome in human health. Not only are gut microbes involved in digestion, metabolism and weight control [It is well known that gut microbiota and the central nervous system are interconnected in a bidirectional fashion, termed the  control –28, they control . There i control –34. As a control . Disturb control . Many of control , 38. RegSeveral psychiatric disorders like stress responsivity, anxiety-like behaviors, sociability, and cognition –41, as wFourteen male children and adolescents with ADHD  and 17 controls  participated in this study. Patients and controls did not differ in age (p = 0.138), BMI (p = 0.728), or IQ  , 49. IntAccording to parental ratings, patients displayed more attention problems, hyperactivity, and impulsivity than did controls  and carried out in accordance with the latest version of the Declaration of Helsinki.TM SPIN KIT FOR SOIL  as per the manufacturer protocol after incubation in 200 ml Tris Lysisbuffer and 25ml proteinase K for 2 hours at 56°C. Extracted DNA was stored at -80°C. For sequencing, DNA was amplified using the primer pair 27F-338R for the variable regions V1 and V2. Normalization of PCR products was done with the SequalPrep Normalization Plate Kit , and products were pooled equimolarly for sequencing on the Illumina MiSeq .Fecal DNA was collected in Sarstedt fecal collection tubes  and stored at 4°C until preparation. Total DNA from fecal samples was extracted using FastDNASequences with a read length less than 200bp and a quality score lower than 25 were rejected. Noise reduction was carried out using Mothur , 59. AmbAlpha diversity of the samples was measured by observed species, the Shannon diversity, and the Chao1 index. The observed species index measures the number of different species per sample which is defined as “richness”. The Chao1 index is also a qualitatively measure of alpha diversity which, beside species richness, takes into account the ratio of singletons (n = 1) to doubletons (n = 2) giving more weight to rare species. However, regarding diversity, not only the qualitative amount of species, but also the abundance of the species must be taken into account. The relative abundances of the different species making up the samples’ richness are defined as “evenness”. The Shannon-diversity index relates both, OTU richness and evenness. The association between microbial diversity and ADHD subtypes was tested via multiple linear regression, with microbial alpha diversity as the dependent variable and attention deficits, hyperactivity, and impulsivity as explanatory variables. For this purpose, we used the parental ratings as assessed by the FBB-HKS, since this questionnaire is designed to determine the severity of these three cardinal symptoms according to the DSM-VI . PairwisOrd < 0.01) with redundancy axes was identified using the envfit functions from the R package vegan [Multivariate statistics were conducted via ANOSIM, ADONIS, and the function “betadisper” from the R package vegan v2.4–1 to analyze microbial beta-diversity which describes the diversity in a microbial community between different samples. ANOSIM is rank-based and tests for similarities, whereas ADONIS tests the homogeneity of dispersion; betadisper tests the similarity of composition among groups. Non-metric multidimensional scaling (NMDS), the most robust, unconstrained and distance-based ordination method, was performed with Bray-Curtis dissimilarity as implemented in the R package vegan v2.4-1. Redundancy analysis is a constrained method based on multiple linear regressions to extract and summarize the variation in a set of response variables which can be explained by a set of explanatory variables. OTU count data were Hellinger-transformed as implemented in the R package vegan v2.4–1. The community composition data matrix that results from deep-sequencing diversity counting is usually characterized by a multitude of zero and single counts of OTUs. To generate data containing many zeros suitable for analysis by linear methods, such as redundancy analysis (RDA), transformation of data like the Hellinger transformation is recommended , 61. Helge vegan .To determine potential biomarker OTUs, which differ in abundance and occurrence between sample groups, full linear discriminant analysis (LDA) effect size (LEfSe)  analysisAll downstream computations were performed in R v3.2.2. P-values in multiple testing scenarios were corrected by false discovery rate.After a rigorous quality check and preprocessing, which removed about 20% of the sequences, all sequences were normalized to 7000 sequences per sample, resulting in a total of 187,807 OTUs with a median of 1970 OTUs per sample.Observed = 0.25) and Chao1 richness estimator (pChao1 = 0.17), while Shannon diversity was significantly decreased in ADHD compared to controls (pShannon = 0.036). Regarding the parents, mothers of ADHD patients also showed a reduction in alpha diversity , while fathers of ADHD patients and controls did not differ significantly . To get more precise information about the differences between the two sample groups, tests for similarity of composition (ADONIS) and homogeneity (betadisper) were performed. Both showed significant differences between ADHD children and controls . Inter-personal variation patterns of different phylogenetic levels can be found in ADONIS = 0.037) and control children (pADONIS = 0.005). A RDA plot comparing the family member can be found in As rank-based approaches, NMDS and ANOSIM were applied in order to test for dissimilarities in the microbial composition between ADHD patients and controls. NMDS results are displayed in Bacteroides , while controls share equal amounts with fathers and mothers . Abundances of possible biomarkers found by LEfSe analysis between patients and controls can be found for all family members in The LEfSe test for biomarkers was used in order to find significantly imbalanced OTUs, which showed the strongest effects for group differentiation. Analysis at the OTU level uncovered two ADHD-associated species belonging to the genera see also . EvaluatBacteroides spec.) correlated with levels of hyperactivity and impulsivity  with mircobial alpha diversity. Levels of hyperactivity were significantly correlated with a change in alpha diversity . There were no significant correlations between the microbiome and clinical symptoms assessed by the CBCL questionnaire . Beta diversity and correlated species were examined by RDA on Hellinger transformed data (tb-RDA). An RDA analysis at the species level revealed that OTU_7  as potential biomarkers for the ADHD group. Members of the genus Bacteroides are usually beneficial for the gut microbiota, but they are also capable of producing extraordinarly complex mixtures of amyloids, lipopolysaccharides, enterotoxins and neurotoxins, which can affect the blood brain barrier´s structure as well as the central nervous system [The LEfSe analysis results showed that the family Prevotellaceae was significantly more abundant in controls, while the ADHD patients showed elevated levels of Bacteroidaceae. Bacteroidetes generate essential vitamins and cofactors, and processing constituents such as fiber, making them beneficial in support of human immunity, physiology, biochemistry, and neurochemistry , 74. Thes system .Assuming a causal relationship, the reduced alpha diversity that we found in ADHD patients might reflect a bacterial community involved in deviant neural transmission. Many bacterial species are able to produce GABA , which iBy including parents in the analyses, we also observed that mothers of ADHD patients compared to mothers of healthy controls showed a reduced alpha diversity. Accordingly, ADHD self-ratings revealed that mothers of patients displayed more ADHD symptoms in the past and present than mothers of healthy controls did. There were no differences in alpha diversity and self-ratings between the fathers of patients and controls. With a heritability of about 76%, ADHD is a familial disorder, and its relative risk is about 5–9 in first-degree relatives , 13, 80.Patients and controls did not differ with respect to BMI and the reported intake of meat, fruits/vegetables, yoghurt, other milk products, or fast food during the month prior to stool donation. All these foods are well-known to influence the intestinal microbiota –85. TherA limitation of the study is the concomitant medication: Ten of 14 patients had taken methylphenidate (MPH), the first-line treatment of ADHD, for more than one year. Nine of them discontinued taking the medication at least 48h  prior to sample collection. MPH increases the availability of dopamine by blocking the dopamine transporter in the CNS –92, reduAnother limitation is the small sample size of 14 patients and 17 controls. Studies with larger cohorts are required not only to replicate our findings in a medication-controlled sample but also to investigate possible differences in alpha diversity between subtypes of ADHD. Moreover, including females is mandatory to investigate possible gender effects as indicated by the parental microbiome. In addition, future studies can be designed to develop effective dietary guidelines or treatment strategies with beneficial bacterial species (probiotics)  or speciNeisseria and elevated levels of Bacteroides spec. are associated with juvenile ADHD.Taking the small sample size and the concomitant medication into account, our findings support the hypothesis of an ADHD-specific microbiota. We suggest that the genus S1 FigObserved = 0.017) and Shannon diversity (pObserved = 0.029), while fathers show no significant difference.Alpha-diversity, measured by observed species (A) and Shannon diversity Index (B) is plotted for parents from ADHD patients (red) and parents from controls (green); IP, index patients; control, healthy controls. The line inside the box represents the median, while the whiskers represent the lowest and highest values within 1.5 interquartile range (IQR). Outliers as well as individual sample values are shown as dots. Statistical testing showed a difference in alpha diversity for mothers from ADHD patients and control mothers in observed species (p(JPEG)Click here for additional data file.S2 FigAlpha-diversity, measured by observed species (A) and Shannon diversity Index (B) is plotted for ADHD patients  and controls (green); IP, index patients; control, healthy controls. The line inside the box represents the median, while the whiskers represent the lowest and highest values within 1.5 interquartile range (IQR). Outliers as well as individual sample values are shown as dots. No significant differences were found.(JPEG)Click here for additional data file.S3 FigBar Plot showing the relative proportion of the bacterial phyla within all participants; P, patients; C, healthy controls.(JPEG)Click here for additional data file.S4 FigBar Plot showing the relative proportion of the top 20 bacterial families within all participants; P, patients; C, healthy controls.(JPEG)Click here for additional data file.S5 FigBar Plot showing the relative proportion of the top 20 bacterial genera within all participants; P, patients; C, healthy controls.(JPEG)Click here for additional data file.S6 FigBar Plot showing the relative proportion of the top 20 bacterial OTU within all participants; P, patients; C, healthy controls.(JPEG)Click here for additional data file.S7 FigBox plot showing the abundances of bacterial phyla stratified by group (ADHD vs. controls).(JPEG)Click here for additional data file.S8 FigBox plot showing the abundances of bacterial families stratified by group (ADHD vs. controls).(JPEG)Click here for additional data file.S9 FigBox plot showing the abundances of bacterial genera stratified by group (ADHD vs. controls).(JPEG)Click here for additional data file.S10 FigBox plot showing the abundances of bacterial OTU stratified by group (ADHD vs. controls).(JPEG)Click here for additional data file.S11 FigBeta diversity as Hellinger-transformed redundancy analysis of ADHD samples versus healthy controls. The axes show the first three constrained axes from redundancy analysis .(PNG)Click here for additional data file.S12 FigBoxplot showing the abundance of significant bacterial families found by LEfSe analysis for participants and their parent; IP, index patients; control, healthy controls.(JPEG)Click here for additional data file.S13 FigBoxplot showing the abundance of significant bacterial genera found by LEfSe analysis participants and their parents; IP, index patients; control, healthy controls.(JPEG)Click here for additional data file.S14 FigBoxplot showing the abundance of significant bacterial OTUs found by LEfSe analysis for participants and their parents; IP, index patients; control, healthy controls.(JPEG)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.