Attention deficit hyperactivity disorder (ADHD) is the most common neuropsychiatric disorder in children and is increasing in prevalence. There has also been a related increase in prescribing stimulant medication despite some controversy whether ADHD medication makes a lasting difference in school performance or achievement. Families who are apprehensive about side effects and with concerns for efficacy of medication pursue integrative medicine as an alternative or adjunct to pharmacologic and cognitive behavioral treatment approaches. Integrative medicine incorporates evidence-based medicine, both conventional and complementary and alternative therapies, to deliver personalized care to the patient, emphasizing diet, nutrients, gut health, and environmental influences as a means to decrease symptoms associated with chronic disorders. Pediatric integrative medicine practitioners are increasing in number throughout the United States because of improvement in patient health outcomes. However, limited funding and poor research design interfere with generalizable treatment approaches utilizing integrative medicine. The use of research designs originally intended for drugs and procedures are not suitable for many integrative medicine approaches. This article serves to highlight integrative medicine approaches in use today for children with ADHD, including dietary therapies, nutritional supplements, environmental hygiene, and neurofeedback. Attention deficit hyperactivity disorder (ADHD) is characterized as a psychiatric condition of heightened impulsivity, inattention, and hyperactivity . ADHD isIt is crucial that children with ADHD are treated appropriately as they can develop significant psychosocial, educational, and neuropsychological impairment . They arMedication for ADHD is associated with its own set of problems. Twenty to 35% of patients with ADHD do not respond to medication. In addition, medications have significant adverse effects with the most common side effects including delayed onset of sleep and decreased appetite both known to potentiate symptoms of ADHD. This results in discontinuation of medication as a result of side effects and the parents’ perception that the medication is ineffective; another factor in medication cessation is the cost burden for families ,16. ThusThe 2007 National Health Interview Survey discovered that approximately 12 percent of children in the United States have used or been given a CAM therapy . More thWhat children eat has a profound effect on their health. Although controversial, dietary therapies have been suggested to play a major role in ADHD and should be considered in the evaluation and management of children with ADHD ,21,22. NAlthough adherence is difficult and time consuming, RED has been shown to benefit children with ADHD. The Impact of Nutrition in Children with ADHD (INCA) was a double-blind crossover research study that showed children ages 4–8 years old improve on the abbreviated Conners’ scale by 11.6 after being placed on a tailored RED for five weeks . FurtherPIM physicians begin with a whole foods, non-processed diet for children with ADHD . ChildreGluten-free diets have been implemented for children with neuropsychiatric disorders, including autism and ADHD . Gluten-et al. did not show any association between CD and ADHD [In the 1950s and 1960s, physicians first began reporting neurologic and psychotic symptoms in patients with CD . There hand ADHD ,50,51. Hand ADHD ,53.The mechanism of action behind gluten’s effect on neuropsychiatric symptoms is still inconclusive, but gastrointestinal immune dysregulation can lead to chronic pro-inflammatory immune dysregulation. This chronic inflammation may be one of the underlying links in the development of ADHD ,30. It iDespite conflicting evidence of CD and its association with ADHD, clinicians should focus on a broad array of symptoms when evaluating children with ADHD, including both gastrointestinal and neuropsychiatric symptoms to rule out CD or NCGS as an exacerbating condition. Screening for and diagnosing CD and NCGS remains a challenge. A research trial assessed the usefulness of screening for CD when children presented with common CD symptoms  . The stuIf the decision is made to choose a gluten-free diet, this should be implemented by a team of healthcare professionals, including a trained dietitian, as it is easy to replace wheat/gluten-containing products with gluten-free products high in refined carbohydrates, but low in fiber and nutrients. And if a gluten-free diet is not a good fit, a whole foods diet rich in fresh unprocessed foods is ideal. Most patients with ADHD would benefit from a whole foods diet.The value of nutrition is increasingly becoming recognized as a means for achieving optimal health, as poor diet and nutritional intake are implicated in chronic disorders . Most stversus placebo in children with ADHD. Bilici et al. demonstrated that zinc decreased motor activity and improved impulsivity, but did not increase attention [et al. showed improvement in total ADHD scores with the addition of elemental zinc 13 mg/day to methylphenidate 1 mg/kg/day versus placebo and methylphenidate [Zinc is known to a play a role in neuropsychiatric disorders and is a micronutrient recognized as participating in metabolism relevant to neurotransmitters, hormones, nutrients, and immune function ,65. It attention . Akhondzhenidate . In a sthenidate . There iIron status is commonly evaluated in children with neuropsychiatric disorders likely due to its role as a cofactor in monoaminergic neurotransmitter metabolism . ResearcInsufficient Vitamin B6, also known as pyridoxine or pyridoxal-5-phosphate, affects metabolism of polyunsaturated fatty acids, hemoglobin synthesis, and neurotransmission . It was Magnesium is the fourth most abundant mineral and is involved as a cofactor in over 300 enzymatic reactions in the body, including fatty acid, glucose and energy metabolism . Some ofPolyunsaturated fatty acid (PUFAs) disturbance has also been implicated in ADHD, as they are necessary for nerve cell membrane fluidity and neuronal function to support neurotransmission. However, the typical American diet contains an imbalance of omega-6 to omega-3 PUFAs. An elevated Omega-6 to Omega-3 ratio is implicated in inflammation ,89. OmegOmega-3 fatty acids, found in flaxseeds, chia seeds, and fatty fish, metabolize downstream to anti-inflammatory prostaglandins and leukotrienes ,91. WithPractitioners, including dietitians, can help guide families of children with ADHD to increase balanced portions of healthy fats and decrease the inflammatory fats. As research has identified elevated inflammatory markers in children and adolescents with ADHD, maintaining a healthy omega-6 to omega-3 ratio with a diet containing healthy balances of PUFAs, saturated fats, and phospholipids may be a promising integrative medicine approach to treatment of ADHD .However, some reports using placebo-controlled randomized controlled trials (RCTs) evaluating PUFAs efficacy in treating ADHD differ in conclusions ,97,98,99In clinical practice, evaluation of fatty acids using a comprehensive serum fatty acid panel or RBC fatty acid panel to guide supplementation may be an effective strategy for optimizing treatment outcomes. Fatty acid synthesis and metabolism also involves several other micronutrients as cofactors for the desaturase and elongase enzymes and these may be evaluated by laboratory testing . Individl-carnitine is an abundant short chain ester of carnitine, which has an effect on brain metabolism and neurotransmission, specifically the cholinergic and dopaminergic pathways [Carnitine is a fatty acid transporter that transfers long chain fatty acids across mitochondrial membrane for beta-oxidation. Acetyl pathways ,106. Thrpathways ,108,109.et al. surveyed parents to compare symptoms of their child with ADHD when they were engaging in leisure activities in a windowless room versus in an outdoor setting where natural production of vitamin D will occur. The survey results demonstrated that the outdoor setting was more likely to be chosen by parents to reduce inattentive symptoms [Vitamin D is now implicated in ADHD . Vitaminsymptoms . SunlighFor women of childbearing age, iodine insufficiency is approximately 15% in the United States. Pregnant women in the United States are at slight risk of iodine insufficiency especially in the first trimester [114. Iodine deficiency can be measured by a low urine iodine of less than 100 microg/L  In womenThe recommended daily allowance of up to 150 micrograms/day is meant to prevent goiter or thyroid disease . It shouHumans have 100 trillion microbes residing in the gastrointestinal tract, some of which may be commensal, while others are pathologic . ConsumpAs part of the practice at University of Kansas Pediatric Integrative Medicine, evaluation of children with neurodevelopment and mood disorders includes stool analysis for intestinal dysbiosis. Medications, such as antibiotics, can disrupt the gut microbiome by reducing diversity and allowing overgrowth of some pathogenic microorganisms . Gut micvs. conventional diets in children, attesting that organic diets have a protective effect against exposure to organophosphate pesticides [Industry produces over 83,000 chemicals greater than one million pounds per year with little information about the potential effects on neurodevelopment ,127. Appsticides . Recommesticides ,135.In addition to obtaining a thorough diet history in patients with ADHD, environmental exposure history needs to be evaluated and addressed. Some families may have a history of using herbicides, insecticides, chemicals including cleaning supplies, and volatile organic compounds, such as air fresheners in their homes. Education is of utmost importance to keep chemical exposure at a minimum. In fact, healthy housing initiatives are gaining popularity among medical communities to help control and prevent chronic disease in children. Advising families to opt for more natural solutions, such as “green” cleaning products that do not contain toxic industrial chemicals and using sticky traps for pests may be healthier alternatives. Limiting plastic products, such as plastic water bottles and microwaveable plastic containers, may also protect against ADHD, as there are positive associations between ADHD and bisphenol A (BPA) and phthalates commonly found in plastics .Of the heavy metals, lead toxicity has been associated with ADHD and other neuropsychiatric disorders. It is well known that lead can cause cognitive impairment and attention dysregulation ,138. In i.e., brain map), utilized as initial baseline testing of brainwaves, should also be considered in future research studies as an evaluation tool for identifying responders and non-responders to ADHD medication [One of the most promising treatments in ADHD is neurofeedback, which is an EEG-based form of biofeedback. Neurofeedback is a safe, non-invasive therapy that restores the brain’s ability to function through altering brain wave patterns by operant conditioning . The inddication . Other idication .In 2004, Slow Cortical Potential (SCP) neurofeedback was found to improve ADHD symptoms . SCP neuA recent RCT research study on 23 children with ADHD carried out 40 sessions of theta/beta training sessions or methylphenidate and compared the two therapies with a six-month follow-up. Although this study had a limitation of small sample size, and 8 out of 12 participants in the neurofeedback group started medication, neurofeedback training was comparable to methylphenidate in reducing ADHD primary symptoms and associated functional impairment. Academic performance significantly improved in the neurofeedback group compared to the group receiving methylphenidate .Despite promising results among research studies and anecdotal evidence, neurofeedback treatment is not yet accepted as a standard therapy for children with ADHD. This is possibly due to limitations of scientific research in neurofeedback accompanied by the difficulty in conducting a well-designed randomized controlled trial. Sham neurofeedback groups are designed as control groups in neurofeedback research. However, attention by neurofeedback technicians may result in positive gains in the control group. Subjective treatment effects can threaten the validity of neurofeedback research and these include participants, parents, and teachers’ expectations of beneficial outcomes, provider training and experience, environmental stressors surrounding the participant, and participants’ engagement during training. In a research update by Loo and Makeig, although the evidence is growing for scientific quality of research in neurofeedback, more research is needed before neurofeedback can be recommended as a first-line, stand-alone treatment modality .In addition, neurofeedback participants must be encouraged to strive toward mastering engagement and regulation . ParticiSome of the research related to integrative therapies for children with ADHD has shown mixed results. Of the integrative medicine approaches, the best evidence is for restricted elimination diets, neurofeedback, omega-3 fatty acid supplements, vitamin B6, and zinc. More rigorous and applicable research trials need to be funded in order to identify effective integrative therapies as standard practice. Some researchers suspect the “placebo effect” is responsible for the improvement seen with some integrative therapies. On the other hand, the “placebo effect” can account for perceived improvement from medications and other conventional therapies.Unfortunately, research is limited in integrative medicine approaches to ADHD. This may be due to funding of and difficulty in designing randomized controlled trials (RCTs), considered the gold standard of evaluating treatment interventions. Evidence-based medicine was initially designed for drugs and procedures, but has been applied to nutrients and other integrative therapies. The high internal validity of RCTs can impede its external validity, meaning that it may not be generalizable to real-life situations, and applicability to the individual may be questionable. Hence, RCTs may not be the best tool to evaluate integrative therapies compared to other types of study designs, such as a pragmatic study design. For example, a single neurofeedback protocol for the general population may not be appropriate for the individual due to differences in brainwaves, genetics, diet, and environmental conditions. Pragmatic trials may be more efficacious in assessing integrative therapeutic approaches in real-life settings, compared to the controlled environments that do not take place outside of the research trial . EvidencWith adverse effects of and poor outcomes in long-term safety studies evaluating ADHD medications, more families are turning to integrative modalities to help decrease symptoms and improve quality of life in their children with ADHD. Integrative medicine may provide an alternative approach to treating ADHD, especially for those classified as non-responders. Because a significant percentage of the population with ADHD is classified as non-responsive, integrative medicine approaches to ADHD have gained popularity among families and practitioners. Because research is limited and outcomes show conflicting results, it is difficult for conventional practitioners to advise ADHD patients and their families when questions about integrative therapies arise. Partnering with an experienced pediatrician of integrative medicine may provide an outlet for treatment options and a trusted source for unanswered questions. Since the numbers of pediatricians of integrative medicine are growing, there is an expanding workforce that can provide guidance to conventional practitioners and families and help with design of future clinical trials in ADHD. Healthcare providers need to be aware of integrative modalities in use by families of children with ADHD in order to keep open communication when providing advice on possible benefits and/or limitations based on current research. This will help foster improved communication between families and patients with ADHD and their practitioners.


These frequency bands also showed statistically significant differences in long-distance interactions, mainly among anterior and central regions, as well as among anterior, central, and other areas. These differences might reflect alterations during brain development in children with ADHD. Our results support the role of frontal abnormalities in ADHD pathophysiology, which may reflect a delay in cortical maturation in the frontal cortex.The aim of the present study was to explore the interchannel relationships of resting-state brain activity in patients with attention-deficit/hyperactivity disorder (ADHD), one of the most common mental disorders that develop in children. Magnetoencephalographic (MEG) signals were recorded using a 148-channel whole-head magnetometer in 13 patients with ADHD (range: 8–12 years) and 14 control subjects (range: 8–13 years). Three complementary measures  were calculated in the conventional MEG frequency bands: delta, theta, alpha, beta, and gamma. Our results showed that the interactions among MEG channels are higher for ADHD patients than for control subjects in all frequency bands. Statistically significant differences were observed for short-distance values within right-anterior and central regions, especially at delta, beta, and gamma-frequency bands ( Attention-deficit/hyperactivity disorder (ADHD) is a persistent neuropsychiatric disorder, which changes with development from childhood through adulthood . It is cIn the recent years, neuroimaging research has evolved rapidly, providing several ways to examine the pathophysiology of ADHD and the biological effects of medical treatments. The earliest neuroimaging studies used single-photon emission computed tomography (SPECT) and positron-emission tomography (PET), showing frontostriatal abnormalities in ADHD , 6. LateTo understand dynamic cognitive processes, neurophysiological techniques, such as electroencephalography (EEG) and magnetoencephalography (MEG), are required. Both allow acquiring neural activity with higher temporal resolution than SPECT, PET, and fMRI . EEG andAll the aforementioned research works analyzed EEG/MEG activity by studying local activation patterns . However, several authors considered that higher brain functions depend on a balance between local specialization and global integration of brain processes . TherefoMost of the previous coupling studies used a single measure, mainly coherence, to analyze brain dynamics in ADHD. In this study, three complementary measures have been applied in order to get a comprehensive characterization of the interchannel relationships in ADHD. To begin with, coherence (COH) is a normalized linear measure widely used to explore connectivity at a particular frequency. In essence, it quantifies linear correlations in the frequency domain . Secondlδ, θ, α, β, and γ). We attempt to address the following research questions: (i) How does ADHD affect the neural interaction patterns? (ii) Can our methodology reflect the regional abnormalities of ADHD? (iii) Can the proposed measures provide complementary information about neural dynamics?The aim of our study is to provide further insights into the underlying brain dynamics associated with ADHD. For this purpose, COH, PLV, and ED were calculated in the conventional MEG frequency bands  located in a magnetically shielded room at the MEG Center Dr. Pérez-Modrego (Spain). During MEG recording, subjects were lying comfortably on a patient bed, in a relaxed state, and with their eyes closed. They were instructed to stay awake and to avoid eye and head movements in order to reduce the presence of artifacts in the recordings. Their behavior was controlled during the recording procedure by means of a video-camera. Additionally, technicians may communicate with children during MEG acquisition using a loud-speaking intercom. Participants included in the final sample did not exhibit significant difficulties in maintaining their immobility.For each subject, five minutes of MEG data was acquired at a sampling frequency of 678.17 Hz. A process of downsampling by a factor of four was carried out, resulting in a sampling rate of 169.55 Hz. Data were then digitally filtered using a 1–65 Hz band-pass filter and a 50 Hz notch filter. To minimize the presence of oculographic, cardiographic, and myographic artifacts, both visual inspection and independent component analysis (ICA) were performed . FinallyIn this study, MEG data were acquired from 27 subjects. The clinical group comprised 13 children with ADHD . Inclusion criteria included a full DSM-IV  diagnosis of ADHD combined type with associated impairment in at least two settings and Conners' Parent Rating Scale (CPRS) hyperactivity rating greater than two SD above age- and sex-specific means . The DSMp values > 0.05, Mann-Whitney U test) between ADHD patients and control subjects in terms of age and years of education  and they all were strictly right-handed. Written informed consent and assent to participate in the study were obtained from parents and children, respectively. The Institutional Review Board approved the research protocol.The control group was formed by 14 healthy children  without past or present neurological disorders. There were not significant differences  and y(t), COH can be calculated as follows [Sxy(f) is the cross-power spectral density and Sxx(f) and Syy(f) are the respective autopower spectral densities [In particular, mean square coherence has been calculated. It is a real-valued function defined as the square cross-spectrum normalized by the product of the two power spectra. Having two MEG channels,  follows :(1)COHxyensities . COH valx(t) and y(t), while their amplitudes could be uncorrelated [PLV is a highly sensitive measure of neural synchronization that specifically identifies whether frequency transient phase locking exists. It quantifies the relationship between the phases of rrelated .φxband(t) and φyband(t), from the two signals, x(t) and y(t), respectively. Thus, the instantaneous phase difference for epoch n was obtained as follows [To calculate PLV, firstly it is necessary to constrain the spectrum by filtering on the frequency band of interest. Then, Hilbert transform was used to extract the instantaneous phase,  follows :(2)ΔφxybN is the number of samples per epoch and |·| indicates the modulus of the complex value. The range of PLV values varies between 0  and 1 (perfect phase synchrony).Finally, PLV was calculated as the variability of the instantaneous phase difference:The concept of distance between two probability distributions was initially developed by Mahalanobis in 1936 . It is wPx(f) = (1/N)Sxx(f) and Py(f) = (1/N)Syy(f) are the normalized power spectra for a given frequency band from the signals x(t) and y(t), respectively. ED ranges from 0 to 1, corresponding to the highest and lowest similarity, respectively. Therefore, to have a direct relation with COH and PLV, In this study, ED was used to evaluate the differences between the spectral contents in the normalized power spectra of two MEG sensors. ED is defined as follows :(4)EDxybδ (1–4 Hz), θ (4–8 Hz), α (8–13 Hz), β (13–30 Hz), and γ (30–65 Hz). Results for the artifact-free epochs were averaged. The result of computing the three measures  for each method, where each entry Mij contains the interaction value between the channels i and j. To facilitate the interpretation of the results, these values were grouped into (left and right) anterior, central, lateral, and posterior regions to obtain long-distance intra- and interhemispheric (within one hemisphere and homologue regions of two hemispheres) and short-distance local measures [In this study, the three methods  method. Statistically significant results have been considered when p values were lower than 0.05. Detailed results for each measure are shown in p values (Mann-Whitney U test) at each frequency band are displayed in (c). It is important to note that connections across regions in (c) were only displayed when statistically significant differences between groups were obtained.Statistical analyses were performed to study the differences in the interchannel relationships between ADHD and control groups using Mann-Whitney δ and β), left-central , right-central , and left-posterior (γ) areas. This effect was more pronounced in central region . Moreover, interhemispheric long-distance values revealed a significant interaction increase between anterior , central , and lateral areas (δ). Finally, intrahemispheric long-distance values also showed a significant increase between anterior and central regions  and between central and lateral areas . Additionally, significant differences were also found between central and posterior regions (δ) and between left-anterior and left-posterior areas (δ and γ).To begin with, short-distance COH values showed statistically significant increases in ADHD patients compared to controls in the right-anterior (δ) and right-central (δ and γ) regions. Furthermore, interhemispheric long-distance values also showed statistically significant differences between central regions (δ and γ). Finally, intrahemispheric values revealed significant differences between anterior and central regions (δ and γ), central and lateral areas (δ), and central and posterior regions (δ).PLV results supported the patterns discussed previously. Short-distance values were higher in ADHD children compared to controls, and these differences were statistically significant within left-central , left-central , right-central , right-lateral (β), and right-posterior (β and γ) regions. Long-distance values also showed significant differences in the same frequency bands and regions. Interhemispheric long-distance values were statistically significant between anterior , central , lateral (β), and posterior regions (β). Finally, δ, β, and γ showed several significant differences in intrahemispheric neural relationships. Significant differences were found between anterior and central regions , central and posterior regions , and right-anterior and right-posterior regions (β and γ). Additionally, it is noteworthy that γ revealed statistically significant differences among all regions with the exception of the connection between left-lateral and left-posterior areas.In the same way as COH and PLV values, short-distance δ band for short-distance values at the right-central region: 88.89% . In the case of PLV, an accuracy of 81.48%  was obtained for central interhemispheric values, also in δ band. Lastly, the highest accuracy value for Finally, receiver operating characteristic (ROC) curves, with a leave-one-out cross-validation procedure, were used to assess the ability of COH, PLV, and δ, α, β, and γ bands. The main differences were found in δ, β, and γ, especially in central and anterior regions. Furthermore, these findings indicate an increase of the neural interactions in ADHD patients in comparison to control subjects. Consequently, this study provides evidence for brain hyperconnectivity in ADHD, which has been associated with higher levels of fluctuations in brain signals [The first research question pointed out in Introduction addressed the characterization of ADHD neural interaction patterns. Previous researches revealed that neural oscillations are an essential instrument for enabling coordinated synchronous activity during normal brain functioning . They al signals .δ and β [β (14–30 Hz) and γ (30–56 Hz) than controls. These coupling alterations may arise from an aberrant balance of excitation and inhibition in local neural circuits of ADHD children [Our findings are consistent with different previous researches. For instance, Montagu  showed aδ and β . Additioδ and β  applied children .γ. In sum, our findings agree with previous studies about children with ADHD, which showed an increase in neural coupling in comparison to controls. Notwithstanding, our results revealed alterations also in high frequency bands.It should be noted that most of the studies that examine functional connectivity in children with ADHD focused on low frequencies. However, several experiments have suggested that gamma-frequency activity also plays an important role in attention and memory . Indeed,β and γ), especially in γ. Furthermore, our results showed that frontal and central regions are the most affected in ADHD patients in comparison to controls.The second research question posed the issue about whether there is a relationship between our results and ADHD regional abnormalities. Our results revealed several coupling differences, mainly in anterior and central regions. These changes differ depending on the interaction measure, the frequency band, and the region considered. Regarding short-distance values, COH and PLV values showed higher connectivity in ADHD compared to controls in central and anterior regions at low frequencies, while Over the past two decades, structural and functional imaging studies have associated ADHD with abnormalities in frontal brain regions . A receWe raised the third research question about whether the proposed measures provide complementary analyses. Several studies suggested that the interactions between pairs of sensors can be useful to understand the underlying neural mechanisms , 48. HowIt should be noted that COH, PLV, and proaches , 43, 44.proaches . Besidesδ frequency band. On the other hand, β and γ bands, providing further significant differences. Hence, our results also suggest that the three measures are complementary.To sum up, different interaction patterns were found in our study depending on the frequency band, the region of cortex, and the measure considered. As shown in Figures Several concerns of this research merit consideration. Firstly, the size of the sample is small to be useful as a diagnostic tool. Therefore, this study should be extended on a larger patient population. Secondly, the current study was carried out during a resting-state condition. This condition has the limitation of being subjective, since it relies on the particular introspective capabilities of each subject . The couδ, β, and γ frequency bands. Moreover, COH provided a maximum accuracy of 88.89% to discriminate between ADHD patients and controls. To the best of our knowledge, this is the first study to analyze the interchannel relationships in ADHD with three complementary measures, providing original and global insights of neural interactions in this disorder. Our results agree with previous researches that have associated ADHD with abnormalities in frontocentral brain region. Furthermore, these findings support the hypothesis that brain development in children with ADHD diverges from that of controls. Changes seem to be associated with a delay in cortical maturation in the frontal region of ADHD patients.This study investigated COH, PLV, and ED of MEG signals in children with ADHD. Our results suggest that ADHD is associated with a neuronal hyperconnectivity in central and right anterior regions for


The framework of graph theory provides useful tools for investigating the neural substrates of neuropsychiatric disorders. Graph description measures may be useful as predictor variables in classification procedures. Here, we consider several centrality measures as predictor features in a classification algorithm to identify nodes of resting-state networks containing predictive information that can discriminate between typical developing children and patients with attention-deficit/hyperactivity disorder (ADHD). The prediction was based on a support vector machines classifier. The analyses were performed in a multisite and publicly available resting-state fMRI dataset of healthy children and ADHD patients: the ADHD-200 database. Network centrality measures contained little predictive information for the discrimination between ADHD patients and healthy subjects. However, the classification between inattentive and combined ADHD subtypes was more promising, achieving accuracies higher than 65%  in some sites. Finally, brain regions were ranked according to the amount of discriminant information and the most relevant were mapped. As hypothesized, we found that brain regions in motor, frontoparietal, and default mode networks contained the most predictive information. We concluded that the functional connectivity estimations are strongly dependent on the sample characteristics. Thus different acquisition protocols and clinical heterogeneity decrease the predictive values of the graph descriptors. Attention-deficit/hyperactive disorder (ADHD) is a neurodevelopmental disorder with a prevalence of around 5.3% in children and adolescents . It is cGraph theoretical analysis is an emerging component in the field of connectomics and brain network analysis based on neuroimaging data , 7. DescPattern recognition methods based on machine learning techniques have shown to be a promising approach to the analysis of neuroimaging data . SupportGraph theory descriptors can be used as predictor variables  in a machine-learning framework. Merging graph theoretical approaches and machine learning techniques might provide a better-adjusted way to scrutinize the impairment of RSNs in ADHD as well as mapping predictions to a single individual case. In this study, we investigated the use of network centrality measures as predictive features to discriminate between typical developing children and ADHD patients with both inattentive and combined presentations. In addition, we investigated possible differences between inattentive and combined ADHD groups. The ADHD-200 dataset  formed tThe publicly available resting-state fMRI data from the ADHD-200 Consortium were used in the present study. The images were acquired at five different sites: Peking University, Kennedy Krieger Institute, NeuroIMAGE sample, New York University Child Study Center, and Oregon Health & Science University (OHSU). The subject sample consisted of 609 subjects, 340 controls , and 269 patients with ADHD according to DSM-IV-TR criteria . Among the total number of ADHD patients, 159 fulfilled the criteria for the inattentive type , while 110 were classified as the combined type .All research protocols from institutes contributing to the ADHD-200 Consortium received local approval by their respective IRB. All the data distributed via the International Neuroimaging Data-sharing Initiative (INDI) are fully anonymized in accordance with HIPAA Privacy Rules. Further details concerning the sample and scanning parameters can be obtained by request to the ADHD-200 Consortium.http://neurobureau.projects.nitrc.org. Step-wise data preprocessing was previously conducted by the NeuroBureau community using the Athena pipeline and consisted in the systematic and homogeneous processing of all resting-state fMRI data. The following steps were carried out: exclusion of the first four EPI volumes; slice time correction; deobliquity of the dataset; head motion correction using the first volume as a reference; exclusion of voxels at non-brain regions by masking the volumes; averaging the EPI volumes to obtain a mean functional image; coregistration of this mean functional image to the subjects' correspondent anatomical image; spatial transformation of functional data into template space; extraction of BOLD time series from white matter and cerebrospinal fluid using masks obtained from segmenting the structural data; removing trend and motion effects through linear multiple regression; temporal band-pass filtering; spatial smoothing using a Gaussian filter.All preprocessed images are available at the website http://www.nitrc.org/plugins/mwiki/index.php/neurobureau:AthenaPipeline. The Pearson correlation coefficient between each pair of ROIs was calculated and regarded as a proxy of functional connectivity. The correlation matrix was equated with the adjacency matrix of an undirected and weighted graph. Meanwhile, binary adjacency matrices were built for each subject by applying three different cut-off values  to the correlation matrix. The cut-offs were defined within this particular range since the network becomes too fragmented and granular to allow a proper graph analysis for higher cut-off values [A representative set of 400 brain-wide regions of interest (ROIs) was chosen for defining the network nodes used for connectivity analysis and the construction of the graphs. The ROIs were determined by using the method developed by Craddock et al.   based onf values . We evalf values , betweenf values , eigenvef values . The degN is the set of all nodes and edges within a network and n is the number of nodes. An edge between two nodes i and j is represented by ai,j. In the undirected graph case, aij = 1 if there is a connection between the nodes i and j; otherwise, aij = 0. In betweenness definition, ρhj is the number of shortest paths between h and j, and ρhj(i) is the number of shortest paths between h and j passing through i. In eigenvector definition, l is a constant. Note that eigenvector and Burt's constraint are definable only for weighted graphs.The mathematical definitions of these measures are described in  R platform for Computational Statistics  (http://www.r-project.org/) using the R igraph package.Degree is a straight and intuitive way to quantify nodes centrality, and it is defined as the number of edges connected to a particular node. The closeness centrality is the average distance between a given node and all other nodes of the network. Betweenness quantifies the influence of a node and is defined as the number of shortest paths passing through it. The basic rationale underlying eigenvector centrality is that connections with more central nodes increase the nodes influence in the network. Hence, different weights are attributed to a vertex depending on the centrality of the connected nodes. Finally, Burt's constraint value is inversely proportional to the number of connections of a node and increases with the number of strong mutual connections . The useThe centrality measures of each graph's nodes were used as features  in an independent classification analysis. Classification was performed using a linear support vector machine (SVM) algorithm . The ratFinally, in order to identify the most discriminative regions, we built brain maps highlighting the 5% brain regions with greater predictive values. We used the approach proposed by Mourão-Miranda et al.  and SatoInterestingly, the mean score (across sites) and the score from whole-sample classification were very similar, except when using betweenness and degree in unweighted graphs . In thisRegarding the identification of the brain regions with greater contribution to prediction, we chose only the classifications with accuracy above 70%. At present, resting-state fMRI is a well-established tool for the assessment of spontaneous brain activity. Graph theoretical measures provide a suitable framework for the investigation of the structures of complex neural networks. In addition, the application of machine-learning algorithms has been of great impact on developing more advanced neuroimaging studies of psychiatric disorders . In the When the whole sample was used, none of the centrality measures had a relevant predictive power beyond chance. However, significant prediction values were observed at the OHSU site. Thus both within- and between-site variability have a negative impact on the extraction of predictive information and consequently on classification. In the OHSU sample, betweenness centrality measures contained predictive information for the classification of ADHD and control subjects with a score of 73%. After an extensive analysis of sample characteristics and acquisition parameters, we hypothesize that the classification score at OHSU was higher than the other scores for two main reasons: (i) the sample was approximately balanced between typically developing controls (42 subjects) and ADHD patients (35 subjects), while the group sizes were very different at the other sites; (ii) OHSU EPI acquisition has the largest voxel size (3.8 mm) and the 3T system was equipped with a 12 channels head coil (as opposed to 8) which increases the signal-to-noise ratio.When the 5% nodes with greater predictive values were mapped, a sparse pattern of brain regions was observed. In fact, widespread brain alterations in ADHD are supported by findings of impaired interregional connectivity between the nodes of large-scale functional networks (reviewed in ), and boA promising finding was observed for the degree centrality in the whole sample analysis on the classification of the disorder types. In the within-site analyses, relatively high scores were observed for degree, betweenness, and eigenvector centralities. However, as the sample size is smaller in these cases, variability is increased. Moreover, the mean scores of within-site analyses were almost identical to the ones from the whole sample analysis. Brain regions mapped for betweenness measures included nodes of the right frontoparietal network. This network has been implicated in attentional and executive processes and is thought to be impaired in ADHD. Cubillo et al.  have shoThe measure of degree centrality, when applied to the separation between ADHD types, produced the highest classification scores in areas of the sensory-motor network and of the DMN, mainly in parietal cortex and the precuneus. These findings are in agreement with our hypothesis, based on consistent results in the literature . In factIn conclusion, a novel approach of applying graph theoretical measures was shown to be useful for testing our hypothesis regarding resting-state network impairment in ADHD disorder. In particular, distinct patterns of network dysfunction were evident for both inattentive and combined ADHD subtypes. The classification scores for discriminating between ADHD and healthy subjects were close to chance. Clearly, within-site analysis improves prediction levels when compared to whole sample analysis, suggesting that heterogeneity across the sites may strongly limit the application of the method as a potential clinical support. The functional connectivity estimation is strongly dependent on the samples' characteristics. Thus, in order to advance the pathophysiological knowledge of ADHD, we emphasize the importance of further multicentric studies with more homogeneous acquisitions.


According to the results, elevated levels of serum MMP-9 in boys with HKD may be associated with clinical impulsivity domain .The serum levels of matrix metalloproteinase-9 (MMP-9) in neuropsychiatric disorders of adults have been widely investigated. So far, no studies have been conducted on the relationship of MMP-9 and cognitive domains in children with two phenotype models, attention deficit/hyperactivity disorder and hyperkinetic disorder (ADHD/HKD). The aim of this research was to evaluate and test the hypothesis that serum MMP-9 levels are associated with the severity of symptoms in children with ADHD/HKD and to compare the results in two models of this disorder. The study group comprised 37 Caucasian boys aged 7–12 years with HKD, being a subset of the combined ADHD subtype. Intellectual functions were measured using Wechsler Intelligence Scale for Children-Revised. The analysis of serum concentrations of MMP-9 was based on a quantitative sandwich ELISA. The statistical regression analysis revealed a correlation between increased serum MMP-9 levels and severity of symptoms in the ADHD ( Matrix metalloproteinases (MMPs) (zinc-dependent proteinases) play important roles in extracellular matrix (ECM) remodelling in physiological and pathological processes –6. The EMatrix metalloproteinase-9 (MMP-9), a typical ECM protease (gelatinase), is characterized by a wide range of substrates in vitro , 6. ReceCognitive deficits and behavioural symptoms in ADHD have been studied intensely and are well documented –34. TheyNeuropsychological studies concerning ADHD show a variety of cognitive deficits, altered level of motivational processes and irregularities in the stages of processing and storage of information in the brain , 38–40. Despite of intense research in recent decades, the brain processes and mechanisms which underlie the cognitive deficits of ADHD are unclear. It can be assumed, that the reorganization of the brain responsible for behavioural and cognitive symptoms of ADHD/HKD, and changes in the activity of MMP-9 may be the result of ECM reconfiguration in this disorder. To date, serum MMP-9 levels in children with ADHD/HKD have not been studied. Therefore, the aim of this study was to evaluate associations between serum MMP-9 levels and the symptoms severity in children with ADHD/HKD. We hypothesized that the activity of MMP-9 may be related to intensity of symptoms and the degree of cognitive dysfunction in children with ADHD/HKD.The study group comprised 37 Caucasian boys with Combined ADHD subtype and HKD, aged between 7 and 12 years . The study group was selected among patients with hyperactivity symptoms from primary care, who had been referred to the specialized psychiatric diagnosis and therapy at the Child and Adolescent Psychiatry Unit of the University Children’s Hospital in Bialystok (Poland).The presence of several inattentive or hyperactive, impulsive symptoms in two or more situation  is required for the diagnosis of ADHD/HKD –37. AtteBoth classifications were used to identify a homogeneous phenotype of HKD/ADHD. The diagnosis of Combined ADHD subtype was ascertained using current DSM-IV criteria , statingEach examination comprised observation of the family and other informants, attention being paid to parents/guardians personality traits and attitudes to the child, relationships of parents, the child’s behaviour towards his/her parents/guardians, the child’s behaviour and spontaneous play and group functioning. Exclusion criteria of the study were the coexistence of other psychiatric or neurodevelopmental disorders , epilepsy, mental retardation and somatic diseases. None of these boys had history of pharmacological treatment.All participants in the study were administered the Wechsler Intelligence Scale for Children-Revised  . The intVenous blood samples of each child with ADHD were taken during the run psychiatric and psychological diagnosis. After centrifugation, the serum was frozen and stored at −70 °C until the time of the signs. Matrix metalloproteinase-9 activity in the serum was determined by ELISA, using a kit Human ELISA System Biotrak  and expressed in μg/l. To minimize assay variance, serum levels of MMP-9 from each subject were measured on the same day. The assay was based on a two-site ELISA sandwich format using two antibodies directed against different epitopes of MMP-9. Protocols were performed according to the manufacturer’s instructions. The sensitivity defined as two standard deviations above the zero dose binding was determined as 0.6 μg/l, assay range 4–128 μg/l.U test and Spearman’s rank correlation. The values of the variables are presented as mean ± SD or median, first and third quartile. The regression analysis was performed. The univariate linear regression models were created. The p value <0.05 was considered statistically significant.All statistical analyses were performed using the Statistica 10.0 PL (StatSoft). Since many variables were not normally distributed according to the Shapiro–Wilk test, the analysis used non-parametric tests: the Kruskal–Wallis test with post hoc test, Mann–Whitney The study was approved by the Ethical Committee of the Medical University of Bialystok, in accordance with the principles of Guidelines for Good Clinical Practice R-I/003/168.Rs = 0.95; p < 0.001) and VADPRS  , however the relationship between the impulsivity of HKD and ADHD domain was not significant . There was a highly significant correlation between inattention of HKD and inattention of ADHD  (Table Both models (HKD and combined ADHD subtype) were identified in every boy in the study group. Total scores were similar for the clinical model of HKD  and ADHD . No significant differences were noted between the average values of VADPRS (mean ± SD: 39.95 ± 6.81) and VADTRS (mean ± SD: 38.14 ± 6.14) in the study group. There were significant positive correlations between ICD-10 and DSM-IV symptoms  , the Verbal IQ-102 (25th–75th percentile: 91–108) and the performance IQ-90 (25th–75th percentile: 87–106) were determined in all the study subjects. The Vocabulary subtest and the Comprehension subtest with the inattention domain showed a negatively significant correlation    of MMP-9 levels 49.13 (15.86) μg/l and median of MMP-9 levels 50.82 μg/l  in study group amounted. The General Regression Models (GRM) were used to assess the effect of MMP-9 on HKD/ADHD symptoms  , 58. It The extracellular proteolysis by MMP-9 is important for the functional and structural synaptic plasticity –7, 9, 10The MMP-9 concentration increases in inflammation, hypoxia or injuries of brain, where the blood–brain barrier is damaged –17, 86. Though the relevant scientific studies gained great interest in recent decades, the etiopathogenesis and underlying mechanism of ADHD/HKD remain still unclear. So far, laboratory, psychological or biological studies that could be specific enough to allow explicit in diagnosis of ADHD/HKD have been missing . The strTo understand the processes engaged in cognitive dysfunction in ADHD/HKD, it is necessary to unravel signalling pathways, complex interaction networks and metabolic alterations involving many anatomical components. However, we are aware that the role of MMP-9 in neurodevelopmental damage still remains unclear, and the present study is the first to show the elevated levels of serum MMP-9 in boys with HKD correlated with severity of the disorder. The results indicate that the increased levels of serum MMP-9 in boys with HKD are associated with the clinical domain of impulsivity.


DAT1/SLC6A3 gene was previously reported to be associated with various phenotypes involving disturbed regulation of dopaminergic neurotransmission.ADHD and alcoholism are psychiatric diseases with pathophysiology related to dopamine system. DAT1 belongs to the SLC6 family of transporters and is involved in the regulation of extracellular dopamine levels. A 40 bp variable number tandem repeat (VNTR) polymorphism in the 3′-untranslated region of DAT1/SLC6A3 gene in this study . Using miRBase software, we have performed a computer analysis of VNTR part of DAT1 gene for presence of miRNA binding sites.A total of 1312 subjects were included and genotyped for 40 bp VNTR polymorphism of DAT1/SLC6A3 gene (P < 0.01). The 9/9 genotype appeared to reduce the risk of ADHD about 0.4-fold (p < 0.04). We also noted an occurrence of rare genotypes in ADHD (frequency different from controls at p < 0.01). No association between alcoholism and genotype frequencies of 40 bp VNTR polymorphism of DAT1/SLC6A3 gene has been detected.We have found significant relationships between ADHD and the 40 bp VNTR polymorphisms of DAT1/SLC6A3 gene and ADHD in the Czech population; in a broad agreement with studies in other population samples. Furthermore, we detected rare genotypes 8/10, 7/10 and 10/11 present in ADHD boys only and identified miRNAs that should be looked at as potential novel targets in the research on ADHD.We have found an association between 40 bp VNTR polymorphism of  DAT1/SLC6A3, has been among the most often studied candidate genes in relation to ADHD [Dopamine and dopaminergic signalling have been implicated in aetiologies of several psychiatric and neurological disorders including attention-deficit/hyperactivity disorder (ADHD) and alcoholism. Dopamine transporter DAT1 is thought to be the main regulator of synaptic and extracellular levels of dopamine and, consequently, the corresponding gene  to ADHD , 3.+ and Cl− dependent transporter of the solute carrier family 6 (SLC6A3). The transport process is active, deriving its free energy from the transmembrane concentration gradients of Na+ and Cl− that are generated by Na+/K+ATPase and potassium chloride symport [DAT1 is a Na symport , 5. The Dopamine transport is a well-established target of potent psychoactive compounds including cocaine, amphetamines and PCP derivatives suggesting that the removal of dopamine from extracellular space is a very important physiological process –8. WhereDAT1/SLC6A3 gene encoding DAT1 comprises 15 exons spanning 60 kb on chromosome 5p15.32, encodes 620 amino acids with 12 putative transmembrane domains and a large second extracellular loop with several putative N-glycosylation sites that need to be glycosylated to transport dopamine (review: [DAT1/SLC6A3 polymorphism is a 40 bp VNTR (variable number tandem repeat) repeated between 3–13 times with the 9 and 10-repeat allele being the most frequently found in the human population. This polymorphism has been linked to various disease phenotypes involving disturbed regulation of dopaminergic neurotransmission [The 3′-untranslated region (3′-UTR) DAT1 is primarily expressed in dopamine neurons. DAT1 immunoreactivity is concentrated in somatodendritic and terminal fields of mesencephalic dopamine neurons with most pronounced immunoreactivity in striatum and nucleus accumbens , 11. BotANKK1 (DRD2) gene polymorphism and other candidate genes [COMT gene [In our previous studies we found associations between ADHD, te genes –15 and aOMT gene . In our OMT gene .DAT1/SLC6A3 gene polymorphism in a subset of a population sample numbering a total of 1312 persons.In the present study we have been using well characterized groups of Czech subjects similar to those used in our previous studies –16. We nvide infra) and 253 control subjects of corresponding age (47 girls and 208 boys). All subjects were apparent Caucasians. Children treated for ADHD (in both outpatient and inpatient settings) in the Child Ward of the Department of Psychiatry of the University Hospital in Brno, in the Children’s Psychiatric Hospital in Velká Bíteš, and in Department of Child Psychiatry, 1st Faculty of Medicine, Charles University, Prague were included in the study group. Control group were students collected as volunteers from elementary schools in Brno (Brunn) and Praha (Prague).The total of 471 mutually unrelated persons of Czech ethnicity were included into ADHD study, 218 of them children with ADHD . In each case a written informed consent was obtained from the child’s legal guardian. Excluding criteria for participation in the study for either of the groups were congenital genetic disorders, epilepsy, mental retardation, schizophrenia, pervasive developmental disorders and serious medical illness.In the group of ADHD-patients a detailed child-psychiatric clinical examination was conducted with a diagnostic assessment based on criteria of the 10th revision of International Classification of Diseases – ICD 10  as well The following questionnaires were used in the selection and evaluation of the control subjects CTQ – Conners Teacher Rating Scale  and CPQ Test diskriminácie tvaru; [We used the Shape Discrimination Test  for assTest nachádzania známych obrázkov; [Impulsivity was tested by the TE-NA-ZO test . This tIn addition we used four tests from the neuropsychological battery for psychological assessment of the Neurobehavioral Evaluation System (NES2) : Finger Samples of buccal tissues were collected using Flocked Swabs  after the diagnosis was established. DNA from swabs was isolated by using the UltraClean Tissue DNA  commercial kit.Alcohol dependent subjects were recruited from General University Hospital in Prague, and several psychiatric hospitals . In total, 441 alcohol-dependent subjects  were diagnosed to be alcohol-dependent . The diagnosis of alcohol dependence was based on a clinical assessment according to DSM-IV criteria , the selThe control group comprised 400 mentally healthy subjects  who declared themselves free of tobacco smoking, alcohol drinking and illegal drug use . As an additional safeguard, all subjects were examined by a structured questionnaire based on criteria specified in DSM-IV . This woSamples of venous blood (containing EDTA to prevent coagulation) were collected from all of the alcoholic and control subjects. DNA was extracted from 200 μL of whole blood using the UltraClean Blood DNA Isolation Kit .DAT1 gene was performed using a method described by Shinohara et al. [The detection of 40-bp VNTR polymorphism of a et al. . PCR reaU Test.CSS Statistica Software  was used for most of the statistical evaluations. Contingency tables were constructed from the genotyping data. The chi-square test was used for the comparison of genotype frequencies in the groups; Fisher’s exact test was used for the allelic frequencies comparison. The statistical difference between genotype frequencies in ADHD group was calculated using Fisher’s exact test by R software ; this teDAT1 gene by miRBase software [DAT1 from NCBI (Reference Sequence NM_001044.4) has been analysed.We have performed a guided computer analysis of software . VNTR paP < 0.01). The 9/9 genotype appeared to reduce the risk of ADHD about 0.4-fold . We also detected a difference in the rare genotypes frequency between ADHD and controls (p < 0.02). There was no significant difference in 10/10 genotype frequency between ADHD patients and controls (p = 0.2).Genotyping was successful in all 471 ADHD patients and controls  in the cerebrum. Furthermore, the dorsolateral prefrontal cortex (DLPFC), anterior cingulate cortex (ACC) as well as their connections with striatal and parietal cortical regions appear to be hypoactive   [It has been shown that distinct variations in  in ADHD  and mole(review: ). Specifreviews: , 39) and00-1.27) . Interes00-1.27) . TranslaThe above considerations might appear less conclusive in the context of larger genome wide association studies (GWAS), particularly after meta-analysis ; see alsDAT1/SLC6A3 genotype frequencies distribution between ADHD children and controls. Allele 10 has not been shown to be a “risk” allele for ADHD, as previously indicated by meta-analysis [DAT1/SLC6A3 gene and individual variations in DAT1 availability in the human striatum [p < 0.01) while the 9/9 genotype turned out to be protective. It is interesting to note that rare genotypes 8/10, 7/10 and 10/11 were identified in only six ADHD boys while in other 1306 persons  these genotypes were completely absent. The results of VanNess et al. [DAT1 3′UTR region was associated with a decreased DAT1 transcription, mRNA stability and translation. The presence of 10-repeat allele resulted in the DAT1 binding site density elevated by approximately 50 % compared to the 9-repeat allele. Fuke et al. [HESR1, HESR2 and HESR3 were identified as the hairy/enhancer split-type basic helix-loop-helix genes. These genes have been shown to be direct transcriptional targets of the NOTCH signalling pathway that is linked to neurodevelopment. HESR1 and HESR2 inhibit DAT1 expression depending on the VNTR allele by 25–50 %. The same authors reported that the presence of androgen receptor influenced inhibition of DAT1 by HESR1 and HESR2 depending on DAT1 VNTR polymorphism. Allele 9 decrease and 10 allele increase DAT1 expression in the presence of androgen receptor [We have detected significantly different analysis . Additiostriatum . We havee et al.  suggestee et al. ). An inte et al. . They foreceptor . HESR doDAT1 VNTR polymorphism and ADHD is related to the regulation of gene expression by miRNA. Recent developments very much broadened our knowledge and understanding of the mechanisms necessary for orderly gene expression . Each c review: . These r review: ).DAT1 it has been predicted that it is targeted by 49 miRNAs identified in miRDB database [DAT1 gene that is transcribed as part of mRNA. VNTR part of DAT1 from NCBI (Reference Sequence NM_001044.4) has been analysed by miRBase software [DAT1 gene: mir-1972, miR-30b-5p, miR-1301 and miR-6070. Mir-1972 was cloned in acute lymphoblastic leukaemia but its role is not known [et al. [DAT1/SLC6A3 expression which would then be influenced by the VNTR polymorphism identified in the present study as associated with ADHD.In the case of database . Accordisoftware . We haveot known . MiR-130ot known . MiR-607 [et al.  as being [et al. . These met al. [DAT1/SLC6A3 on the cell surface and/or lower affinity of DAT for dopamine; given that these changes were produced by early postnatal events, it is very likely that epigenetic mechanisms, including DNA methylation, were involved  in the population samples that we have analysed and this is in agreement with a previous meta-analytical study by van der Zwaluw [Human neuroimaging studies have identified neuroadaptive changes in the brain in response to chronic alcohol consumption. The relevant neural circuits are anchored in midbrain areas and their dopaminergic and glutamatergic projections , 62 to oDAT1/SLC6A3 gene encoding DAT1 dopamine transporter. The differences were of a VNTR polymorphism type in an untranslated region of the gene. Preliminary in silico analysis indicated that the region is potentially rich in miRNA’s binding sites. These miRNA’s may be of importance in the regulation of DAT1 expression. The present study has detected no significant differences between alcoholic patients and controls thus suggesting that the studied variations of DAT1/SLC6A3 (and possible resulting changes in DAT1 expression) could be more closely associated with ADHD than alcoholism.We have identified significant differences between juvenile ADHD patients and corresponding controls in


One criterion for a diagnostic and statistical manual of mental disorders (DSM-IV) diagnosis of attention deficit hyperactivity disorder (ADHD) and oppositional defiant disorder (ODD) is that symptoms are present in at least two settings, and often teacher ratings are taken into account. The short Conners’ Teacher Rating Scales—Revised (CTRS-R) is a widely used standardized instrument measuring ODD and ADHD behavior in a school setting. In the current study CTRS-R data were available for 7, 9 and 12-year-old twins from the Netherlands Twin Register. Measurement invariance (MI) across student gender and teacher gender was established for three of the four scales  of the CTRS-R. The fourth scale (ATT) showed an unacceptable model fit even without constraints on the data and revision of this scale is recommended. Gene-environment (GxE) interaction models revealed that heritability was larger for children sharing a classroom. There were some gender differences in the heritability of ODD and ADHD behavior and there was a moderating effect of teacher’s gender at some of the ages. Taken together, this indicates that there was evidence for GxE interaction for classroom sharing, gender of the student and gender of the teacher.The online version of this article (doi:10.1007/s10519-015-9712-z) contains supplementary material, which is available to authorized users. Attention deficit hyperactivity disorder (ADHD) is characterized by difficulties of both inattention and hyperactivity or impulsiveness that interfere with a child’s daily functioning. At school, children have, for example, difficulty remaining in their seats and paying attention for a longer period of time. Oppositional defiant disorder (ODD) is characterized by hostile and defiant behavior towards figures with authority, going beyond normal childhood behavior. Children argue with their teacher and often lose their temper (American Psychiatric Association The American Psychiatric Association (APA) estimates that 3–7 % of all school-aged children are diagnosed with ADHD, while estimates of the prevalence of ODD in children range from 2 to 16 % . Absence of MI for student’s gender may lead to what is known as scalar sex limitation, the effect of the genetic and environmental factors may, for example, be larger in boys than girls , established around 1987 by the Department of Biological Psychology at the VU University Amsterdam, registers approximately 40 % of all multiple births in the Netherlands. A survey about the development of the children is sent to the parents of the twins every 2 years until the twins are 12 years old  or attended specialized education, special schools are available for children with extra needs . Surveys were excluded if they were filled out by more than one teacher , filled out by someone other than the regular teacher , or if familiarity with the student was below average . This resulted in a total sample for the MI analyses of 8,611 surveys for 7-year-olds, 8,021 surveys for 9-year-olds and 5,954 surveys for 12-year-olds.The sample for the GxE interaction analyses included complete phenotype data for most twin pairs . Incomplete data are due to only one of the teachers returning the survey. The sample consisted of 1,208, 1,102, and 762 twin pairs of opposite sex for respectively age 7, 9 and 12. For the same-sex twin pairs , determination of zygosity status was based on blood or DNA polymorphisms  or on the basis of parental report of items on resemblance in appearance and confusion of the twins by parents and others . This last method established zygosity with an accuracy of approximately 93 %  and ‘Short attention span’ . As a consequence, the more stringent MI models did not converge due to multicollinearity when including this item. For the GxE interaction analyses, a sum score of a scale was computed when there was at most one missing item  or at most two missing items (ADHD) for a scale. Missing items were imputed by the rounded averaged item score of the scale for that child. The sum scores of the scales showed an L-shaped distribution and therefore the data were square root transformed prior to the analyses.The short Conners’ Teacher Rating Scale—Revised (CTRS-R) is a measurement instrument to asses ODD and ADHD behavior at school. Teachers had to indicate whether a child displayed a certain type of behavior currently or in the prior month. The short version of the CTRS-R consists of 28 items scored on a 4 point scale from 0 (not true or never) to 3 (completely true or very often)  with an Oblimin rotation. The number of latent factors was decided based on the scree plot and eigenvalues (larger than 1) of the factors. To test whether the scales of the CTRS-R were MI across student (‘boy’ or ‘girl’) gender and teacher  gender, multigroup (4 groups) confirmatory factor analyses (CFA) for ordinal item level data were carried out Dolan  using MpDifferent levels of MI were tested by constraining the model parameters step by step. The first level is configural invariance , where the factor structure is the same across groups. Factor means are fixed to zero for identification purposes while factor variances, thresholds, loadings and residual variances of the continuous latent response variables are group specific. One of the factor loadings is constrained to be equal to 1 for scaling purposes. A stricter model is strong factorial invariance (strong MI), where differences in latent response means are the result of differences in the latent factor means. This model is tested by constraining both the factor loadings and thresholds to be equal across groups. The factor mean of the first group is fixed to zero and freely estimated in the other groups. The last model, strict factorial invariance (strict MI) implies that the differences in the latent response means reflect true differences in the latent factor means and variances. This is tested by constraining the factor loadings, thresholds and residual variances of the continuous latent response variables to be equal across all groups. The factor mean is still fixed to zero in the first group and freely estimated in the other groups Dolan .2 values between two nested models when using the WLMSV χ2 values is not distributed as a χ2 and as a consequence regular χ2 testing is not appropriate when using the WLSMV estimator  and the comparative fit index (CFI) were chosen as indices of model fit. A RMSEA value smaller than 0.05 indicates a good fit as does a CFI value of 0.97 or higher  which calculates the difference in −2log-likelihood (−2LL) between two models and evaluates this χMI of the four scales  of the CTRS-R was tested across gender of the student (‘boy’ or ‘girl’) and gender of the teacher  at age 7 (Age: Mean = 7.44 and SD = 0.47), age 9 (Age: Mean = 9.92 and SD = 0.53) and age 12 (Age: Mean = 12.15 and SD = 0.30), resulting in a 4 group comparison. Information on the gender of the teacher was available for 8,611 7-year-olds , 8,021 9-year-olds  and 5,954 12-year-olds . Table 2 between the models with increasing equality constraints were rather small and, for the strong MI level, did not exceed more than three times the number of degrees of freedom. However, for the strict MI level, the difference in a χ2 for OPP at age 9 and HYP at age 7 and 12 was somewhat larger than this criterion, but these differences were accompanied by minor changes in RMSEA and CFI. Inspection of the modification indices revealed that they were larger for female teachers compared to male teachers for both boys and girls. Taken together, we could accept MI for the scales OPP, HYP and ADHD, for all ages, with respect to gender of the student and, more tentatively, for gender of the teacher. The fit of the MI models was acceptable to mediocre for ATT in 7-year-olds while the fit of the models was unacceptable for 9 and 12-year-olds. Even the models without constraints on the factor structure did not fit the data very well. Increasing MI levels led to a large decrease in model fit for all ages. Therefore, we could not accept MI across gender of the student and teacher for the ATT scale.Results for the tests of the three levels of MI are reported in Table S1. For OPP, HYP and ADHD the configural, strong and strict invariance models all showed an acceptable to good fit, based on the RMSEA and CFI, for all age groups. Differences in χTable Twin correlations for each gender by zygosity group rated by the same teacher or by different teachers are given in Table Correlations between twins rated by the same teacher could not be constrained to be equal to correlations between twins with different teachers. Constraining the variance components to be equal across same and different teachers also resulted in a significant deterioration of the model fit. A model with correlated errors was fitted to the data to check whether the differences between the same teacher and different teacher groups could be explained by rater bias. For none of the scales did the correlated errors model provide a better fit. In general, the proportion of the variance explained by genetic effects (heritability) was higher, at all ages, for children taught by the same teacher (ST) than for children rated by different teachers (DT) for OPP in boys  and girls , HYP in boys  and girls  and ADHD .For the scales OPP and HYP, the contribution of the variance components differed between boys and girls at all ages, while this was not the case for the ADHD scale. Heritability of OPP was higher for boys  than girls . The influence of common environmental effects was, at most ages, negligible in boys  while it had some influence in girls . Heritability of HYP was slightly higher for boys  than girls . Differences between boys and girls on this scale could mainly be attributed to differences in the influence of dominant genetic effects.Moderation by gender of the teacher was significant for OPP at age 9 and 12, HYP at age 12 and ADHD at age 7. For OPP at age 9, the relative influence of genetic effects was larger in boys with female teachers  than with male teachers  while it was somewhat larger for girls with male teachers  compared to with female teachers . For OPP at age 12, the opposite was true; heritability was larger in boys with male teachers  than with female teachers  while heritability was somewhat larger when girls were taught by a female teacher  compared to when they were taught by a male teacher . For HYP at age 12, heritability was almost equal in boys and girls with male and female teachers, but the extent to which dominant genetic effects played a role differed across gender of the teacher. For ADHD at age 7, heritability was larger for children with male teachers  compared to with female teachers .Three  of the four scales of the short Conners’ Teacher Ratings Scale—Revised (CTRS-R) Conners , used inHeritability of ODD and ADHD behavior, measured with the OPP, HYP and ADHD scales of the CTRS-R is substantial. Common environmental effects had some influence on ODD behavior while dominant genetic effects had an influence on ADHD behavior. The finding of common environmental effects is consistent with earlier studies of ODD behavior using parental ratings  is highly recommended in order to be useable in clinical practice. The heritability of ODD and ADHD behavior was lower for children in different classrooms compared to children sharing a classroom, suggesting that different behavior is elicited by different classroom environments. Apparently, teachers, the classroom and/or peers are important environmental factors that influence the expression of ODD and ADHD behavior in primary school. The direction of the moderation of the heritability of ODD and ADHD behavior by gender of the teacher was not consistent, which makes interpretation difficult. Finding environmental factors with a moderating influence on the heritability ODD and ADHD might help improve learning environments at school to prevent manifestation of ODD and ADHD behavior in children with an increased genetic vulnerability for these disorders.Supplementary material 1 (DOCX 46 kb)Below is the link to the electronic supplementary material.


We propose a fruitful scheme for exploring multiphoton entangled states based on linear optics and weak nonlinearities. Compared with the previous schemes the present method is more feasible because there are only small phase shifts instead of a series of related functions of photon numbers in the process of interaction with Kerr nonlinearities. In the absence of decoherence we analyze the error probabilities induced by homodyne measurement and show that the maximal error probability can be made small enough even when the number of photons is large. This implies that the present scheme is quite tractable and it is possible to produce entangled states involving a large number of photons. Undoubtedly, entanglement123791011121314151718192021222324252627−2 even by using electromagnetically induced transparency49et al.51π-radian conditional phase shifts created by the cross-Kerr effect. In these cases, with the increase of the number of photons it is usually more and more difficult to study multiphoton entanglement in the regime of weak nonlinearities.Generally, a spontaneous parametric down-conversion (PDC) source29313233343536383940414243444647In this paper, we focus on the exploration of multiphoton entangled states with linear optics and weak nonlinearities. We show a quantum circuit to evolve multimode signal photons fed by a group of arbitrary single-photon states and the coherent probe beam. Particularly, there are only two specified but small phase shifts induced in the process of interaction with weak nonlinearities. This fruitful architecture allows us to explore multiphoton entangled states with a large number of photons but still in the regime of weak nonlinearities.Before describing the proposed scheme, let us first give a brief introduction of the Kerr nonlinearities. The nonlinear Kerr media can be used to induce a cross phase modulation with Hamiltonian of the form t. In order to distinguish different cases, one may perform a homodyne measurementX homodyne measurement; while for P homodyne measurement.where Let Without loss of generality we may suppose that each input port is supplied with an arbitrary single-photon state. Then, the total input state readswhere θ and 2θ are induced on the coherent probe beam, respectively. We here introduce a single phase gate X homodyne measurement on the probe beam. n and n, are phase shifts on the signal photons based on the measured values of x via the classical feed-forward information. At last, at the ports n or n.Each polarizing beam splitter (PBS) is used to transmit n is even, after the interaction between the photons with Kerr media and followed by the action of the phase gate, the combined system evolves asWe describe our method in details. For X homodyne measurement38x of the X homodyne measurement is obtained, then the signal photons becomeIn order to create the desired multiphoton entangled states, we here perform an whereX homodyne measurement. Note that the peaks of these Gaussian distributions locate at We now consider the phase shifts After these feed-forward phase shifts have been implemented and the signal photons pass through the PBSs, one can obtain the desired states as follows. Clearly, for for and for n, we haveSimilarly, for odd Also,n, except for Here, the functions forandAs an example of the applications of interest for the present scheme, we introduce a class of remarkable multipartite entangled stateswhereandcat-like state, and especially for n-partite Greenberger-Horne-Zeilinger (GHZ) state. In the present scheme, obviously, for n and n, where the qubits are encoded with the polarization modes are two orthonormal states, namely Dicke states53et al.There are two models commonly employed in the process of Kerr interaction, single-mode model and continuous-time multi-mode modelθ and 2θ instead of a series of related functions of the number of photons in the process of interaction with Kerr nonlinearities. This implies that the present scheme is quite tractable especially for creating entangled states with a larger number of photons. In addition, the error probabilities n and n, the maximal error probability It is worth noting that, there are only small phase shifts generalized cat-like states. Evidently, three aspects are noteworthy in the present framework. First, since there are no large phase shifts in the interacting process with weak Kerr nonlinearities, our scheme is more feasible compared with the previous schemes. Second, the system is measured only once with a small error probability and it means that the present scheme might be realized near deterministically. Finally, the fruitful architecture allows us to explore a group of multiphoton entangled states involving a large number of photons, i.e., to produce entangled states approaching the macroscopic domain.In summary, based on linear optics and weak nonlinearities we have shown a fruitful method for exploring a class of multiphoton entangled states, the How to cite this article: He, Y.-Q. et al. Exploration of multiphoton entangled states by using weak nonlinearities. Sci. Rep.6, 19116; doi: 10.1038/srep19116 (2016).


Secale cereale L.) possesses many valuable genes that can be used for improving disease resistance, yield and environment adaptation of wheat (Triticum aestivum L.). However, the documented resistance stocks derived from rye is faced severe challenge due to the variation of virulent isolates in the pathogen populations. Therefore, it is necessary to develop desirable germplasm and search for novel resistance gene sources against constantly accumulated variation of the virulent isolates. In the present study, a new wheat-rye line designated as WR49-1 was produced through distant hybridization and chromosome engineering protocols between common wheat cultivar Xiaoyan 6 and rye cultivar German White. Using sequential GISH (genomic in situ hybridization), mc-FISH (multicolor fluorescence in situ hybridization), mc-GISH (multicolor GISH) and EST (expressed sequence tag)-based marker analysis, WR49-1 was proved to be a new wheat-rye 6R disomic addition line. As expected, WR49-1 showed high levels of resistance to wheat powdery mildew  pathogens prevalent in China at the adult growth stage and 19 of 23 Bgt isolates tested at the seedling stage. According to its reaction pattern to different Bgt isolates, WR49-1 may possess new resistance gene(s) for powdery mildew, which differed from the documented powdery mildew gene, including Pm20 on chromosome arm 6RL of rye. Additionally, WR49-1 was cytologically stable, had improved agronomic characteristics and therefore could serve as an important bridge for wheat breeding and chromosome engineering.Rye ( Blumeria graminis f. sp. tritici (Bgt) is one of the major serious diseases threatening the production of wheat (Triticum aestivum L.) in many regions around the world. Although fungicides controlling the disease are available, host resistance has proved to be the most economical and environmentally safe approach to reduce yield losses. However, as co-evolution of pathogen virulence and host resistance, cultivars carrying a single isolate-specific resistance gene, successively lost resistance to pathogens a few years after extensive growing [Powdery mildew caused by  growing . In rece growing . To combSecale cereale L., 2n = 2x = 14, RR), a close relative of common wheat, has already been proved to be a desirable donor of valuable genes for wheat improvement [Pm) gene Pm8, stripe rust (Puccinia striiformis Westend. f. sp. tritici) resistance gene Yr9, stem rust (P. graminis Pers. f. sp. tritici) resistance gene Sr31 and leaf rust (P. triticina Eriks. f. sp. tritici) resistance gene Lr26, all derived from chromosome arm 1RS of Petkus rye; Pm17 came from 1RS of Insave rye; Pm7 derived from 2RL of Rosen rye and Pm20 from 6RL of Prolific rye [Pm8 [Pm7 and Pm17 were also no longer effective. Thus, to meet the challenge of the rapid loss of resistance, it is essential to continually develop new resistance germplasm and identify novel resistance gene sources from other rye genotypes against new virulent isolates.Rye  [Pm genes or gene combinations were used as controls, including TAM104/Thatcher with Pm20 derived from rye chromosome arm 6RL, CI14189 with Pm7 from 2RL, Kavkaz with Pm8 and Amigo with Pm17 both from 1RS. Twenty-three single-pustule-derived powdery mildew virulent isolates was used to test different wheat genotypes in the same way to differentiate their reaction patterns.The winter wheat cultivar Xiaoyan 6 was derived from hybridization of common wheat and 0x = 70) . Wheat-rin situ hybridization (GISH) and multicolor fluorescence in situ hybridization (mc-FISH) detection of WR49-1. The following lines were used as controls to detect rye chromatin in WR49-1 by PCR (polymerase chain reaction) analysis, including two T1BL·1RS wheat-rye chromosome translocation lines Lovrin 10 and Lovrin 13 [Total genomic DNA isolated from wheat cultivar Chinese Spring   was usedBgt isolates prevalent in northern China. Six plants with powdery mildew resistance were selected to make backcross with the wheat parent Xiaoyan 6. The plants of BC1F1 were continually screened for resistance to powdery mildew, and the resulting resistant plants as females were continually back-crossed with Xiaoyan 6. The BC2F1 plants were again tested for resistance to powdery mildew and karyotyped by cytological examination. Resistant plants of putative rye monosome addition lines with chromosome numbers 2n = 43 were allowed to self-pollinate. The plant with somatic cell chromosome number 2n = 44 was selected and continually self-pollinated for an additional five generations. During development, each individual plant was selected based on resistance to powdery mildew, wheatlike plant type and high seed set, and then karyotyped by cytological examination. Finally, a fertile and genetically stable line with homogeneous resistance, designated as WR49-1, was selected.The following procedure was used for the production of resistant wheat-rye addition line WR49-1. Wheat cultivar Xiaoyan 6 was crossed with rye cultivar German White in 1996 and 16–18 d following pollination, inflorescences were collected and stored at 4°C for 48–72 h. Hybrid embryos were dissected from young panicles and 12 plants were obtained by embryo rescue on MS medium. Following chromosome doubling with a solution containing 0.05% colchicine, 1.5% dimethylsulfoxide, and 5% MS medium [GISH technique was used for detection of rye chromatin in wheat-rye line WR49-1. The mitotic chromosomes of root tips cells of WR49-1 were prepared and observed by the method of An et al. . Total gAs1 (or pHvG38) labeled with digoxigenin-11-dUTP and pSc119.2 labeled with biotin-11-dUTP, respectively [Sc119.2 and pAs1 as probes enabled all the R-genome chromosomes of rye, 17 of 21 chromosome pairs of hexaploid wheat to be distinguished from each other [As1 combined with pHvG38 could identify all 21 wheat chromosome pairs [FISH analysis was used to characterize rye and wheat chromosomes in WR49-1. After rinsing the GISH hybridization probes signals, mc-FISH was conducted by using two highly repeated DNA sequences pectively . Detectich other , whereasme pairs .T. Urartu , Aegilops speltoides  and Ae. Tauschii, respectively [T. urartu were labeled with fluorescein-12-dUTP, and total genomic DNA of Ae. tauschii was labeled with Texas-red-5-dUTP, while total genomic DNA of Ae. speltoides was used for blocking [Total genomic DNA was isolated from young leaves of ectively . Total gblocking . Detectiblocking .Seven primer pairs were used to identify rye chromosome in WR49-1: one EST-STS (expressed sequence tag-sequence tagged site) marker CGG143 specific for chromosome 6R of Imperial rye, and six EST-SSR (simple sequence repeat) markers specific for chromosome arm 6RL of Imperial rye, including SWES78, SWES206, SWES231, CGG23, CGG59 and DUPW111 . PCR reaPm genes or gene combinations  was performed to characterize rye and wheat chromosomes in WR49-1. The additional rye chromosome in WR49-1 had five specific hybridization bands when labeled with pSc119.2, including one strong terminal band and one faint subterminal band on the short arm, as well as two strong intercalary bands and one faint terminal band on the long arm Sc119.2 aTo further confirm the genomic identity of the chromosomes in WR49-1, mc-GISH analysis was done by using the total genomic DNA of the various diploid progenitors of common wheat and rye as probes or blockers. The A-, B-, and D-genome chromosomes were detected by yellow-green fluorescence, brown or gray, and red or pink fluorescence, respectively as a result of cross-hybridization by the different genomic probes , while tAmong the seven EST-based markers specific for chromosome 6R or 6RL of Imperial rye used, CGG143, SWES78, SWES206 and SWES231 could amplify about 110, 260, 200 and 260 bp DNA fragments from German White rye, respectively . The corBased on comprehensive analyses of GISH, mc-FISH, mc-GISH and PCR, WR49-1 was determined to be a 6R chromosome disomic addition line involving two small translocations between B- and D-, B- and A-genome chromosomes and therefore its chromosome composition was 2n = 44 = 21″W+1″R (6R).Pm gene or gene combinations were tested for their reactions to 23 Bgt isolates , E31 and E32, but susceptible to E20, whereas TAM104/Thatcher showed reverse reaction patterns to these 8 isolates. Therefore, the resistance to powdery mildew by WR49-1 appeared to be controlled by new Pm gene(s) different from documented Pm genes or gene combinations.The seedlings of WR49-1, German White rye, Xiaoyan 6, Mingxian 169, Huixianhong and 38 wheat genotypes carrying documented isolates . The susisolates . WR49-1 isolates . By compinations . For exaPm20, Kavkaz with Pm8, CI14189 with Pm7 and Amigo with Pm17, were tested with the mixture of Bgt isolates collected from northern China. Xiaoyan 6 and the five controls, Kavkaz, CI14189, Amigo, Mingxian 169 and Huixianhong, showed all susceptible with IT from 8 to 9, whereas German White developed no mildew symptoms with IT 0. WR49-1 and TAM104/Thatcher with Pm20 showed IT 1, therefore, were considered as highly resistant [In three consecutive wheat growing seasons, WR49-1, German White and Xiaoyan 6, along with six controls, including Mingxian 169, Huixianhong, TAM104/Thatcher with esistant .P<0.05), whereas no significant difference with German White (P<0.05). WR49-1 possessed superior fertility than both parents for its desirable performance on sterile spikelet number per spike  [H. filipjevi nematode [Mayetiola destructor) [1Bx 14 and 1By 15, early maturity, stress tolerance and wide adaptation, but showed vulnerable to powdery mildew. In order to improve the powdery mildew resistance of Xiaoyan 6 and in view of the multiple potentially useful genes on rye chromosome 6R, we conducted wide hybridization and chromosome manipulation between Xiaoyan 6 and rye cultivar German White, developed and characterized a new wheat-rye 6R disomic addition line WR49-1.Chromosome 6R of rye possessed many genes for favorable traits, such as resistances to powdery mildew , stripe  avenae) , H. filinematode , Hessiantructor) , as welltructor)  and droutructor) , which wBgt isolates prevalent in northern China at the adult stage and showed high resistance to 19 of 23 Bgt isolates at the seedling stage. It had different reaction patterns with Pm7, Pm8, Pm17 and Pm20 derived from rye (Bgt isolates than TAM104/Thatcher with Pm20 from 6RL. WR49-1 may possess new Pm gene(s) and is an additional source for powdery mildew resistance, thus can be used in broadening mildew resistance in wheat. Additionally, it is necessary for identifying the resistance genes in WR49-1 to conduct genetic and molecular mapping studies.WR49-1 conditioned absolute resistance against the composite of from rye  and a wiPm genes at 49 loci and more than 20 temporarily named Pm genes have been reported in common wheat and its wild relatives [Pm7, Pm8, Pm17 and Pm20 [Pm8 from T1BL·1RS translocation line, Pm17 from T1AL·1RS translocation line, and Pm20 from T6BS·6RL translocation line showed highly resistant to 5, 1 and 13 of 23 Bgt isolates tested in this study, respectively. Currently, the cultivars with Pm8 and Pm17 have already successively lost the resistance to powdery mildew [Pm7 from T4BS·4BL-2RL translocation line, also exhibited highly susceptible to all the 23 Bgt isolates tested  substitution line, had located on 6RL of rye, further was mapped 3.7 cM distal from the RFLP locus, XksuF37 [H. filipjevi was a newly identified pathogenic species of cereal cyst nematode that has severely damaged wheat in central China. A two-year field test demonstrated that the substitution line 6R (6D) was also highly resistant to the nematode [H25 was located on the 6RL telocentric chromosome of Balbo rye and transferred into hexaploid wheat via X-irradiation-induced terminal and intercalary chromosomal translocations [The gene  XksuF37 ,34. H. fnematode . A Hessiocations .T. timopheevii cytoplasm. Using a set of CS-Imperial disomic addition lines, QTLs (quantitative trait locus) controlling agro-physiological indicators of drought tolerance were also detected on chromosomes 6R [Alt1 controlling aluminum tolerance was located on 6RS of rye and two isozyme loci linked to Alt1 gene was also identified on 6RS [Curtis and Lukaszewski  reportedsomes 6R . A loci d on 6RS .Rhizoctonia cerealis), which may improve the progress of sharp eyespot resistance breeding in wheat. However, for different resistances of WR49-1, more genetic and molecular mapping studies are needed to identify the resistance genes and determine gene interactions.Resistance genes to wheat stripe rust were also located on 6RL of rye . Our resBgt isolates in China (The alien chromosome disomic addition lines were not only valuable genetic resources for studying the origin and evolution of species, genetic relationships between different genomes, gene interactions and expression, but also important intermediate materials for developing substitution lines and translocation lines . To datein China , but hadin China .Chromosome instability and genome rearrangements in wheat-rye addition lines have been well documented ,41. Amonin situ hybridization techniques, PCR-based markers have also become a convenient and efficient tool to identify the presence of alien chromosomes and chromosomal segments in wheat [In addition to in wheat ,44. In t60Coγ radiation, ph1b-induced homoeologous recombination, gametocidal chromosome originating from Aegilops [The wheat-rye chromosome addition, substitution and translocation lines were valuable source materials for development of more highly engineered introgression lines. Comparing with addition and substitution lines, wheat breeders preferred translocation lines in view of the smaller amount of alien genetic material, less linkage drag, regular meiotic behavior and even direct utilization . To furtAegilops , and bac