Previous evidence supports the role of noradrenergic systems in ADHD, and norepinephrine transporter (NET) is critical in regulating the noradrenergic system. The present study aimed to investigate the association between NET gene polymorphism and the performance measures of the Continuous Performance Test (CPT) in Korean ADHD children.Eighty-seven children (mean age = 9.23 ± 1.99 years) with ADHD were recruited from a university hospital. Genotypes of G1287A of the NET gene (SLC6A2) were analyzed. All participants completed the CPT, with performance measures of omission errors, commission errors, reaction time and reaction standardization computed. The relationship between G1287A polymorphisms and CPT performance measures was examined.There were 46 subjects with the G/G genotype, 35 subjects with the G/A genotype and 6 subjects with the A/A genotype. Among the three groups, there were no significant differences in the performance of CPTs. When dichotomized according to whether the subjects have the rare allele or not, subjects with the homozygous G/G genotype showed significantly lower commission errors compared to those without G/G genotypes .Our study found a significant association between commission errors of the CPT and the G1287A genotype of the NET gene in Korean ADHD children. These findings suggest a protective role of the G/G genotype of the NET polymorphisms in the deficits of response inhibition in ADHD children. ADHD is a highly prevalent childhood psychiatric disorder that is characterized by inattention, impulsivity and hyperactivity. Neurobiological studies of ADHD point to the role of genetic factors with its high heritability estimates-3. ConsiContinuous performance test (CPT) is one of the most widely used neuropsychological tests in ADHD. The test assesses several measures including sustained attention in response to target stimuli and inhibitory control in response to non-target stimuli. These measures are continuously quantifiable and probabilistically predictive of ADHD, with reSeveral lines of genetic research have suggested the role of noradrenergic pathways in the pathophysiology of ADHD. The noradrenergic system is known to be involved in the modulation of attention and arousal. Its activation has marked effects on the function of the prefrontal cortex (PFC), with particular relevance to ADHD. Low to In regards to the CPT, only a few studies have explored the association with the NET gene. In a study by Cho et al., ADHD suEighty-seven children with ADHD were recruited from a child psychiatric outpatient clinic in a university hospital in South Korea. Inclusion criteria were as follows: 1) subjects diagnosed as having ADHD according to the DSM-IV diagnostic criteria, 2) subjects between the ages of 6-15 years, 3) those who gave informed consent, and 4) those who have never taken psychostimulants. Exclusion criteria were as follows: 1) subjects diagnosed with autism, mental retardation, language difficulties or developmental problem including learning difficulties, 2) those with a history of a brain damage or seizure disorders, 3) those without an informed consent form from their parents or guardians. The study was approved by the Institutional Review Board. Korean version of the Kiddie-Schedule for Affective Disorder and Schizophrenia Present and Lifetime Version (K-SADS-PL) was used as a diagnostic tool. K-SADS-PL is a semThe ARS is the ADHD symptom severity scale designed by DuPaul et al. composedAll participants completed the computerized CPT. The Korean version of the CPT used in this study was developed and standardized by Shin et al.. SubjectThe details about four indices were as follows: 1) Number of cases where a response is missed - namely, omission error, as indicator of inattention; 2) Number of cases where a response occurs in the presence of non-target stimuli - these are commission errors, an indicator of hyperactivity or impulsivity; 3) Mean reaction time that measures the speed of process handling as a hit response time toward target stimuli; 4) Standard deviation of reaction time that measures vigilance. The resGenomic DNA was extracted from the blood lymphocytes of all participants with a genomic DNA extraction kit . Detection of the SNP was done by the analysis of the primer extension products generated from the previously amplified genomic DNA using a chip-based Matrix-Assisted Laser Desorption/Ionization - Time of Flight (MALDI-TOF) mass spectrometry platform . The general methods were performed according to the protocol of the producer.http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi. The forward and reverse PCR primers (5'- ACGTTGGATGAGACCCTAATTCCTGCACCC and 5'- ACGTTGGATGTTCAGGACCTGGAAGTCATC) were used to generate PCR products. The PCR was performed in a volume of final volume of 5 μl containing 1X PCR buffer , 2.5 mmol/L MgCl2, 0.2 mmol/L of each deoxyribonucleotide triphosphate (dNTP), 0.1 U HotStar Taq Polymerase , 8 pmol/L of each primers and 4.0 ng of the genomic DNA. The PCR reaction consisted of the following processes: denaturation at 95°C for 15 min, 45 cycles of 95°C for 20 sec, 56°C for 30 sec and 72°C for 1 min, with the final extension step at 72°C for 3 min.PCR primers were generated using the Primer3 program To remove the unincorporated dNTPs, 0.3 U of Shrimp alkaline phosphatase were added and incubation was performed for 20 min at 37°C, followed by 5 minutes at 85°C to inactivate enzyme.The oligonucleotide sequence of the hME extension primer was 5'- GCATGGAGGCTGTCATCAC, which was designed manually. We selected the forward or the reverse DNA strand depending on several factors including the characteristics of the polymorphism and the GC content. The final volume of each reaction was 9 μl, containing hME enzyme , the termination mix and 5 μmol/L of the extension primer. The primer extension reaction was started with the initial denaturation at 94°C for 2 min, followed by 55 cycles of 94°C for 5 sec, 52°C for 5 sec, and 72°C for 5 sec. The reaction product was desalted with SpectroCLEAN . The samples were distributed on 384 well SpectroCHIP , using SpectroJET . The SpectroCHIPs were analyzed by MALDI-TOF MassARRAY system . After an overall automatic measurement, assays with bad peaks were checked manually.For statistical analyses, one-way analysis of variance (ANOVA) was used to evaluate the association between the three genotype groups  and the four CPT measures. Due to the small sample size of those with A/A genotypes, subjects were dichotomized according to whether they have the rare A allele or not. Independent T-test was used to compare the differences in the CPT measures between the two groups. The level of significance was held at 0.05. All analyses were done by the Statistical Package for the Social Science  for Windows.Eighty-seven ADHD children were enrolled, consisting of 72 males (82.8%) and 15 females (17.2%). The mean age of the ADHD subjects was 9.23 years of age (SD = 1.99), and the average total IQ was 104.79 (SD = 16.2). Among the 87 ADHD subjects, 39 (44.8%) were classified as ADHD combined type, 40 (46.0%) as the inattentive type and 8 (9.2%) as the hyperactive/impulsive type. The average total score of the ADHD symptom rating scale measured by the parents was 32.26 (SD = 7.91). There were no significant differences among groups of G/G, G/A and A/A genotypes in the demographic and clinical characteristics (Table The distribution of the genotypes for SLC6A2 was as follows: 46 subjects (52.9%) with the G/G genotype, 35 subjects (40.2%) with the G/A genotype and 6 subjects (6.9%) with the A/A genotype. The results were similar to the previous studies in Korean ADHD children  The distribution of the genotypes was in Hardy-Weinberg equilibrium.Among the three SLC6A2 genotype groups, there were no significant differences in the performance of CPTs. When dichotomized according to whether the subjects have the rare allele or not, subjects with the homozygous G/G genotype showed significantly lower commission errors compared to those without G/G genotypes  (Figure Our study found a significant association between commission errors of the CPT and the NET G1287A genotype in Korean ADHD children. The finding suggests that there may be a protective role of the G allele regarding the response inhibition deficit in ADHD. Furthermore, the results emphasize the possible role of the noradrenergic system underlying the pathophysiology of ADHD.The commission errors are an indicator of deficits in response inhibition. Barkley proposedAtomoxetine selectively increases catecholamine neurotransmission in the prefrontal cortex, without the associated effects of psychostimulants on striatal dopamine transmission. In the study by Chamberlain et al., a singlIn other lines of research, Jonsson et al. reportedStober et al.  reportedThere are several limitations to be mentioned in our study. First, although the level of performance of CPT and the frequencies of the NET polymorphisms in this study are similar to previous reports, we did not compare between ADHD subjects and controls on the CPT performances and NET polymorphisms. Secondly, the sample size of the current study was very small, so the results cannot be generalized to the general population and also should be carefully interpreted. Thirdly, we did not a multiple comparison test by independent T-test to compare the four CPT measures between two groups, even though we performed a multiple comparisons test by one-way ANOVA to compare the four CPT measures among three groups. Fourthly, when we performed non-parametric test to evaluate the significance level on two group difference, even if our CPT scores were normally distributed, we found that the significance level became lower. In addition, the outlier in the G/A + A/A group might drive the more significant group difference on the commission error measure. Therefore, to eliminate some effect of the outlier of the commission error measure, we reanalyzed the comparison statistics of the commission errors between two groups without the extreme scores of the commission errors, which are considered as the outlier group in both group. However, the results still showed the significant group difference on the commission error measure. Nevertheless, we will need to perform this correlation study in the larger sample in the near future. Lastly, the differences in the male and female ratio were not controlled. Majority of participants were male in this study, and we cannot exclude the possible effects of gender differences. In future studies, these limitations should be taken into consideration to retest the current results.In conclusion, our study found a significant association between commission errors of the CPT and the G1287A genotype of the NET gene in Korean ADHD children. The findings suggest a protective role of the G/G genotype of the NET polymorphisms in the deficits of response inhibition in ADHD children.KC proposed the idea of this study. KC, KJ and DS collected and evaluated the subjects with ADHD for this study. KJ and DS carried out the molecular genetic studies, and drafted the manuscript. JS and KC participated in the design of the study and performed the statistical analysis. DS participated in its design and coordination. DS and KC completed to write the final manuscript. All authors read and approved the final manuscript.All authors of this work have no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within three years of beginning the work submitted that could inappropriately influence bias our work.


Innate immunity is the ancient defense system of multicellular organisms against microbial infection. The basis of this first line of defense resides in the recognition of unique motifs conserved in microorganisms, and absent in the host. Peptidoglycans, structural components of bacterial cell walls, are recognized by Peptidoglycan Recognition Proteins (PGRPs). PGRPs are present in both vertebrates and invertebrates. Although some evidence for similarities and differences in function and structure between them has been found, their evolutionary history and phylogenetic relationship have remained unclear. Such studies have been severely hampered by the great extent of sequence divergence among vertebrate and invertebrate PGRPs. Here we investigate the birth and death processes of PGRPs to elucidate their origin and diversity.We found that (i) four rounds of gene duplication and a single domain duplication have generated the major variety of present vertebrate PGRPs, while in invertebrates more than ten times the number of duplications are required to explain the repertoire of present PGRPs, and (ii) the death of genes in vertebrates appears to be almost null whereas in invertebrates it is frequent.PGRP genes may have an impact on the availability of the repertoire and its function against pathogens. These striking differences in PGRP evolution of vertebrates and invertebrates should reflect the differences in the role of their innate immunity. Insights on the origin of PGRP genes will pave the way to understand the evolution of the interaction between host and pathogens and to lead to the development of new treatments for immune diseases that involve proteins related to the recognition of self and non-self.These results suggest that the emergence of new  Innate immunity is the ancient defense system of multicellular organisms against microbial infection. The basis of this first line of defense resides in the recognition of unique motifs or components conserved in microorganisms, and absent in the host. The innate immune system uses sets of pattern recognition receptors to recognize such foreign or non-self motifs. Proteins in the immune system can be located intracellularly, on the cell surface, or secreted into the bloodstream, ready to signal the presence of an intruder in every compartment. In systems lacking the adaptive arm of immunity, the pattern recognition concept serves well to explain the general triggering of the system as well as providing receptors for the limited specificity shown by innate immunity -4.PGRP-S, PGRP-L, PGRP-Iα and PGRP-Iβ), while thirteen loci in Drosophila [Anopheles [Toll, or Imd pathways to induce an expression of anti-bacteria peptides, induce prophenoloxidase cascade, or directly cause phagocytosis and lysis [Peptidoglycan (PGN) is the major structural component of the cell wall of almost all bacterial species. PGN is a large, repetitive macromolecule that forms the rigid cell wall of bacteria. PGN recognition is mediated by the PGRP (PGN recognition protein) family of receptors ,6. PGRPsosophila ,11, whicnopheles . Severalnd lysis -18. On tnd lysis . While Pnd lysis -22.PGRPs by systematically analyzing PGRP genes from a set of diverse eukaryotes, and discuss the role of selection and diversification of this gene family.Vertebrates have the acquired immunity system in addition to the innate immunity system, while in insects only the latter is a self-defense system. It is of interest whether possessing acquired immunity has effects on the evolution of molecules involved in innate immunity. Here, we investigate the birth and death processes of PGRP genes in vertebrates and invertebrates separately by using the neighbor-joining (NJ) and minimum evolution (ME) methods. There was no conflict in the topologies obtained with these methods.To detect lineage-specific expansions 40 sequences of the PGRP family from 21 vertebrate species and 42 sequences from 6 invertebrate species were studied . The divergence time of these orders is similar to that of vertebrates. Gene duplication and loss are rather frequent and taxon-specific sets of PGRPs are evident. For example, in Drosophila only six of the thirteen are found in different orders, and seven seem to be Drosophila specific. The phylogenetic tree reveals that at least 14 rounds of gene duplication and two of gene losses are required to produce the extant repertoire of PGRPs in Drosophila genome  method with the JTT substitution matrix [2+ ligand-binding in the bacteriophage T7 lysozyme. Since all seven amino acids are conserved in both the proto- PGRP-L and PGRP-S sequences, the ancestor of both proto-PGRPs is likely to possess the amidase activity  analysis as described in Methods [PGRP-L is positively selected among all the vertebrate PGRPs. This site, which is involved in substrate binding, shows a high degree of amino acid variation in PGRP-L of different species.Next important question is whether some kind of selection process has acted on each amino acid site of vertebrate  Methods . Only thPGRP-S, L and I are 0.16, 0.13 and 0.17, respectively, and the overall value for PGRPs is 0.20. This indicates strong functional constraint, suggesting that amino acid sequences of these domains are well conserved among vertebrates.Average values of the ratios of non-synonymous to synonymous substitutions of Drosophila PGRP-LC [PGRPs, because the clustering pattern and phylogenetic relationship among sequences are not ensured and the results of SLAC strongly depend on the tree topology (data not shown).Although there is a report where a few positively selected sites were observed in the PGRP domain of  PGRP-LC , we coulDrosophila PGRP-LB [Drosophila PGRP-SA [We further examined parallel and convergent evolution at the amino acid level to infer the operation of natural selection. We aligned 39 vertebrate PGRP sequences for each locus and deduced the ancestral amino acids  at all i PGRP-LB , Drosoph PGRP-SA , and Hum PGRP-SA  showed tPGRP-L . Serine is the only amino acid residue present in this site in PGRP-L except for these fishes. Moreover, exclusively non-polar character of this site indicates that it may have an important role in the function of these proteins especially because this residue is involved in substrate binding. Site 78 has suffered one parallel and one convergent substitution. The chemical profile of this site in mammal PGRP-S is exclusively hydrophobic. The variability which results from the change of I (Ile) to the polar T (Thr) in pig and cow may suggest an effect on the structure since this amino acid is located in the hydrophobic core. At the site 123 there is a convergent change of A (Ala) or Y (Tyr) to V  in pig and frog PGRP-S, respectively located in the hydrophobic core. Both substitutions create a possible adaptive site in PGRP-S gene.Among the six parallel sites and ten convergent sites, which occurred with a significance level of 1%, sites 45, 78, and 123 may be potential adaptive sites. Site 45 has three independent parallel changes of S (Ser) to A (Ala) only in fish PGRP-I genes, but showed it in PGRP-S and PGRP-L genes [In vertebrates, Tajima's relative test did not show rate heterogeneity in -L genes   species, the phylogenetic trees clearly show different patterns. In invertebrates each PGRP shows a relatively longer branch than those in vertebrates, suggesting a relatively ancient origin of each PGRP in invertebrates. In addition, in invertebrates the clustering pattern was rarely orthologous among In vertebrate PGRP proteins we observed changes as consequence of parallel and convergent amino acid substitutions, with significance greater than the random chance expectation. The changes may be either due to conservation of chemical property of amino acids at the site or due to modification of their properties. Convergent or parallel substitutions can provide evidence for the action of natural selection for keeping the function or structure .PGRP proteins play an important role in innate immunity, which requires updated and immediate responses, because pathogens may change frequently. As a consequence, a high turnover rate is expected to happen. Actually, in invertebrates, the frequent turnover of PGRP repertoire was observed. On the other hand, several motifs indispensable for peptidoglycan recognition should be conserved through evolution of PGRPs in both vertebrates and invertebrates. In addition, we have observed that the amino acid residues that are located on the hydrophobic groove have high degree of conservation and do not show any parallel or convergent amino acid substitution.PGRPs, compared with invertebrates, namely insects, PGRPs.This study provides for the first time a description of the origin and mode of evolution on vertebrate PGRPs are proposed to be a family of genes that evolved by birth and death process with different rates in vertebrates and invertebrates. In the model of the birth and death process [PGRPs have experienced several rounds of gene duplications and some duplicated genes have been deleted from the genome. This lineage specific birth and death process has been observed both in vertebrates and invertebrates. process ,34, someThe PGRP proteins are involved in innate immunity, which responds to protect the organisms from invading pathogens. Therefore, several motifs in PGRP domains are, of course, indispensable for pathogen recognition and have been conserved through the vertebrate and invertebrate evolution. However, since vertebrates possess acquired immunity, the significance of innate immunity might be more relaxed than in insects whose immune systems depend solely on innate immunity. This difference in the evolutionary patterns could be related to the plasticity of the receptors to detect a broad spectrum of microbial pathogens and it is clearly reflected in the birth and death process of the PGRP molecules in vertebrates and invertebrates.PGRP gene family reveals an example of genetic and functional variation of which roles in the immune systems are understood through an analysis of comparative genomics. Especially the analysis reveals that the mode of PGRP evolution was characterized by birth and death process. Vertebrates and invertebrates show striking differences in the evolutionary tempo and mode of PGRP genes. Broad repertoire of pathogen recognition proteins is advantageous in invertebrates, due to the absence of adaptive immunity, in contrast to the moderate repertoire in vertebrates. This reveals that the mode of evolution of a system strongly depends on other systems, which interact with the former both directly or indirectly.http://www.ncbi.nlm.nih.gov, the Ensembl database http://www.ensembl.org and the TIGR database http://www.tigr.org/tdb/tgi/ using TBLASTN and PSI-BLAST http://www.ncbi.nlm.nih.gov/BLAST/. Search was performed using each exon of human PGRP-S, PGRP-L, PGRP-Iα, and PGRP-Iβ as a probe. Takifugu rubripes PGRP-L full cDNA and genomic sequences were predicted using Genscan and confirmed by sequence analysis. Takifugu rubripes liver tissue was kindly provided by Dr. Shugo Watabe of the University of Tokyo, Japan. The Eptatretus burgeri PGRP cDNA sequence was kindly provided by Dr. Masanori Kasahara of The Graduate University for Advanced Studies (Sokendai), Hayama, Japan.The sequences were retrieved from the genomic and EST NCBI database The nomenclature used in this study and the accession numbers are listed on Additional Files Based on the partial sequence information of chicken PGRP-L retrieved from databases, we reconstructed the missing 3' region of the transcript using the BD SMART™ RACE cDNA Amplification Kit  according to manufacturer's instructions.http://www.datamonkey.org/. Poisson and gamma genetic distances were determined by using MEGA version 4 [Sequences were aligned using the CLUSTALW version 1.83 computer program with its default parameter setting  and manuersion 4 .The ancestral sequences were determined using the program ANCESTOR , and theThe following abbreviations used in the manuscript are listed here in alphabetical order: ME: (minimum evolution); ML: (maximum-likelihood); NJ: (neighbor-joining); PGN: (peptidoglycan); PGRPs: (Peptidoglycan Recognition Proteins); RACE: (Rapid amplification of cDNA ends); SLAC: (Single Likelihood Ancestor Counting).AMM conceived, designed and performed the experiments. AMM, FT, YS and NT analyzed the data. AMM and YS wrote the paper. All authors read and approved the final manuscript.Table of vertebrate PGRP nomenclature. Nomenclatures and resources of vertebrate PGRP sequences used in this study.Click here for fileTable of invertebrate PGRP nomenclature. Nomenclatures and resources of invertebrate PGRP sequences used in this study.Click here for fileAlignment of vertebrate PGRPs. Alignment of the C-terminal amino acid sequence of PGRPs from various vertebrate species. A dash represents the same amino acid as the above.Click here for fileAlignment of invertebrate PGRPs. Alignment of the C-terminal amino acid sequence of PGRPs from various insects species. A dash represents the same amino acid as the above.Click here for fileReconciled gene tree and species tree of vertebrate PGRPs. NOTUNG analysis predicted 16 duplications and 42 losses. Two of the duplication events are domain duplications and three duplication events are possibly due to allelic divergence. D/L score = 66 [ore = 66 .Click here for fileReconciled gene tree and species tree of invertebrate PGRPs. NOTUNG analysis predicted 30 duplications and 53 losses. D/L score = 98 [ore = 98 .Click here for fileAverage orthologs divergence tree of vertebrate PGRPs. The EvolMAP analysis predicted 14 gains and 14 losses. In-paralogs, diverged in-paralogs and ambiguous gains constituted 36%, 57% and 7% of total gains, respectively. Gene gains (+) and gene losses (-) are depicted for each branch. Number of in-paralogs, diverged in-paralogs and ambiguous gains are indicated below or next to each gene gain [ene gain .Click here for fileAverage orthologs divergence tree of invertebrate PGRPs. The EvolMAP analysis predicted 26 gains and 8 losses. In-paralogs, and diverged in-paralogs gains constituted 27%, and 73% of total gains, respectively. Gene gains (+) and gene losses (-) are depicted for each branch. Number of in-paralogs, diverged in-paralogs and ambiguous gains are indicated below or next to each gene gain [Click here for fileAlignment of PGRP ancestral sequences. Alignment of ancestral sequences of PGRP-L and PGRP-S. A dash means the same amino acid as the above. A blue star indicates the amino acid position responsible for Zn2+ ligand binding, whereas a red star indicates the amino acid position responsible for amidase activity. These sites are inferred from the sequences of T7 lysozyme of bacteriophage.Click here for file


To the Editor: Bartonellosis, a globally emerging vector-borne zoonotic bacterial disease, is caused by hemotropic, gram-negative, aerobic, facultative intracellular Bartonella spp.  has been PCR amplified and sequenced . Of the 86 birds screened, 78 (16 species) were captured by mist net during March 2010–June 2012 and 8 (3 species) were injured birds that were to be euthanized . Each biBartonella DNA was amplified by using conventional Bartonella genus PCR primers targeting the 16S–23S intergenic spacer region: oligonucleotides, 425s (5′-CCG GGG AAG GTT TTC CGG TTT ATCC-3′) and 1,000as (5′-CTG AGC TAC GGC CCC TAA ATC AGG-3′). Amplification was performed in a 25-μL reaction, as described . DNA extraction and PCR-negative controls remained negative throughout the study.Before DNA was extracted from the samples, 10 μL of blood was diluted in 190 µL of phosphate-buffered saline. DNA was automatically extracted by using a BioRobot Symphony Workstation and MagAttract DNA Blood M96 Kit . Bartonella spp. (3 for B. henselae and 2 for B. koehlerae). B. henselae was amplified from 2 Northern Mockingbirds (Mimus polyglottos) and 1 Red-winged Blackbird (Agelaius phoeniceus) (GenBank accession no. KC814161). The DNA sequences were identical to each other and had 99.6% (456/457 bp) sequence similarity with B. henselae San Antonio 2 intergenic spacer region genotype (GenBank accession no. AF369529). B. koehlerae was amplified from a Red-bellied Woodpecker (Melanerpes carolinus) and a Common Loon (Gavia immer) (GenBank accession no. KC814162). The DNA sequences were identical to each other (404/404 bp) and to GenBank sequence AF312490. Lice  were found on 5 Boat-tailed Grackles , but no ectoparasites were observed on Bartonella spp.–positive birds. Hemoparasites (Haemoproteus and Plasmodium spp.) were detected in 7 of 86 birds, indicating exposure to hematophagous ectoparasites, but hemoparasites were not detected in the Bartonella spp.–positive birds. No bacteria were visualized in Bartonella PCR–positive blood smears.Results are summarized in the Bartonella spp. are increasingly associated with animal and human illnesses; thus, the identification of reservoirs and increased understanding of Bartonella spp. disease ecology are of public health importance. Our finding of 2 pathogenic species not previously reported in birds has expanded the potential sources for zoonotic infection.Rickettsia spp. (Borrelia spp. (Anaplasma and Bartonella spp.) (Bartonella spp. to birds should be investigated, and additional studies that investigate the reservoir host range of Bartonella spp. and the transmission of these bacteria to non–host species will improve epidemiologic understanding of bartonellosis and will identify additional risk factors for Bartonella spp. transmission to new hosts, including humans.There is growing evidence that migratory birds serve as reservoirs and/or mechanical vectors for pathogens such as tick-borne encephalitis virus and


Attention deficit hyperactivity disorder Controlled Trial Investigation Of a Non-stimulant) is a multi-center, double-blind, randomized cross-over trial of the non-stimulant medication, Atomoxetine, in children and adolescents with attention deficit hyperactivity disorder (ADHD). The primary aims are to examine the efficacy of atomoxetine for improving cognition and emotional function in ADHD and whether any improvements in these outcomes are more pronounced in participants with comorbid anxiety; and to determine if changes in these outcomes after atomoxetine are more reliable than changes in diagnostic symptoms of ADHD. This manuscript will describe the methodology and rationale for the ACTION study.The ACTION study , Session 2 , and Session 3 . The standardized touch-screen battery, "IntegNeuro™", will be used to assess cognitive and emotional function. The primary measure of response will be symptom ratings, while quality of life will be a secondary outcome. Logistic regression will be used to determine predictors of treatment response, while repeated measures of analysis will determine any differences in effect of atomoxetine and placebo.The methodology for the ACTION study has been detailed.The ACTION study is the first controlled trial to investigate the efficacy of atomoxetine using objective cognitive and emotional function markers, and whether these objective measures predict outcomes with atomoxetine in ADHD with and without comorbid anxiety. First enrollment was in March 2008. The outcomes of this study will be a significant step towards a 'personalized medicine' (and therefore a more efficient) approach to ADHD treatment.ANZCTRN12607000535471.Australian and New Zealand Clinical Trials Registry  Attention deficit hyperactivity disorder (ADHD) affects at least one child or young person in every classroom worldwide, with prevalence estimates ranging between 2 and 16% , as wellStimulant medications are the most common pharmacologic treatment for ADHD , acutelyATMX has been approved by the US Food and Drug Administration for the treatment of ADHD , and hasThe ACTION study will be a double-blind, randomized, controlled cross-over trial, to assess ADHD at baseline, and after treatment with either ATMX or placebo. At each assessment point clinical ratings of symptoms will be acquired. The ACTION study will assess cognition using a standardized, computerized battery called 'IntegNeuro™" . IntegNeuro™ has been validated and established in ADHD ,31,32, tThe findings from the ACTION study will help support a more personalized approach to treatment options available for children and adolescents with ADHD by examining the efficacy of ATMX in improving both subjectively-rated symptom measures and objectively-assessed performance in ADHD. It will also provide evidence about which objective cognitive markers relate to ATMX response in ADHD, and how anxiety moderates ATMX response. An evidence-base of this kind may help support clinicians in making decisions about if and when to consider non-stimulants as an alternative to stimulants or non-pharmacologic treatments in each individual child or adolescent.The primary aims of the ACTION study are to:1. Assess whether ATMX has efficacy in improving cognition and emotional function in children and adolescents with ADHD.2. Assess whether presence of co-morbid anxiety influences the response to ATMX in children and adolescents with ADHD in relation to cognition and emotional function.3. Ascertain the reliability of cognitive and emotional function changes after ATMX compared to clinical symptoms of ADHD, and how these correlate with symptom changes. Please refer to Section 5 for more detail.The methodology for the ACTION study will be detailed in this manuscript.Eligible and consenting participants will complete rating scales and a computer-based test battery to assess cognitive performance. Figure Randomization will be performed by a hospital biostatistician using a centralized, pre-determined pair-wise randomization technique, ensuring an even allocation of participants to receiving ATMX or placebo first; while also taking into account the expected ratios across the five weight groups Figure . Once thThe dose schedule for ATMX will be based on recommendations from clinical practice and previous literature Figure  18,33].,33.18,33Figure Diagnostic and Statistical Manual of Mental Disorders - Fourth edition criteria [Potentially eligible patients will undergo a clinical examination by a participating clinician. A primary diagnosis of ADHD will be based on criteria . The ADHcriteria  will be criteria , validitcriteria , and medcriteria  in childParticipants deemed eligible for the ACTION study will undergo a two-week washout period prior to Baseline testing (Session 1) if they are taking any ADHD medication at the time of Screening. Subsequent visits occur at weeks six and 13. Appointments will be confirmed over the telephone at least one day prior.Prior to attending assessment sessions, participants will be asked to refrain from consuming caffeine that day .Assessments will include cognitive and emotional function tasks using IntegNeuro™, body mass, and validated parent-reported scales for ADHD symptom severity and self-report questionnaires (Section 4). On average, assessments will take the participants approximately two hours to complete. Upon completion of Session 1, parents will be given their child's first course of study capsules and a "Take Home Package" booklet as mentioned above in 1.1.The assessments undertaken at Baseline (Session 1) will be repeated at Session 2. Parents will be reminded to return their child's capsule blister packs and "Take Home Package" booklet, so that the investigators can record compliance to the study drugs during each study phase. As with Session 1, on completion of Session 2 parents will be provided with their child's next course of study capsules and a second "Take Home Package" booklet for use during Phase B of the study.The assessments undertaken at Session 2 will be repeated at Session 3, with the exclusion of the provision of capsule blister packs and "Take Home Package" booklets.The ACTION study will be conducted according to the principles of the Declaration of Helsinki 2008, and International Conference on Harmonization (ICH) guidelines. Investigators ensure that "Good Clinical Practice" principles will be adhered to, as outlined in 21 FDA Code of Federal Regulations CFR 312, subpart D, "Responsibilities of Sponsors and Investigators," 21 CFR, part 50, 1998, and 21 CFR, part 56, 1998.Institutional Review Board (IRB) approval is obtained prior to patient enrolment at any clinical site. All protocol modifications will be submitted to each IRB for approval before implementation. Prior to undertaking any study-related procedures, investigators will obtain written informed consent from each study participant after verbal and written explanation of study aims, methods, and potential hazards and benefits.• Sydney: Brain Dynamics Centre laboratory located at Westmead Hospital (lead site). It is a center of the University of Sydney Medical School and has a partnership with the Centre for Research into Adolescents' Health  and Westmead Hospital).• Adelaide: Cognitive Neuroscience Laboratory located at School of Psychology, Flinders University. It has a partnership with a large community adolescent mental health practice in Adelaide.• Melbourne: Department of General Paediatrics at the Royal Children's Hospital, Melbourne.Clinical Trial Coordinators (CTCs) will be appointed at all three sites.Clinical sites were selected based on the likelihood of meeting recruitment goals and executing the protocol. During a site initiation visit, CTCs at each site will be trained in protocol implementation and data collection methods. CTCs will work closely with participants and clinicians, ensure that all instruments are completed by participants, and function as study coordinators .Patients are referred to the ACTION study by participating clinicians, by faxing completed Clinical Package forms  to their local CTC. The site's CTC will contact the interested patient's parent to formally invite them to the study, and provide additional information and consent forms.Attention Deficit Hyperactivity Disorder Rating Scale-IV (ADHD-RS IV) [D-RS IV)  The ADHDAnxiety Disorders Interview Schedule for DSM-IV: Child Version (ADISC: Parent Interview Schedule) [Clinicians will also undertake the chedule) . Scores chedule) ); or ADHAt each testing session parents will complete the ADHD-RS IV, as a measure of ADHD symptom severity.Parents will also rate symptom severity on the Conners' Parent Rating Scale - Revised  . The CPRThe secondary outcomes of ACTION will be the relationships between the primary cognitive measures and ratings obtained from the ADHD-RS IV and CPRS-R.Negative feelings of anxiety, depression and stress will also be assessed via self-report, using previously established scales. The Depression Anxiety and Stress Scale (DASS)  has beenTo assess state as well as trait anxiety, the State-Trait Anxiety Inventory (STAI)  or STAI To complement the scales assessing negative feelings, an established measure of self-regulation (the BRISC) will be used, which captures risk for brain health versus resilience and capacity for seeking social support . The BRITo further explore the secondary aims of the study, other measures of ADHD symptoms will be included at each assessment session.The CGI Severity scale will be used at Session 1, based on the opinion of the investigator; while the CGI Improvement scale will be used at both Session 2 and Session 3, taking into account the opinions of both the investigator and the parent. Rating scores for each time-point will be recorded.The PedsQL - Parent reports for young children (8 to 12 years old) and adolescents (13 to 18 years old) were utilized at all three time-points to assess the health-related quality of life, based on the opinion of the parent. The two summary (mean) scores of Physical health  and Psychosocial health  will be recorded.The PedsQL - Child report will be given to participants aged 8 to 12 years, while the PedsQL - Teenager report will be provided to those aged 13 to 18 years of age. Similarly to the PedsQL - Parent report, the summary scores for Physical health and Psychosocial health will be recorded for analysis.This questionnaire was developed by Eli Lilly Pty. Ltd. for monitoring morning and late afternoon/evening behavior in children and adolescents with ADHD who are being treated using ATMX . Six copThe IntegNeuro™ cognitive test battery is made up of 13 different tasks which require a total of approximately 50 minutes to complete. The 13 tasks which make up the IntegNeuro™ test battery were designed and validated to challenge participants in six different cognitive domains: i) Sensori-motor; ii) Learning and memory; iii) Language; iv) Attention and working memory; v) Executive function/planning; and vi) Emotion identification ,32. TablIn the IntegNeuro™ cognitive battery, participants will hear the test instructions through a pair of headphones, and respond to the tasks by either speaking into the microphone (attached to the headphones) or using the touch-screen for non-verbal tasks. Participants will be supervised throughout the test via CCTV (closed-circuit television) from an adjacent room. Additional instructions will be given during the test if necessary. Participants will provide consent to be monitored via CCTV by signing a Surveillance Authority Form. The CCTV will only be used for the purpose of monitoring the patient during the visit and no recordings will be made.To minimize the effect of familiarization and practice effects at subsequent testing sessions, IntegNeuro™ has a set of parallel forms for use in repeat testing sessions, whereby the sequences/words/patterns used in each of the tasks have been programmed to be different between the time-points. IntegNeuro™ will be set up to have the participant complete the test battery corresponding to their ACTION time-point.The primary endpoint will be clinically significant improvement in cognition and emotional function after ATMX treatment assessed using IntegNeuro™ Table ; and in Part of the novelty of this trial lies in our utilization of objective assessments for cognition and emotional function. As such, we will also determine if our objective measures predict response to ATMX in ADHD, and in patients with comorbid anxiety. Treatment response is defined in Section 7.3.2.The secondary endpoints will be improvements after ATMX treatment in quality of life (PedsQL - self and parent); emotional states and self-regulation (DASS-BRISC); and state and trait anxiety . Previous ADHD treatment with stimulant medication and comorbid anxiety will be included as covariates.Parents are advised to contact the CTC in the case of any adverse events - defined as any physical, behavioral/psychological, or physiological problems potentially related to the study treatment or testing sessions. In the case of mild adverse reactions, the parent will be asked to continue on the study treatment and monitor the condition, informing the CTC if the problem persists or worsens. The study clinician will also be available for the CTC to obtain a sound medical opinion if required. If the adverse reaction is deemed an emergency, the parents will be advised to take the participant to the Emergency department of their nearest hospital. In these situations, the participant's group allocation will be revealed in order to inform the hospital of what the participant was administered. All adverse events will be recorded.The investigators will be responsible for monitoring the safety of study participants and to take appropriate action concerning any event that seems unusual. They will ensure that appropriate medical care is maintained throughout the study and after the study to follow adverse events.The data used for the power calculation of the inhibition function primary outcome measures are from a sample of 175 ADHD individuals assessed off and then on stimulant medication . The power calculation of the emotional function primary outcome measures are based on a sample of 30 ADHD participants from the same sample.Power was set at 0.9, alpha level at 0.05, and the mean difference (change in off-versus on-medication) and standard deviations for the mean of the n = 175 group was used. The means for the n = 30 group were set at '0', since there was no change expected in these outcome measures for the placebo group. After applying the pilot data means from each primary outcome measure the highest sample size estimate we obtained was n = 152 for each group under two-tailed conditions. Therefore, a minimum total of 152 participants are required in this cross-over study to achieve the desired level of probability in detecting a statistically significant difference across the two primary outcome measures.A computerized protocol will quantify cognitive data in a standardized manner. Responses obtained from the questionnaires will be paper-based, and entered into computer-based scoring spreadsheets which have been set up to automatically score each questionnaire. Raw and composite IntegNeuro™ data for all sites will be extracted and stored by the Brain Resource Company Ltd., which will be periodically forwarded to the Sydney site (once per month).IntegNeuro™reports (from Brain Resource Company Ltd.) will be generated and sent to the referring study clinicians after Session 1 and Session 3 testing. These reports will present performance at Session 1 compared to normative data, and after Session 3 .p value of <0.05 will be considered statistically significant.The ACTION study will adopt an all available data analytical approach, meaning that all data obtained will be included in analyses, regardless of adherence to the study. Accordingly, there will be no imputation of missing data. For all analyses, a Continuous data at baseline will be checked for normality of distribution and log-transformed if required. Logistic regression models will be used to determine whether or not the cognitive and emotion measures (using the composite scores) predict the presence of comorbid anxiety (as determined using the ADISC questionnaire).predictive relationship of cognition/emotion measures on ADHD symptom measures, changes after ATMX treatment in ADHD symptom measures will be categorized depending on whether or not they responded to the treatment. Analyses will be performed using two different definitions of treatment response: 1) ≥25% improvement and 2) ≥40% improvement; while non-response will be defined as 3) <25% improvement and 4) <40% improvement [Simple linear regression models will be used to observe any relationships between cognition/emotion composite scores at baseline and questionnaire scores after ATMX treatment to see if the objectively-measured cognition/emotion measures are related to the subjectively-measured ADHD symptom measures . To examine the rovement ,17,19,52moderate response to ATMX in ADHD symptom measures. These investigations will initially be performed on the total ADHD cohort, before examining any differences between those with and without comorbid anxiety.Absolute change scores (continuous ADHD symptom data) will be used to see if the cognition/emotion measures Repeated measures analysis of variance (ANOVA) will be performed on continuous variables (see Section 5), comparing the results obtained between baseline and ATMX, baseline and Placebo, and between ATMX and Placebo. Cohen's d effect size will be calculated using the mean differences between baseline and ATMX, and baseline and Placebo. Analyses will be performed in the total cohort, and then comparing those with and without comorbid anxiety. Presence/absence of previous stimulant treatment for ADHD, and an interaction term between presence/absence of comorbid anxiety and previous stimulant treatment will be used as covariates.The ACTION study will be a multi-site, double-blinded, randomized cross-over study examining the effects of ATMX on cognitive and emotional functions in children and adolescents with ADHD, whilst also identifying objective predictors of ATMX response in this group, including those with and without comorbid anxiety. The cross-over design of the study enables participants to act as their own controls when testing whether cognitive and emotional function at baseline is able to predict changes in symptom ratings after medication treatment. Enrolment and testing commenced in March 2008. Potential limitations in the study design pertain to the one-week washout period between treatment phases, and the absence of an "anxiety only" arm for examining comorbid anxiety outcomes. Although our selection of a washout period of one-week's duration was based on previous research -55, it iThe detailed methodology for the ACTION study has been presented. The novelty of this trial lies in its utilization of objective measures for cognition and emotion; and in its combination of objective and subjective, commonly-used assessment tools to determine the efficacy of ATMX in ADHD with and without comorbid anxiety. This research will further our knowledge on the effective use of ATMX in improving symptoms in child and adolescent ADHD; providing additional evidence to aid in personalized treatment approaches.ACTION: ADHD Controlled Trial Investigation Of a Non-stimulant; ADISC: Anxiety Disorders Interview Schedule for DSM-IV: Child version (Parent interview schedule); ADHD: Attention Deficit Hyperactivity Disorder; ADHD-RS IV: Attention Deficit Hyperactivity Disorder - Rating Scale IV; ANOVA: Analysis Of Variance; ATMX: Atomoxetine; BRISC: Brain Resource Inventory of Social Cognition; CCTV: Closed Circuit Television; CFR: Code of Federal Regulations; CGI: Clinical global impression scale; CHW: Children's Hospital at Westmead; CPRS-R: Conners' parent rating scale - revised (long version); CTCs: Clinical trial coordinators; DASS: Depression; anxiety and stress scale; DSM-IV: Diagnostic and Statistical Manual of Mental Disorders - Fourth edition; FDA: U.S. Food and Drug Administration; ICH: International Conference on Harmonization; ID: Identification; PedsQL: Pediatric quality of life inventory; STAI: State-trait anxiety inventory; STAI-C: State-trait anxiety inventory for children; WPREMB-R: Weekly parent rating of evening and morning behavior - revised.MRK is a member of the Strattera (Atomoxetine) Advisory Board for Eli Lilly Pty. Ltd. The atomoxetine used in the ACTION study was purchased from Eli Lilly Pty. Ltd. MRK has received research support from Brain Resource Company Ltd. for previous ADHD studies. CRC has received consulting fees and stock options in Brain Resource Company Ltd., and is a stock holder in Brain Resource Company Ltd. LMW has received consulting fees and stock options in Brain Resource Company Ltd., and is a stock holder in Brain Resource Company Ltd. She has received Advisory Board fees from Pfizer Inc. SDC has received research support from Brain Resource Company Ltd. for previous ADHD studies. The remaining authors declare they have no competing interests.All authors contributed to the conceptualization and/or design of the study. TWT drafted the manuscript, while all other authors provided critical revisions and approved the final manuscript.


Background: α,ω-Difunctional substrates are useful intermediates for polymer synthesis. An attractive, sustainable and selective (but as yet unused) method in the chemical industry is the oleochemical cross-metathesis with preferably symmetric functionalised substrates. The current study explores the cross-metathesis of methyl oleate (1) with cis-2-butene-1,4-diyl diacetate (2) starting from renewable resources and quite inexpensive base chemicals.Results: This cross-metathesis reaction was carried out with several phosphine and N-heterocyclic carbene ruthenium catalysts. The reaction conditions were optimised for high conversions in combination with high cross-metathesis selectivity. The influence of protecting groups present in the substrates on the necessary catalyst loading was also investigated.Conclusions: The value-added methyl 11-acetoxyundec-9-enoate (3) and undec-2-enyl acetate (4) are accessed with nearly quantitative oleochemical conversions and high cross-metathesis selectivity under mild reaction conditions. These two cross-metathesis products can be potentially used as functional monomers for diverse sustainable polymers. Quantitative conversions of methyl 10-undecenoate were obtained with a catalyst loading of 5 mol % [Ru]-1 -1 .1) with cis-2-butene-1,4-diyl diacetate (2). The self-metathesis of methyl oleate (1), which yields octadec-9-ene (5) and dimethyl octadec-9-enedioate (6), is the only concurrent metathesis reaction which had to be suppressed  and undec-2-enyl acetate (4). Both are thus derived from a renewable precursor and are interesting substrates for the synthesis of different types of polymers. Substrate 2 can be considered as an important cross-metathesis partner for the metathetical conversion of methyl oleate 1 in terms of its short chain length. Thus, the chain length of the cross-metathesis products is appropriate for certain polymer applications. Therefore, in contrast to polymers prepared from short-chain monomers, these polymers have a higher flexibility and higher stability against hydrolysis -1 to [Ru]-8 (1) with cis-2-butene-1,4-diyl diacetate (2). The reactions were performed with a fivefold excess of 2 in toluene at 50 °C for 5 h to shift the reaction equilibrium towards the cross-metathesis products 3 and 4.The catalytic activity of the ruthenium complexes o [Ru]-8  using a 1 and yields of the cross-metathesis products 3 and 4 were determined by gas chromatography with internal standard.The differences in metathesis activity of these investigated metathesis initiators are presented in 1 (about 15%) and yields of each of the desired cross-metathesis products 3 and 4 (about 5%) were achieved using the ruthenium phosphine complexes [Ru]-1 and [Ru]-3 ([Ru]-1 and its indenylidene counterpart [Ru]-3. The self-metathesis of methyl oleate (1) mentioned above could not be suppressed. Other side-reactions were not observed. No double-bound isomerisation took place. Promising results were obtained with catalysts bearing N-heterocyclic carbene ligands (1) was converted and the yields of the cross-metathesis products 3 and 4 of ca. 28% were achievable with ruthenium catalysts [Ru]-2 and [Ru]-4  were obtained with ruthenium complexes [Ru]-5–[Ru]-8. Due to their bidentate Schiff base ligands, they must be chemically activated by the addition of 100 equiv of phenyltrichlorosilane [[Ru]-7 and [Ru]-8 were the most active catalysts due to their space-filling isopropyl substituted Schiff base ligands -7 and [Ru]-8 is decisively higher compared to the other catalysts used.Comparable or higher conversions of rosilane –31. With1 (from 15 to 94%) and of yield of each of the cross-metathesis products 3 and 4 (from 2 to 66%) were obtained with a catalyst loading of [Ru]-7 in a range of 0.1 and 1.5 mol %. Cross-metathesis selectivity was increased by 60% to 67%. With a catalyst loading of 1.5 mol % of [Ru]-7, a nearly quantitative conversion of unsaturated fatty ester 1 was achievable (1 were obtained in lower amounts (ca. 14% of each self-metathesis product 5 and 6). From the results, it can be concluded that a catalyst loading of above 1.0 mol % of the ruthenium catalyst [Ru]-7 was necessary for efficient conversion of cis-2-butene-1,4-diyl diacetate (2). Further increasing the quantity of [Ru]-7 led neither to an essentially higher oleochemical conversion nor to higher cross-metathesis yields  was quite high (<96%). The cross-metathesis yields reached its maximum at 77% using an eightfold excess of 2 .Independent of the excess of ess of 2 , entry 4d yields , entry 51) was already converted  appears to be temperature-independent (1) underwent a rapid self-metathesis at low reaction temperatures  is required. On increasing the reaction temperature from 30 to 50 °C an increase in the yields of the cross-metathesis products (up to 77%) was observed. At the same time the self-metathesis reaction of 1 was hindered and only 10% of each of the self-metathesis products 5 and 6 was produced.Moreover, the conversion of methyl oleate  with cis-2-butene-1,4-diyl diacetate (2) were applied to the cross-metathesis reaction of the corresponding fatty acid 7 with the diol 8. The oleic acid (7) was reacted with both cis-2-butene-1,4-diyl diacetate (2) and the diol 8  with the protected diol 2 was nearly quantitative using 1.5 mol % of the ruthenium complex [Ru]-7 at 50 °C within 5 h (7) with 2 were only achieved with the use of 3.0 mol % of the same ruthenium catalyst. Under otherwise similar reactions conditions, 75% of oleic acid was converted  with cis-2-butene-1,4-diyl diacetate (2) was feasible with the relatively low catalyst loading of the Schiff base ruthenium catalyst [Ru]-7 to yield two value-added and sustainable intermediates in one step. Methyl 11-acetoxyundec-9-enoate (3) and undec-2-enyl acetate (4) are both very interesting substrates for polymer synthesis. They could be prepared under mild reaction conditions within 5 h. Moreover, this is an advantageous contribution towards the synthesis of sustainable monomer units because a new α,ω-difunctional substrate class starting from a renewable compound and an inexpensive base chemical was prepared.In conclusion, the cross-metathesis of methyl oleate  (97%), solvents and reagents were purchased from Sigma-Aldrich. Benzylidene ruthenium catalysts [Ru]-1 and [Ru]-2 were obtained from Sigma-Aldrich and the remaining indenylidene ruthenium catalysts [Ru]-3–[Ru]-8 were provided by Umicore AG & Co. KG and were used as received.Sunflower oil with a high oleic content (91.4% oleic acid) was obtained from Emery Oleochemicals. 1) was prepared by transesterifcation of high oleic sunflower oil with methanol using hypostoichiometric amounts of sodium methoxide (30% in methanol). cis-2-Butene-1,4-diyl diacetate (2) was prepared by the pyridine catalysed acylation of the diol 8 with acetic anhydride according to -1–[Ru]-8 were added in the range of 0.1–2.0 mol % to the reaction mixture. In the case of Schiff base ruthenium catalysts [Ru]-5–[Ru]-8, 100 equiv of phenyltrichlorosilane  were also added. The reaction mixture was stirred magnetically at the appropriate temperature (20–50 °C) for the appropriate time (1–9 h). After completion of the reaction, the mixture was cooled to ambient temperature. Conversion and yield analyses were performed by gas chromatography. The metathesis products were isolated after removing toluene in vacuo by flash chromatography on silica gel with cyclohexane/ethyl acetate (from 10/1 to 1/2) as eluent, and subsequently characterised by NMR spectroscopy.A flame-dried Schlenk tube was charged with 0.050 g (0.17 mmol) methyl oleate : δ (ppm) = 0.88 , 1.28 , 1.61 -CH2-CH2-), 2.00 , 2.30 -CH2-), 3.66 , 5.34 . 13C NMR : δ (ppm) = 14.0, 22.6, 24.9, 27.0, 27.1, 28.9, 29.0, 29.1, 29.2, 29.4, 29.6, 29.7, 31.8, 34.0, 51.3, 129.6, 129.9, 174.2. MS : m/z (%) = 296 (4) [M+], 264 (28), 246 (2), 235 (3), 222 (17), 207 (3), 194 (3), 180 (16), 166 (8), 152 (10), 137 (14), 123 (24), 110 (29), 97 (54), 83 (55), 74 (65), 69 (68), 55 (100), 41 (76), 29 (29).1H NMR : δ (ppm) = 1.96 -CH3), 4.57 , 5.64 . 13C-NMR : δ (ppm) = 20.6, 59.7, 127.8, 170.3. MS : m/z (%) = 172 (14) [M+], 113 (7), 99 (1), 82 (2), 70 (46), 61 (4), 53 (2), 43 (100), 39 (6), 27 (6).1H NMR : δ (ppm) = 1.61 , 1.66 -CH2-CH2-), 2.05 -CH3), 2.07 , 2.30 -CH2-), 3.66 , 4.67 , 5.87 . 13C NMR : δ (ppm) = 21.0, 24.9, 28.7, 29.0, 29.3, 29.7, 32.2, 51.4, 65.3, 129.7, 130,9, 170.9, 174.2. MS : m/z (%) = 256 (2) [M+], 213 (3), 196 (3), 182 (23), 164 (31), 154 (8), 135 (14), 122 (14), 107 (8), 98 (15), 81 (29), 67 (24), 55 (34), 43 (100), 29 (9).1H NMR : δ (ppm) = 0.88 , 1.29 , 2.56 , 4.50 , 5.66 . 13C NMR : δ (ppm) = 11.4, 21.0, 22.7, 29.2, 29.2, 29.3, 29.4, 31.9, 32.2, 65.3, 129.8, 136.8, 179.3. MS : m/z (%) = 212 (4) [M+], 170 (5), 152 (3), 141 (4), 124 (12), 110 (8), 96 (19), 82 (26), 67 (25), 54 (31), 43 (100), 39 (11), 29 (14).1H NMR : δ (ppm) = 0.89 , 1.28 , 2.00 , 5.37 . 13C NMR : δ (ppm) = 13.1, 25.9, 26.2, 28.2, 28.5, 28.8, 31.0, 33.4, 129.4. MS : m/z (%) = 252 (5) [M+], 154 (1), 139 (2), 125 (10), 111 (29), 97 (59), 91 (1), 83 (68), 79 (7), 69 (79), 65 (4).1H NMR : δ (ppm) = 1.27 , 1.59 -CH2-CH2-), 1.93 , 2.26 -CH2-), 3.63 , 5.34 . 13C NMR : δ (ppm) = 24.9, 28.9, 29.4, 29.5, 29.6, 32.5, 34.0, 51.3, 130.2, 174.2. MS : m/z (%) = 340 (1) [M+], 308 (7), 290 (3), 276 (16), 265 (1), 207 (1), 165 (7), 151 (11), 133 (12), 121 (13), 109 (18), 95 (38), 81 (59), 74 (44), 67 (58), 55 (100).


This trial was performed to test the efficacy and safety of an extended-release formulation of methylphenidate (MPH ER).N = 84) or placebo (N = 78). The primary efficacy outcome was the Wender-Reimherr Adult Attention Deficit Disorder Scale (WRAADDS) 8 weeks after randomization. Secondary efficacy measures were the ADHD Diagnostic Checklist (ADHD-DC), the Conners Adult Attention Deficit Disorder Scale (CAARS-S:L), the Clinical Global Impression (CGI) and the Sheehan Disability Scale (SDS).A total of 162 adults with ADHD according to DSM-IV were treated for 8 weeks with either two daily individually body weight-adjusted doses of MPH ER up to 1 mg/kg per day (P = 0.0003). The rates of responders were 50% in the MPH ER and 18% in the placebo group (P < 0.0001). Furthermore, similar effects were observed for the secondary efficacy variable: ADHD-DC score (P = 0.004), CAARS-S:L score (P = 0.008) and the SDS score (P = 0.017). 50% of the MPH ER group and 24.4% of the placebo group were improved “much” or “very much” according to the CGI rating (P = 0.0001). MPH ER treatment was well tolerated. At week 2 also the mean heart rate was significantly higher in the MPH ER group as compared to the placebo group (P = 0.01). No differences between the study groups were observed regarding mean blood pressure at any visit.At week 8 a significantly higher decline of the total WRAADDS score was found in the MPH ER group as compared to the placebo group (This clinical trial demonstrated statistically significant and clinical relevant effects of MPH ER in adults with ADHD for several self- and investigator-rated ADHD psychopathology and also functional efficacy measures. Attention-deficit/hyperactivity disorder (ADHD) is one of the most common psychiatric conditions in childhood, with approximately 6–9% of school-age children being affected . StudiesThe longest-used medications for ADHD are stimulants which have been used in the management of childhood ADHD since the 1930s . Methylpd') on the reduction of ADHD core symptoms were calculated from studies of appropriate design and methodological standards. In a first meta-analysis, d’ = 0.9 and no evidence of publication bias. Larger MPH effect sizes were associated with physician ratings of outcome and use of higher doses. More recently, an overall effect size of d’ = 0.42 was reported from a meta-analysis of 16 studies with methylphenidate in adult ADHD patients  than for non-stimulants (d’ = 0.39)  .Beside the reduction of ADHD symptoms according to DSM-IV, which focus on inattention, hyperactivity and impulsivity, recent pharmacological trials have also considered treatment effects on additional symptom domains which are related to ADHD in adult patients like disorganization and problems with emotional regulation. Decline of different measures of emotional dysregulation, oppositional symptoms and disorganization in adults with ADHD have been shown as a result of methylphenidate treatment in several randomized placebo-controlled trials . Also imThe purpose of this study was to test the hypotheses that treatment with an extended release formulation of methylphenidate (MPH ER) not only reduces (1) the core symptom domains of ADHD, inattention, hyperactivity and impulsiveness, but also (2) the broader psychopathological spectrum of adult ADHD according to the Utah criteria, which comprise symptoms of emotional dysregulation and disorganization, and (3) may improve every day functioning in adults with ADHD. In this study we used a twice a day dosing schedule with a sustained release MPH preparation instead of a single dose in the morning in order to adapt treatment effects to the needs of adult ADHD patients. Moreover, this study was the first in adults with ADHD that employed individually body-weight adjusted daily MPH ER doses for an optimal balance of efficacy and side effects.Subjects were outpatients with ADHD aged 18 years and older. For study inclusion the subject had to fulfil the DSM-IV criteria for ADHD (314.00 and 314.01). The diagnosis was established by clinical assessment and by use of a German standardized diagnostic instrument for psychiatric experts ADHD-DC, . A retroBefore inclusion of a subject in the study, each case has been re-evaluated by a central control committee of experienced psychiatrists  for verification of patient eligibility. Anamnestic data, standardized ADHD ratings and diagnostic interviews , were cross-checked for any inconstancies in order to make the diagnoses of ADHD as sure as possible. Inclusion and exclusion criteria were carefully re-examined to avoid protocol violations.The German versions of the SCID-I and -II interviews . Subjects were included in the study after provided written informed consent.The study was approved by the ethical committee of the State of the Saarland and has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. The trial has been registered with the Federal Opium Agency at the Federal Institute for Drugs and Medical Devices as well as at A double-blind, randomized, placebo-controlled study with parallel-group design was conducted at 10 sites. The treatment period was 8 weeks with a 2-week titration and a 6-week maintenance phase.Randomisation was performed by Medice's Galenic Department which included the generation of the randomisation list and the preparation of emergency envelopes. We used block randomisation with a block size of 4. The block size was not mentioned in the investigational plan or the consent given to patients.MPH ER is a MPH preparation manufactured by Medice Company (Germany) with a proportion of 50% immediate release MPH and 50% of extended release MPH. The effective time of action is 7–8 h . The effMedication was individually titrated b.i.d. after breakfast and lunch during the first 2 weeks to an optimal dose on the basis of tolerability and according to the body weight with a maximum daily dose of approximately 1 mg/kg body weight, starting with 10–30 mg/day. Patients were assigned to one of four weight classes  with doses of 40, 60, 80 and 120 mg daily, respectively. The interval between the two doses was 6–8 h. A standardised disease management programme consisting of seven sessions was administered individually to all participants of the study. The programme has already been used in an earlier study  and was Each subject underwent a comprehensive clinical assessment by a certified psychiatrist using standardized rating scales and interviews. The clinical examination included medical history, physical and neurological examination, assessments of vital parameters, body weight, liver function tests, complete blood count, ECG and EEG.The primary outcome measure was the total score of the Wender-Reimherr Adult Attention Deficit Disorder Scale WRAADDS, . The WRAAssessments of the ADHD DSM-IV criteria at screening and at week 8 were performed by use of the German version of the ADHD Rating Scale-IV  was used for the assessment of the degree to which ADHD interferes in function regarding three domains of daily living: work/school, social and family life. The extent of impairment in these domains was rated by the patient on a 0–10 point visual analogue scale. The three items were summed up to a single measure of global functional impairment that ranges from 0 to 30 . SDS scoAdverse events were assessed at each visit. Patients were asked for any complaints and new adverse symptoms. In addition, standardized assessments of adverse events were performed at weeks 1, 2 and 8 by use of the somatic symptom sheet of the AMDP-system . This raThe total score of the WRAADDS after 8 weeks was analysed as primary variable. The total score is the sum of all items (in case of missing items the sum was divided by the number of items answered and multiplied by 28). Missing data were imputed using the last-observation carried forward (LOCF) procedure (only one measurement was missing).F-test of the fixed effect). Additional sensitivity analyses were performed to test the robustness of the primary analyses with regard to the imputed data (under a best- and a worst-case scenario). All secondary endpoints were analysed exploratively either with the Wilcoxon [U-test  or with Fisher's exact test (binary data).The primary analysis was performed on the intent-to-treat (ITT) population using a linear mixed effects model with fixed effect treatment, covariate baseline WRAADDS and random effect centre. We applied a significance level of α = 0.05 (two-sided) for the primary confirmatory test (t-test scenario with a significance level α of 5% (two-sided). Since a dropout rate of about 15% was expected, a total of 75 patients should be recruited for each of the two treatment groups.A sample size of 64 in each treatment group has 80% power to detect a difference in means of 7 assuming that the common standard deviation is 14 under a two-group A total of 162 patients were randomized and included in this trial. 84 patients received MPH ER and 78 placebo during the double-blind study period . Seven sP = 0.32).Prior to randomization, i.e. at the beginning of the treatment phase, we also compared both groups descriptively. There was no evidence for difference in mean age, ADHD symptom score by ADHD-DC and WRAADDS score, CGI severity ratings, SDS score and IQ between the MPH ER and the placebo group at the beginning of the treatment phase. Some evidence for differences was found regarding sex and body weight/BMI with a preponderance of women and subjects with lower body weight/BMI in the MPH ER group . The meaU-test, P = 0.0001). These are equivalent to 0.9 ± 0.2 mg/kg body weight MPH ER and 0.9 ± 0.1 mg/kg body weight placebo, respectively . Note that there were slightly more women in the MPH-ER group and as women weigh on average less than men this resulted in a lower average daily dose in milligrams in the MPH ER group. In addition, dose reductions were descriptively more frequent in the MPH-ER than in the placebo group (19 vs. 2 patients). There was no evidence for group differences regarding compliance (96% in the MPH ER group and 97% in the placebo group).At week 8 the mean daily doses at week 8 were 66 ± 20 mg in the MPH ER group  and 78 ± 17 mg in the placebo group (Wilcoxon d'). The difference between the MPH ER group and the placebo group at week 8 was statistically significant . The results were confirmed by robustness analyses with worst- and best-case imputations  and also by analysis of the per protocol (PP) population, which consisted of 143 patients who completed the study without any major protocol violations . The effect size in this analysis was d’ = 0.59.There was a marked decline of 13.8 points on the WRAADDS in the MPH ER group as compared to 6.2 points in the placebo group during the 8 week double-blind treatment period . At weekd’ = 0.68 was found regarding attention difficulties . Marked effects were also found regarding hyperactivity and impulsivity , which refer together with attention difficulties to the core symptoms of ADHD according to the concept of DSM-IV. The effect sizes were d’ = 0.51 and d’ = 0.46, respectively. Evidence for effects were also found regarding disorganisation  and affective lability .Explorative analyses of the WRAADDS subscales revealed substantial differences at week 8 between patients of the MPH ER and the placebo group with the exception of the subscales hot temper and emotional over-reactivity. The highest effect size of d’ = 0.54; Wilcoxon U-test, P = 0.004). The decline on the ADHD-DC was 5.6 points in the MPH ER group and 2.6 points in the placebo group. Differences between placebo and MPH ER patients at week 8 were found for both symptom domains – inattention  and hyperactivity/impulsivity .Similar differences were found for other secondary measures: At week 8, patients on the placebo and the MPH ER group also differed regarding ADHD-DC symptom scores . However, we observed evidence for a difference between patients treated with MPH and those treated with placebo at week 8 for the secondary outcome CAARS-S:L total score .In the MPH ER group the decrease of the CAARS-S:L total score was 41.5 points over the 8-week treatment period. In the placebo group the decline was 13.1 points . Note thU-test, P = 0.0167). The total score decline was 6.9 points in the MPH ER group and 3.1 points in the placebo group (d’ = 0.40).Finally, we also observed evidence for a difference regarding functional improvement on the SDS at week 8 between the MPH ER and the placebo group (Wilcoxon P < 0.0001). The Spearman correlation coefficients between the expert ratings ADHD-DC and WRAADDS was 0.81 (P < 0.0001). Regarding the SDS total score, Spearman correlation coefficients were 0.67 for the correlation with the WRAADDS total score, 0.57 for the correlation with the ADHD-DC total score, and 0.72 for the correlation with the CAARS-S:L total score .The Spearman correlation coefficient between the total scores of self and investigator ratings in the entire patient group at week 8 were 0.72 for ADHD-DC and CAARS-S:L and 0.76 for WRAADDS and CAARS-S:L (both P < 0.0001).The number of responders according to the criterion of a 30% reduction of the WRAADDS total score at study endpoint was 50% for the MPH ER group and 18% for the placebo group . Marked and moderate therapeutic effects were seen in 50% of the patients treated with MPH and in 24.4% of those treated with placebo .Moreover, the improvement assessed by expert clinical global impression (CGI) also indicated between-group differences. 50% of the MPH ER patients and 24.3% of the placebo group showed “very much” or “much” improvement at week 8 . 63% of all adverse events were rated as “moderate". One serious adverse event was observed in the MPH ER group (rupture of the acromioclavicular joint), and two were reported in one patient of the placebo group (appendicitis and postoperative pain). The relation between SAEs and study medication were rated as “unlikely” or “not related".In the double-blind treatment phase 151 adverse events were reported in 55 patients of the MPH ER group, and 77 in 32 patients of the placebo group . The difference compared to placebo was 4 bpm. At week 8 there was still some descriptive evidence for a difference between the MPH ER and the placebo group .There was a small but clinically not meaningful increase of mean systolic and diastolic blood pressure during the treatment period in both treatment groups . No signIn this randomized clinical trial statistically and clinically relevant effects of MPH ER in adult patients with ADHD were found in comparison to placebo. Differences between patients treated with MPH ER and placebo for 8 weeks were observed for both the primary and the secondary outcomes, i.e. regarding standardized measures of expert ratings  as well as on the patients’ self-reports (CAARS-S:L) of ADHD symptoms, the global clinical situation (CGI) and everyday functioning (SDS). Thus, the results of this study provide evidence for improvement of psychopathology and functional impairment associated with ADHD from the therapists’ and the patients’ perspective, respectively.The findings of this study also suggest that MPH ER treatment results not only in relevant amelioration of ADHD core symptoms, which comprise symptoms of inattention, hyperactivity and impulsiv-ity according to DSM-IV, but also of ADHD according to the Utah-criteria for adult ADHD. This concept has been introduced by The WRAADDS has been used in several clinical trials with adults with ADHD . The resIt is another important finding of this study that MPH treatment has not only a beneficial effect on ADHD symptoms from the expert's view, but also from the patients themselves. We found a decline of the mean CAARS-S:L total score of about one third in the MPH group during the 8 weeks of treatment. This adds additional support to the clinical relevance of the results of this study. In this context it is notable that also the global clinical situation, rated with the CGI and also functional disabilities regarding work/school, social and family life, which have assessed with the SDS, were improved by MPH treatment. We found moderate to high correlations between ADHD self and expert ratings and functional impairment assessed with the SDS, which might suggest that social problems and ADHD are no independent phenomena. Above all, the finding of functional improvement together with clinical symptoms of ADHD underlines the clinical relevance of MPH ER treatment effects found in this study. Thus, our results are in line with other recent studies regarding the efficacy of stimulants in adult ADHD, which showed improvement of executive functions, quality of life and social adjustment . Taken td') found in this study ranged between 0.31 regarding amelioration of affective lability and 0.68 regarding the improvement of attention difficulties. The effect size on the primary outcome measure, the WRAADDS total score was 0.54 in the intent-to-treat and 0.59 in the per protocol population, respectively. The effect size on the primary outcome measure in this study compares to those seen in earlier MPH studies in adult ADHD and exceeds those found in large efficacy studies, e.g., of antidepressants in affective disorders (d’ = 0.39 on the WRAADDS as the primary efficacy parameter was found (The effect sizes (Cohen's isorders . The effas found . In thisAlthough the mean daily doses used in this study were not far from the recommended upper dose limit of 1.0 mg/kg body weight per day, treatment with MPH ER was well tolerated and the observed side effects like reduced appetite and dry mouth resembled those reported from other clinical studies. Moreover, most side effects were observed during the titration phase and were moderate and short-lasting. In contrast to prior short-term investigations with long-acting MPH formulations , we founSome limitations of this study have to be mentioned. The first point refers to the diagnostic process in ADHD patients, as diagnosing ADHD correctly is a crucial issue in any study with adults suffering from this disorder. In this study we used standardized instruments for the diagnostic procedure, which were applied by well trained psychiatrists. Moreover, each case has been re-evaluated by a central control committee before randomization of the subject. Nevertheless, it might be a limitation that – in contrast to ADHD studies with children and adolescents – assessments of ADHD symptoms and diagnoses were mainly based on self-reports of the subjects. In particular, also assessment of childhood ADHD symptoms did not necessarily rely on third person observations in this study. However, diagnostic interviews which were primarily used in this study are the fundamental basis of every diagnostic process in adult psychiatry and it has been shown that adults present competent sources of information and that self-reports may lead to valid diagnoses in subjects with ADHD in particular .Another limitation of this study might be the implementation of a basic disease management program with psychoeducational elements. This program was already used in a further study  accordinIn conclusion this study showed both statistically significant and clinically relevant improvements in ADHD symptoms based on several expert ratings and self-report instruments. Further, treatment with MPH ER was associated with a decline of ADHD symptoms according to DSM-IV as well as according to the broader concept of the Utah criteria. Importantly, also functional impairment of patients was improved after 8 weeks of treatment with MPH ER. Treatment with body weight adjusted mean daily MPH ER doses of 0.9 mg/kg was well tolerated and no severe adverse events related to study medication were observed during the study. Although no clinical relevant changes of vital signs were observed after 8 weeks of treatment, some small drug effects on the heart rate during the titration phase were observed. Therefore, controls of vital signs have to be recommended in the treatment of adults with MPH.


Among the most robust neural abnormalities differentiating individuals with Attention-Deficit/Hyperactivity Disorder (ADHD) from typically developing controls are elevated levels of slow oscillatory activity  and reduced fast oscillatory activity  during resting-state electroencephalography (EEG). However, studies of resting state EEG in adults with ADHD are scarce and yield inconsistent findings.EEG profiles, recorded during a resting-state with eyes-open and eyes-closed conditions, were compared for college students with ADHD (n = 18) and a nonclinical comparison group (n = 17).The ADHD group showed decreased power for fast frequencies, especially alpha. This group also showed increased power in the slow frequency bands, however, these effects were strongest using relative power computations. Furthermore, the theta/beta ratio measure was reliably higher for the ADHD group. All effects were more pronounced for the eyes-closed compared to the eyes-open condition. Measures of intra-individual variability suggested that brains of the ADHD group were less variable than those of controls.The findings of this pilot study reveal that college students with ADHD show a distinct neural pattern during resting state, suggesting that oscillatory power, especially alpha, is a useful index for reflecting differences in neural communication of ADHD in early adulthood. Attention-deficit/hyperactivity disorder (ADHD) is a pervasive mental health condition that is characterized by symptoms of inattention and hyperactivity. About 5% of children are estimated to meet the diagnosis of ADHD worldwide . Adults In the last few years, research investigating ADHD populations has used neurophysiological measures such as EEG oscillatory power to determine whether ADHD can be distinguished by specific neural abnormalities . NeuronaHowever, the specificity of this EEG power profile to adult ADHD, and its meaning, are under debate ,17. Simirelative beta power  because no group differences were found in the beta and alpha bands for absolute power. In contrast, Clarke et al., [Compared to studies of children with ADHD, research examining oscillatory power in adults with ADHD has been scarce and findings have been inconsistent. For example, in keeping with the child ADHD literature, a higher power in slow oscillations has been found for ADHD adults compared to healthy comparison groups in some studies ,25, but  et al.,  found hi et al., , whereas et al., ,27. No s et al., ,25,27,28The present pilot study investigated neural oscillatory power during a resting state in college students with ADHD and their normal healthy peers. To the best of our knowledge, this is the first study to investigate oscillatory power in college students with ADHD. This relatively successful subset, accepted into post-secondary education, continues to manifest cognitive and other functional impairments ,11. In asd = 4.27) were recruited from University Student Services and 17 normal healthy controls were recruited through campus advertisements . Inclusion criteria were 1) current enrollment in a post-secondary program, 2) a previous diagnosis of ADHD, and 3) registration with respective university or college Student Disability Services, which requires supporting documentation of a confirmed diagnosis of ADHD. All participants completed the Adult ADHD Self Report Scale (ASRS) to assess current symptoms of ADHD. Exclusion criteria were 1) uncorrected sensory impairment, 2) major neurological dysfunction and psychosis, and 3) current use of sedating or mood altering medication other than stimulants prescribed for ADHD. Among the clinical sample, 10 subjects (56%) were being treated with medication. Of those 10 subjects, 6 subjects were using stimulants only, 2 subjects were using a combination of stimulants and antidepressants, and 2 subjects were using a combination of stimulants, antidepressants, and other non-prescriptive medications. Participants were asked not to change their medication treatment when visiting the lab for assessment. Three participants had a comorbid learning disability, and one participant was diagnosed with anxiety and depression.Eighteen participants with ADHD  and all participants provided informed written consent prior to the start of the study.Participants were seated in a comfortable chair and fitted with a 129-channel EEG net . Acquisition started after impedances for all channels were reduced to below 50 kΩ in accordance with standard data collection procedures . Data weEach participant completed a number of standard questionnaires and tasks to assess current symptom impairment:The Adult ADHD Self-Report Scale (ASRS v1.1) is a reliable and valid scale for evaluating current ADHD symptoms in adults . The ASRThe Cognitive Failures Questionnaire (CFQ) measures self-reported failures in perception, memory, and motor function in everyday life. Twenty-five questions ask subjects to rank how often these mistakes occur .The Reading Fluency subscale of the Woodcock Johnson-III Tests of Achievement  was admiThe Digit Span subtest from the Wechsler Adult Intelligence Scale- Fourth Edition (WAIS-IV) was used to assess auditory-verbal working memory, as a crude index of executive function . The Digsd = 13.6) in the eyes-closed condition and 43 segments (sd = 12.7) during eyes-open, whereas the control group had an average of 43 segments (sd = 14.2) in the eyes-closed condition and 46 segments (sd = 9.5) during eyes-open. Groups did not significantly differ in segment count (p’s > .25). No subjects were discarded for meeting our cutoff criterion of less than 11 segments.Netstation  was used to filter  and segment the data into 2-second segments . Segments containing artifacts were removed using standard, automatic algorithms for the detection of eye blinks, eye movements, as well as large drifts, and spikes in the data. Segments containing more than 20% bad channels were automatically removed. In addition, all segments were visually inspected by a trained research assistant blind to the hypotheses. Bad channels were replaced by values interpolated from neighboring channel data. Across all subjects, an average of 1 channel needed to be replaced. The ADHD group had an average of 38 useful segments  for further analysis.In MATLAB, the data were average referenced, and a Fast Fourier Transform was run using the pwelch algorithm, with a 128 sample triangular window, to obtain time-frequency domain measures. Mean spectral estimates in various power bands  were computed. As a measure of intra-individual variability in neural activity, the standard deviation of the power in each 2-second trial was calculated for each band for each participant. To reduce unnecessary computation and multiple tests, we chose to extract data for 66 channels based on the standard EGI template.To examine potential outliers, the modified thompson-tau method was used. In addition to absolute power, the relative power of each frequency band  was also computed for the eyes-open as well as eyes-closed conditions to permit comparison with other studies. Independent sample t-tests were used to assess differences at each electrode between the ADHD and control groups. Data were presented in difference plots showing the t-value test statistic between groups at each electrode. Significant effects are indicated on the plots using dots at a .05 level, and using crosses when significance was reached using the conservative Bonferroni correction for multiple comparisons. This manner of presenting data instantly shows the direction as well as the significance of the differences found between groups, and provides an overview of the spatial variability of the data across the scalp.p’s < .001). Furthermore, the CFQ showed that the ADHD group reported significantly more general cognitive failures in their everyday life .The ASRS confirmed that the ADHD group, relative to the control group, exhibited more ADHD symptoms  reported more ADHD symptoms on the ASRS, at a trend level, than those who were not using medication , t(16) = 1.93, p = .07. It is possible that those subjects who were not using medication had less severe symptoms to begin with.Participants on medication (p’s < .05) between groups were found for slow as well as fast oscillations. Slow oscillations (theta) showed increased power for ADHD compared to the control group for anterior and lateral electrodes. However, lower power in slow oscillations was found in more central and posterior electrodes. As expected, the ADHD group exhibited lower power in the fast oscillations for all electrodes. This pattern of results also held when investigating those subjects with ADHD who used medication. A similar pattern of differences was found for the eyes-open condition, however, the increases in slow oscillations seemed less pronounced. Figure For the eyes-closed condition, significant differences using t-tests (p’s < .05) with, as expected, the eyes-closed condition showing increased alpha compared to eyes-open. A significant main effect of Eye Condition was also found for the Beta band, but only for electrode site Pz (p < .05). Significant Group by Eye Condition interactions were found for power in the alpha band at electrode site Fz, F = 7.83, p < .01, as well as Oz, F = 4.63, p < .05. Post-hoc analyses showed that the comparison group had a stronger decrease in alpha power compared to the ADHD group from eyes-closed to eyes-open. This is partly due to the comparison group having higher alpha power during the eyes-closed condition.To test whether the two Eye conditions  differed between ADHD and controls, a 2 (Group) x 2 (Eye Condition) Mixed Model ANOVA looking at electrode sites Fz, Cz, Pz, and Oz was conducted with medication as a covariate. A main effect of Eye condition was found in the alpha band for all electrodes . Mean, sample standard deviation, and intra-individual standard deviation values are presented in additional file 1 for each band and for each electrode site for eyes-closed  and beta (p < .001) band. We note that the strong decrease in alpha power for the ADHD group is indirectly driving the relatively larger contribution of slow wave oscillations shown in computations of relative power.To illustrate the contributions of various bands underlying the relative power differences, a power-by-frequency plot  can coordinate long-distance brain regions and function in larger temporal scales . Furtherrelative power in the slow frequency bands can be attributed to decreases in alpha. Last, the theta/alpha ratio showed even stronger group differences than the theta/beta ratio.Although the child ADHD literature mostly focuses on power in theta and beta bands ,14, it sWe suggest that the lower alpha seen in the ADHD group may be related to problems in attentional self-control. Recently, alpha power has been associated with active inhibition of external stimuli in a variety of tasks . This frless inter-trial variability in power, particularly in fast oscillations such as alpha. These results agree with emerging notions in the field suggesting low variability of metastable brain states are associated with less behavioural stability [The current study also investigated differences in oscillatory power between the eyes-open and eyes-closed conditions as well as the intra-individual variability of that power. Concerning eyes-open and eyes-closed conditions, based on our data we conclude that both conditions are relatively similar between groups, however, the effects of higher slow oscillatory power and lower fast oscillatory power seemed more pronounced during the eyes-closed condition for alpha in the ADHD group. It is possible that the eyes-closed condition is a better, or cleaner, reflection of intrinsic alpha oscillation because visual input from the thalamus during the eyes-open condition may 'disturb' alpha rhythms mediated by cortico-thalamic loops ,43. Conctability ,45. Indetability . We shalA limitation of this study is its relatively low sample size, which constrained our ability to investigate the effects of comorbidities. Furthermore, this study included subjects who were on medication. Questionnaire data indicated that ADHD symptomatology was stronger among those taking medication compared to those in the ADHD group, however, analyses demonstrated that subjects on medication showed a similar pattern of differences with the control group for absolute as well as relative power. Furthermore, we point out that previous studies have found that stimulants tend to normalize EEG oscillatory power on ADHD adults , suggestThese data suggest that the neurophysiological differences found between individuals with ADHD and their peers in childhood are also present in adulthood. The findings may help document the behavioral and neural nature of adult ADHD, which may eventually lead to a better understanding and treatment.ADHD: Attention-Deficit/Hyperactivity Disorder; EEG: Electroencephalography; ASRS: Adult ADHD Self Report Scale; CFQ: Cognitive Failures Questionnaire; WJ-III: Woodcock Johnson-III Tests of Achievement; WAIS-IV: Wechsler Adult Intelligence Scale- Fourth Edition.The authors declare that they have no competing interests.SW, ZL and RT contributed to the conception and design of the study. SW and JJ were responsible for data acquisition, performed the data processing and analyses, and drafted the manuscript. ZL assisted with the power analyses. RT and ZL helped critically revise the manuscript. All authors read and approved the final manuscript.Table A1. Mean  in absolute (mV2) and relative power for theta, alpha, and beta bands between the ADHD and control group for electrodes Fz, Cz, Pz, and Oz in the eyes-closed condition. Table A2. Mean (sample standard deviation) in absolute (mV2) and relative power for theta, alpha, and beta bands between the ADHD and control group for electrodes Fz, Cz, Pz, and Oz in the eyes-open condition. Figure A3. Variability in absolute Alpha power (in mV2) for electrode 40 in the eyes closed condition for ADHD subjects on, or off, medication. Figure A4. Power (in mV2) x Frequency plot for eyes-closed for Controls, ADHD participants who were on medication, and those ADHD participants who were not.Click here for file


In the last decade of the 20ynthesis . The devOrganic chemists are now provided with molybdenum and ruthenium complexes that present good application profiles for a range of metathesis reactions. Most of the ruthenium initiators can be handled in air and are compatible with functionalized substrates, such as unsaturated esters, amides, ketones, aldehydes and even alkenes bearing protic functions, e.g., hydroxy or carboxylic acid groups. While more sensitive towards air and moisture, molybdenum catalysts provide excellent results in stereoselective metathesis and are sometimes more active. Therefore these catalyst families can be used in a complementary fashion. Currently, olefin metathesis   is one oBeilstein Journal of Organic Chemistry, on “Progress in metathesis chemistry”. I would like to thank all authors for their excellent contributions. Enjoy reading them!The papers collected in this Thematic Series nicely highlight many of the aspects mentioned above and will certainly help to update the “state of the art” knowledge in this fascinating field. In this Thematic Series contributions from many of the leading practitioners in the area, which cover a wide range of topics related to alkene and alkyne metathesis, are presented. It is therefore a pleasure to serve as Guest Editor for this Thematic Series in the Karol GrelaWarsaw, November 2010